An improved method for the production and purification of adenoviral vectors

专利摘要:
PURPOSE: Provided is generally fields of cell culture and virus production, more particularly, it concerns improved methods for the culturing of mammalian cells, infection of those cells with adenovirus and the production of infectious adenovirus particles therefrom. CONSTITUTION: In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production with cells grown in serum-free conditions, and in particular inserum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsC1 banding using an ultracentrifuge.
公开号:KR20000057160A
申请号:KR1019990704446
申请日:1997-11-20
公开日:2000-09-15
发明作者:슈안 쟝；카푸시니 쓰윈；장 우；투욘 쵸
申请人:파커 데이비드 엘.；인트로겐 테라페티스, 인코퍼레이티드；
IPC主号:

专利说明:

An improved method for the production and purification of adenoviral vectors}
Adenovirus vectors expressing proteins used for treatment have been clinically evaluated for treating a variety of cancers, including cancers of the lungs, head, and neck. As clinical trials are underway, there is an increasing demand for clinically available adenovirus vectors. The expected annual requirement for clinical trials in 300 patients will be approximately 6 × 10 ¹⁴ PFU.
Typically, adenoviruses are made in commercially available tissue culture flasks or "cellfactories". Virus infected cells are obtained and freeze-thawed to release the virus from the cells in the form of crude cell lysate. The resulting unpurified cell lysate (CCL) is purified using double CsCl gradient ultracentrifugation. The virus obtained in 100 single tray cellfactories is approximately 6 × 10 ¹² PFU. Clearly, such a conventional process does not produce the required amount of virus. In order to meet viral requirements, it is necessary to increase production scale and develop new effective production and purification processes.
Purification via CsCl gradient ultracentrifugation is extremely limited and cannot meet the adenovirus vector requirements required for gene therapy methods. Therefore, in order to produce adenovirus vectors on a large scale, purification methods other than the CsCl gradient ultracentrifugation method need to be developed. Although chromatography is widely used to produce recombinant proteins, purification of the virus using chromatography is extremely limited. There are literatures that use size extrusion, ion exchange, and affinity chromatography to purify retroviruses, encephalitis viruses from ticks, and plant viruses, but their success has been different (Crooks, et al., 1990; Aboud, et al. , 1982; McGrath et al., 1978, Smith and Lee, 1978; O'Neil and Balkovic, 1993). There is also very little research on the purification of adenoviruses using chromatography. The reason for this lack of research is that adenoviruses do not have effective CsCl gradient ultracentrifugation purification.
Recently, Huyghe et al., (1996) report that adenovirus vectors were purified by combining metal chelate affinity chromatography and ion exchange chromatography. It has been reported that virus purity similar to that obtained through CsCl gradient ultracentrifugation was obtained. Unfortunately, only 23% of the virus was recovered after the double column purification process. The low virus recovery rate is due to the use of freeze / thaw steps to lyse the cells to release the virus from the cells and double column purification.
Clearly, there is a need for an effective, mass-produced method of producing adenovirus vectors that can provide high levels of productivity that can be met to meet the growing demand for such products.
Summary of the Invention
The present invention provides a new process for producing and purifying adenoviruses. This new production process not only provides viral purity comparable to that with CsCl gradient ultracentrifugation, but also provides scale and effectiveness.
Accordingly, the present invention provides a method for producing adenovirus, which grows host cells at low irrigation rates, infects host cells with adenovirus, obtains host cells, and hemolyses them to obtain unpurified cell lysate. Obtaining, concentrating the unpurified cell lysate, exchanging lysate buffer, and reducing the concentration of impurity nucleic acid in the lysate.
In certain embodiments, the method further comprises separating the adenovirus particles from the lysate using chromatography. In certain embodiments, the separating process consists essentially of one-step chromatography. In another embodiment, the chromatography step uses ion exchange chromatography. In certain suitable embodiments, ion exchange chromatography is performed using a pH 7.0 to 10.0 range. In a more suitable embodiment, the ion exchange chromatography is anion exchange chromatography. In certain embodiments anion exchange chromatography utilizes DEAE, TMAE, QAE, PEI. In another suitable embodiment, anion exchange chromatography uses Toyopearl Super Q 650M, MonoQ, Source Q, Fractogel TMAE.
In certain embodiments of the invention, the glucose concentration of the medium is maintained at 0.7 to 1.7 g / l. In certain other embodiments, diafiltration was used to exchange the buffer.
In certain embodiments of the invention, the adenovirus consists of adenovirus vectors that encode exogenous gene constructs. In certain embodiments, the genetic construct is linked to a promoter. In an embodiment, the promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late promoter, polyoma F9-1, tyrosinase and the like. In certain embodiments of the invention, the adenovirus is a replication-incompetent adenovirus. In another embodiment, the adenovirus lacks at least a portion of the E1-part. In another aspect, adenoviruses lack some of the E1A or E1B moieties. In certain embodiments, the host cell is 293 cells.
In certain embodiments of the invention, the exogenous gene construct encodes a gene that is therapeutically effective. For example, therapeutic genes include antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fms, antisensejun, antisense trk, antisense ret, antisense gsp, antisense hst, antisense bcl, antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC , NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL Encodes -5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF G-CSF, thymidine or p53.
In certain embodiments of the invention, cells are obtained and lysed in ex situ using low resolution solutions, hypertonic solutions, freeze-thaw, sonication, impingement jets, microfluidization reactions or surfactants. In another aspect, cells are obtained and lysed in situ, using hypotonic solutions, hypertonic solutions or surfactants. As used herein, "in situ" refers to a cell located in a tissue culture device such as CcllCube ^™, and "ex situ" refers to a cell taken out of a tissue culture device.
In certain embodiments, the cells are lysed and obtained using a surfactant. In a suitable embodiment, the surfactant is Thesit ^R , NP-40 ^R , Tween-20 ^R , Brij-58 ^R , Triton X ^R- 100 or octyl glucoside. In another feature of the invention, lysis is carried out through autolysis of infected cells. In another feature of the invention, the cell lysate is treated with Benzonase ^R , Pulmozyme ^R.
In certain embodiments, the step of concentrating with membrane filtration is further included. In certain embodiments, the filtration uses filtration moving in a tangential direction. In a suitable embodiment, the filtration uses 100 to 300K NMWC, regenerated cellulose or polyether sulfone membranes.
The present invention cultures host cells in medium at low irrigation rates, infects adenoviruses with host cells, obtains and lyses host cells to produce crude cell lysates, concentrates them, exchanges buffers of lysates, It provides an adenovirus made according to a process consisting of reducing the concentration of impurity nucleic acids in the lysate.
Another feature of the present invention provides a method for purifying adenovirus, which grows host cells, obtains host cells, contacts surfactants to the cells to obtain cell lysates, and concentrates the lysates. By exchanging a buffer of lysate and reducing the concentration of impurity nucleic acid in the cell lysate.
In certain embodiments, the surfactant may be Thesit ^R , NP-40 ^R , Tween-20 ^R , Brij-58 ^R , Triton X-100 ^R or octyl glucoside. More suitably the surfactant is present in the dissolution solution at a concentration of about 1% (w / v).
In another aspect of the invention, the invention provides for growth of host cells, infection of adenoviruses with host cells, contact with a surfactant to obtain cells, lysis to obtain cell lysates, concentration thereof, and cell lysis. The buffer of material is exchanged to provide an adenovirus produced by a process consisting of reducing the concentration of impurity nucleic acid in the cell lysate.
In another embodiment, the present invention provides a method for purifying adenovirus, which grows host cells in serum-free medium, infects adenovirus with host cells, obtains and lyses host cells, Obtaining, concentrating, and exchanging buffers of cell lysates, thereby reducing the concentration of impurity nucleic acids present in the cell lysates. In suitable embodiments, the cells may be grown in cell suspension culture or fixed-dependent culture, and the like.
In certain embodiments, the host cell is suitable for growing in serum-free medium. In a more suitable embodiment, a method of adapting to growth in serum-free medium is to continuously slow down fetal bovine serum content in the growth medium. More specifically, serum-free medium consists of fetal bovine serum content of less than 0.03% v / v.
In another embodiment, there is further provided a method of separating adenovirus particles from cell lysate using chromatography. In a suitable embodiment, the method of separation consists of one step chromatography. More specifically, the chromatography step is ion exchange chromatography.
The invention also grows host cells in serum-free medium, infects adenoviruses with host cells, obtains and lyses host cells to obtain cell lysates, concentrates them, and exchanges buffers of cell lysates, It relates to an adenovirus produced according to a process for reducing the concentration of impurity nucleic acids present in cell lysates.
The invention also provides 293 host cells suitable for growing in serum-free medium. In certain aspects, a method to be suitable for growth in serum-free medium is to continuously slow down fetal bovine serum content in the growth medium. In certain embodiments, the cells are suitable for growing in suspension culture. In certain embodiments, cells of the invention are referred to as IT293SF cells. These cells were deposited with the American Tissue Culture Collection (ATCC) under the Budapest Agreement, which allowed microorganisms to be deposited with international depositors for patent purposes. These cells were represented in 1997 by Introgen Therapeutics, Inc. (Houston, Tx). It was deposited by Shuyuan Zhang. The IT293SF cell line was derived from the adaptation of the 293 cell line to serum free suspension culture as described herein. These cells are grown in IS 293 serum free medium (Irvine Scientific. Santa Ana, Ca) supplemented with 100 mg / l heparin, 0.1% pluronic F-68 and can be infected with human adenovirus.
Other objects, features and advantages of the invention will be apparent from the following detailed description. However, it will be appreciated that the description, the specific embodiments, are illustrative and that various changes and modifications are possible within the scope of the invention.
This application is a continuation of US Patent Application 60 / 031,329, filed November 20, 1997. The above is incorporated by reference in its entirety.
The present invention relates to the field of cell culture and virus production. More specifically, it relates to improved methods of culturing mammalian cells, infecting these cells with adenoviruses, and producing infectious adenoviruses from these cell cultures.
The following drawings, which are part of the invention, are attached to illustrate the features of the invention. The present invention will be better understood with reference to the drawings in conjunction with the detailed description provided herein.
1A and 1B show HPLC profiles of viral solutions according to medium irrigation rate. Figure 1A is a high speed, Figure 1B is a low speed.
FIG. 2 shows HPLC of unpurified cell lysate (CCL) of CellCube ^™ (straight A ₂₆₀ ; dashed A ₂₈₀ ).
3A, 3B, 3C, 3D, 3E show HPLC profiles from CellCube ^™ using different surfactants. 3A; Thesit ^R. 3B; Triton ^R X-100; 3C; NP-40 ^R. 3D; Brij ^R 80. Figure 3E; Tween ^R 20 is used. Surfactant concentration was 1% (w / v), dissolution temperature; Room temperature (straight line A ₂₆₀ ; dotted line A ₂₈₀ )
4A and 4B show the HPLC profiles of the viral solution before benzonase treatment (FIG. 4A) and after treatment (FIG. 4B) (straight line A ₂₆₀ ; dashed line A ₂₈₀ ).
5 shows the HPLC profile of the viral solution after benzonase treatment in the presence of 1 M NaCl (straight line A ₂₆₀ ; dashed line A ₂₈₀ ).
Figure 6 shows purification of AdCMVp53 virus in Buffer A [20 mM Tris + 1 mM MgCl ₂ + 0.2 M NaCl, pH = 7.5].
Figure 7 shows AdCMVp53 virus purification in buffer A [20 mM Tris + 1 mM MgCl ₂ + 0.2 M NaCl, pH = 9.0].
8A, 8B, 8C show HPLC analysis of aliquots obtained during purification. 8A is fraction 3, FIG. 8B is fraction 4, and FIG. 8C is fraction 8. FIG. (Straight line A ₂₆₀ ; dotted line A ₂₈₀ )
Figure 9 shows purification of AdCMVp53 in the presence of buffer A [20 mM Tris + 1 mM MgCl ₂ + 0.3 M NaCl, pH = 9].
10A, 10B, 10C, 10D, and 10E show HPLC analysis of CsCl purified virus and virus aliquots obtained during purification. 10A shows an unpurified virus solution; 10B shows a penetrating fluid; 10C shows Peak # 1; 10D shows peak # 2; 10E shows virus purified using CsCl (straight A ₂₆₀ ; dashed A ₂₈₀ ).
11 shows the HPLC purification profile obtained on a 5 cm id column.
Figure 12 shows the major adenovirus structural proteins detected in SDS-PAGE.
Figure 13 shows BSA concentration in purified virus as detected by Western blot test.
FIG. 14 shows chromatography of unpurified cell lysate made on CellCube ^™ .
Figure 15 shows the elution profile of the treated virus solution purified by the method of the present invention using Toyopearl SuperQ resin.
16A and 16B show HPLC analysis of virus fractions obtained from the purification process. 16A is an HPLC profile of virus fraction taken in the first purification step. 16B is an HPLC profile of virus fraction taken in the second purification step (straight line A ₂₆₀ ; dashed line A ₂₈₀ ).
Figure 17 shows the virus solution obtained by purification with 1% Tween ^R in a slow medium irrigation.
18 shows HPLC of virus aliquots under slow medium irrigation.
19A, 19B, 19C show column analysis of purified virus. 19A shows the SDS-PAGE analysis, and FIG. 19B shows the Western blot of BSA. 19C shows nucleic acid slot blots for determining contaminated nucleic acid concentrations.
20A, 20B, 20C, 20D, 20E, and 20F show performance studies of Toyopearl SuperQ 650M resins. 20A shows 1: 1 flow and drop ratio, FIG. 20B shows 1: 1 drop ratio of purified virus, and FIG. 20C shows 2: 1 flow and drop ratio. FIG. 20D shows a 2: 1 loading ratio of purified virus and FIG. 20E shows a 3: 1 flow and drop ratio. 20F shows a 3: 1 loading ratio of purified virus (straight line A ₂₆₀ ; dashed line A ₂₈₀ ).
Figure 21 is an equal density CsCl centrifuge column purified virus.
FIG. 22 shows HPLC profiles of native virus present in column purified virus A, native virus B, defective virus (straight line A ₂₆₀ ; dashed line A ₂₈₀ ).
23 shows a flow chart of the production and purification process of AdCMVp53.
Adenovirus vectors have been successfully used for eukaryotic gene expression and vaccine development. Recently, recombinant adenoviruses have been used for gene therapy through animal studies. Recombinant adenoviruses have been administered to a variety of other tissues, demonstrating the usefulness of adenovirus vectors for treatment. These findings have led to the use of these vectors in human clinical trials. There is an increasing demand for the use of adenovirus vectors for various treatments. Currently available technologies do not meet these demands. Accordingly, the present invention provides a method for producing a large amount of adenovirus that can be used for such treatment.
The present invention is a process developed to produce and purify replication defective recombinant adenoviruses. The production process uses Cellcube ^™ bioreactors for cell growth and virus production. The given irrigation rate used during cell growth, the virus production phase for culturing, has been found to have a significant effect on virus later purification. More specifically, lowering the medium irrigation rate improves virus production. In addition, using a lysis solution consisting of a buffered surfactant used to lyse the cells in Cellcube ^™ at the end of virus production can improve the process. With these two advantages, the obtained crude virus solution can be purified via single ion exchange chromatography after concentration / diafiltration and nuclease treatment to reduce the impurity nucleic acid concentration in the crude virus solution. . Virus purified by column is similar to the purity of virus purified using double CsCl gradients. The overall recovery of the viral product is 70% ± 10%. This is a significant improvement over the results reported by Huyghe et al. (1996). Compared with the dual CsCl gradient ultracentrifugation, column purification has the advantage of being consistent, scalable, valid, faster and less expensive. This new process is a significant improvement in the production of adenovirus vectors for use in gene therapy.
Accordingly, the present invention aims to provide these advantages in large scale culture systems with the aim of producing and purifying adenovirus vectors. Various components in such a system, methods for producing adenoviruses using the same are described in detail below.
1. Host cell
A) cells
In a suitable embodiment, the development and proliferation of adenovirus vectors varies depending on the unique helper cell line (called 293), wherein 293 is structurally E1 transformed from human embryonic kidney cells with adenovirus serotype 5 (Ad5) DNA fragments. Express proteins (Graham et al., 1977). Since the E3 moiety is absent from the Ab genome (Jones and Shenk, 1978), with the help of 293 cells the current Ad vector has foreign DNA in E1 or E3 or E1, E3 (Graham and Prevec, 1991; Bett et al. , 1994).
One aspect of the invention is to provide a recombinant cell line that expresses part of the adenovirus genome. These cell lines can support the replication of helper viruses and adenovirus recombinant vectors that have defects in certain adenovirus genes, which means, for example, "allowing" the growth of these viruses and vectors. Such recombinant cells are sometimes referred to as helper cells because of their ability to compensate for or support replication of a replication-adaptive adenovirus vector. The prototype of the adenovirus helper cells is the 293 cell line, which contains the adenovirus E1 moiety. 293 cells support the replication of adenovirus vectors that lack E1 function by providing in trans the E1-active elements required for replication.
The helper cells according to the invention can be derived from mammalian cells, suitably from primate cells such as human embryonic kidney cells. Although various primate cells are suitable, human or human embryonic kidney cells are most suitable, and cell types capable of supporting viral replication may also be used in the present invention. Other cell types include, but are not limited to, Vero cells, CHO cells, or any eukaryotic cells that can be made using tissue culture techniques in which the cells are adenovirus acceptable. By "adenovirus tolerant" is meant that an adenovirus or adenovirus vector can complete the entire intracellular viral life process within the cellular environment.
Helper cells can be derived from existing cell lines such as 293 cells or made from de novo. Such cell lines express adenovirus genes essential for compensating for in trans deletions in the adenovirus genome or support replication of other defective adenovirus vectors such as E1, E2, E4, E5 and later functions. A specific portion of the adenovirus genome, ie the E1 portion, was used to create complementary cell lines. Regardless of the combined or episomal type, if a portion of the adenovirus genome lacking a viral origin for replication is introduced into a cell line, the cell cannot replicate even if it is infected with the wild type adenovirus. In addition, the late function of adenovirus is not fully expressed in the cell line because Major Late Unit transcription occurs after viral DNA replication. Thus, the E2 portion that overlaps with late function (L-5) is provided by the helper virus and not the cell line. In general, the cell line according to the present invention expresses E1 or E4.
As used herein, a "recombinant" cell refers to a cell into which a gene derived from an adenovirus genome or the genome of another cell is introduced into the cell. Thus, recombinant cells are distinguished from naturally-occurring cells that do not have a gene introduced by recombination. Recombinant cell refers to a cell having a gene introduced through a "human hand".
The uninfected cell layer or cells are contacted with one or more helper viruses or virus particles and the cells are cultured to determine replication. The formation of viral flocs or cell free zones in the cell layer is due to the lysis of cells by the expression of certain viral products. Cell lysis indicates that the virus has replicated.
Other useful mammalian cell lines used to convert host cells to competence for replication competence viruses or for replication defective viruses include Vero, HeLa cells, Chinese hamster ovary cell lines, W138, BHK, COS-7, HepG2, 3T3, RIN, MDCK cells.
B) Growing on Selective Medium
In certain embodiments, it is beneficial to use a selection system that excludes unwanted cell growth. Selective markers can be used to permanently transform a cell line or to infect or transduce a cell line with a viral vector encoding a selective marker. In any case, culturing transformed or transfected cells with the appropriate drug or selection compound results in collecting only a population of cells that have only the marker.
Such markers include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase genes in tk-, hgprt- and aprt- cells, respectively. Also dhfr, which provides resistance to methotrexate, gpt, which provides resistance to mycophenolic acid; Neo which provides resistance to aminoglycoside G418; Anti-metabolite resistance, such as hygro, which provides resistance to hygromycin, can be used.
C) Growth with serum removed
Recombinant proteins (Berg, 1993) and viral vaccines (Perrin, 1995) were used to produce serum-free methods to adapt fixed dependent cells to serum-free suspension culture. Until recently, little has been reported about the adaptation of 293A cells to serum-free suspension culture. Gilbert reports that 293A cells were adapted to serum-free suspension culture for adenovirus and recombinant protein production (Gilbert, 1996). Using similar adaptation methods, A549 cells were adapted to serum-free suspension culture for adenovirus production (Morris et al., 1996). The cell specific virus production produced in the adapted suspension cells was about 5-10 times less than that obtained in the attached parental cells.
Using a similar serum removal procedure, the inventors have successfully adapted 293A cells to serum-free suspension culture (293SF cells). In this process, 293 cells were adapted to commercially available 293 medium while continuously lowering the FBS concentration in the T-flask. Briefly, the initial serum concentration in the medium was 10% FBS DMEM medium in a T-75 flask, which was subsequently adapted to cells in serum-IS 293 medium in a T-flask by lowering the FBS concentration in the medium. After six passages to the T-75 flask, FBS% was identified as about 0.019%, 293 cells. Pass them two or more times in the T flask before transferring to the spinner flask. From the results described below, it can be seen that the cells are sufficiently grown in serum-free medium (IS293 medium, Irvine Scientific, Santa Ana, Calif.). The average doubling time of the cells is 18-24 hours, which is the time to reach a concentration of 4-10 x 10 ⁶ cells / ml of stagnant cells without medium exchange.
D) Adaptation of Cells to Suspension Cultures
Using two methods, 293 cells are adapted to suspension culture. Graham was passaged three times in nude mice to adapt 293A cells to suspension culture (293N3S cells) (Graham, 1987). Suspension 293N3S cells have the ability to support E1 adenovirus vectors. However, Garnier et al., (1994) found that 293N35 cells have a relatively long initial lag phase in suspension, slow growth rate and strong tendency to aggregate.
The second method used is the gradual adaptation of 293A cells to suspension growth (Cold Spring Harbor Laboratories, 293S cells). Garnier et al. (1994) report using 293S cells to produce recombinant proteins from adenovirus vectors. The authors found that 293S cells were less prone to aggregation in calcium-free media and significantly increased protein production when exchanged with fresh media at the time of virus infection. It was also found that glucose was a limiting factor in culture without medium exchange.
In the present invention, 293 cells adapted to grow in serum-free conditions are also adapted to suspension culture. Cells are transferred to a 250 ml spinner suspension incubator (100 ml working volume) without serum for suspension culture between an initial cell density of between 1.18E + 5 vc / ml and 5.22E + 5 vc / ml. Heparin is added to the medium to prevent cell aggregation. Such cell culture systems increase cell density to some extent while maintaining cell viability. Once these cells have grown in culture, passage the cells at least about seven times in a spinner flask. The doubling time of the cells was gradually reduced to about 1.3 days by the end of the serial passage, which is similar to 1.2 days of doubling of the cells in 10% FBS medium in the attached cell culture. In the case of serum-free IS 293 medium supplemented with heparin, the cells present in the suspension culture are hardly aggregated and are present individually.
2. Cell Culture System
The ability to produce infectious viral vectors has emerged in the pharmaceutical industry, particularly in gene therapy. Over the last decade, advances in biotechnology have produced a number of important viral vectors that can be used for therapy, vaccines, and protein production. The use of these viral vectors in mammalian culture has advantages when compared to bacteria or other life-threatening proteins, such as complex structures that occur after translation such as folding structures and glycosylation following disulfide bonds. Protein structure can be made.
Biochemistry and cell mechanisms associated with the development of molecular biology techniques such as design and construction of highly effective vector systems for mammalian cell cultures, useful screening marker batteries, gene amplification processes, and preparation of final biologically active molecules from introduced vectors. By better understanding, cell cultures for producing viral vectors have been developed.
Frequently, prior to selecting a cell line as a host for the expression system, no factors influencing downstream processes (in this case after cell lysis) of increasing production scale were taken into account. In addition, the development of bioreactor systems capable of maintaining cultures at very high densities for long periods of time did not meet the desire to increase production at low cost.
The present invention provides the advantages of recently available bioreactor technology. By growing the cells in a bioreactor according to the present invention, it is possible to make a large scale of cells with biologically complete activity that can be infected by the adenovirus vector of the present invention. By operating the system at a low irrigation rate and applying other procedures for purifying infected particles, the present invention provides a purification method that can easily produce large quantities of high purity products.
Bioreactors have been widely used to produce biological products from suspended and fixed dependent animal cell cultures. The most widely used cells for producing adenovirus vectors are fixed dependent human embryonic kidney cells (293 cells). Bioreactors developed for the production of adenovirus vectors are characterized by large volume-specific culture surface areas, resulting in high density production cells and high virus yields. Microcarrier cell cultures in stirred tank bioreactors have been used to make viral vaccines, providing a large volume specific culture surface area (Griffiths, 1986). It has also been demonstrated that stirred tank reactors can industrially increase production scale. The multi-plate Cellcube ^™ cell culture system manufactured by Corning-Costar also has a very large volume-specific culture surface area. Cells grow on both sides of the culture plate and are sealed in the shape of a compressed cube. Unlike tank bioreactors with agitation, Cellcube ^™ culture units can be discarded after use. This is highly desirable for early stage production of clinical products, as the cost savings, quality control and quality assurance costs are reduced for processable systems. Given the apex provided by the other systems, the adenoviral virus was produced, and the stirred tank bioreactor and the Cellcube ^™ system were evaluated.
A) Fixed non-dependent culture for fixed dependence
Animal and human cells can be propagated in two ways; Freely cultivate non-fixed dependent cells in suspension via mass culture; Or attaching fixed dependent cells in solid support (for monolayer cell growth) for their proliferation.
The most commonly used as a means for mass production of cells and cell products is to use non-fixed dependence or suspension culture in continuous chemotaxis cells. Large scale suspension cultures using microbial (bacterial and yeast) fermentation techniques are clearly beneficial for producing mammalian cell products. This process is relatively simple to operate and scale up. A homogeneous state is provided to accurately monitor and control the temperature, dissolved oxygen, pH, etc. in the reactor and a representative sample of the culture can be taken.
However, suspension cultured cells are not always used to make biological material. Suspension cultures still have tumor potential, and in production, their use as substrates is limited to those used in humans and animals (Petricciani, 1985; Larsson, 1987). In contrast to fixed-dependent cultures, viruses propagated in suspension cultures, in some cases, rapidly change viral markers, thereby reducing immunogenicity (Bahnemann, 1980). Finally, recombinant cell lines secrete significant amounts of product when grown in fixed-dependent cultures as compared to suspensions of the same cells (Nilsson and Mosbach, 1987). For this reason, various types of fixed-dependent cells are widely used to produce other biological products.
B) Suspension Reactor and Process
Mammalian cultures were subjected to large scale suspension culture in a stirred tank. Bioreactor mechanisms and controls have adopted the fermenter design used in the relevant microbial field. However, in culturing mammalian cultures slowly, as the demand for contamination control increases, the improved sterilization design is quickly implemented, improving the dependence of the reactor. The instrument and control method are essentially the same as for other fermenters, including stirring, temperature, dissolved oxygen and pH control. More advanced probes that can measure turbidity (function on existing particles), capacity (function on living cells present), glucose / lactate, carbonate / bicarbonate, carbon dioxide, etc. on- and off-line; Analyzers are available. The maximum cell density obtainable in suspension culture is about 2-3 × 10 ⁶ cells / mL, which is relatively lower than the number obtained in microbial fermentation (which is less than 1 mg of dry cell weight per mL).
Two suspension culture reactor designs, agitated reactors and airborne reactors, are most widely used in the industry because they are simple and robust. Stirring reactor designs have been used to increase the capacity up to 8000 L to produce interferon (Phillips et al., 1985; Mizrahi, 1983). Height; Cells are grown in stainless steel tanks with a diameter ratio of 1: 1 to 3: 1. Cultures are mixed using one or more stirrers in the form of sharp disks or propellers. The stirrer has less shearing force than using a blade. Stirring is done either directly or in an indirect manner driven by coupling magnets. Indirect drive can be sealed on the stirrer shaft to reduce the risk of microbial contamination.
Airborne reactors used early in microbial fermentation and in mammalian culture utilize airflow to mix the culture and provide oxygen. Airflow enters and circulates in the vertical portion of the reactor. The gas on the culture surface causes dense bubbles, free of liquid, to move down the reactor. The main advantage of this design is that it is simple and does not require mechanical mixing. In general, the ratio of diameter to height is 10: 1. Increasing the size of the airborne reactor is relatively easy, good mass transfer by gas, and relatively low shear forces occur.
Most large-scale suspension cultures are operated in batch or fed-batch processes because they are the simplest to scale up and operate. However, it is possible to use a chemostat or a continuous process using the irrigation principle.
The batch process is a closed system where a typical growth profile can be seen. Followed by the lag followed by the exponential, stagnant and declining phases. In such systems, the environment is constantly changing because nutrients are depleted and metabolites accumulate. This allows the analysis of factors affecting cell growth and productivity, optimizing the process. By regulating the supply of key nutrients, the growth process can be extended to increase the productivity of the batch process. This fed-batch process is still a closed system because cells, products and waste are not removed.
In the case of a closed system, cells are maintained in a variety of devices (fine mesh spin filter, porous filter, flat plate membrane filter, settling tube) to irrigate the fresh medium to the culture. Spin filter cultures produce a density of about 5 × 10 ⁷ cells / ml. In practice, open systems and simple irrigation processes are material environmental control devices that introduce media and cells and products. The culture medium is fed into the reactor at a predetermined constant rate that maintains the dilution ratio of the culture at a value less than a certain maximum production rate of the cells (to prevent cell material from being washed out of the reactor). Supply. Culture fluids containing cells, cell products, and by-products are also removed at the same rate.
C) irrelevant attachment systems
Traditionally, fixed-dependent cell cultures grow at the bottom of small glass or plastic containers. Traditional techniques have limited volume ratios for surfaces suitable for laboratory scale, hindering the production of cells and cell products in large quantities. Several techniques have been proposed in an attempt to provide a system that provides a large surface area for cell growth at small culture volumes; Roller bottle system, stacked plate propagator, spiral film bottles, hollow fiber system, packed bed, plate exchanger system ), Membrane tubing gel. Because these systems are non-homogeneous in nature, in some cases based on a multi-step process, there are several drawbacks: limitations in increasing scale, difficulty in obtaining cell samples, limitations in measuring and controlling Chuo process variables, uniformity during incubation Difficulties in maintaining environmental conditions.
Despite these drawbacks, the roller bottle system is a commonly used process for the production of fixed-dependent cells on a large scale. Like large T-flasks of different shapes, the simplicity of the system is very reliable and attractive. A fully automated robot can control thousands of roller bottles per day, reducing the risk of contamination and significantly reducing manpower. If medium is changed frequently, roller bottle cultures yield cell densities close to 0.5 x 10 ⁶ cells / cm 2 (which is equivalent to 10 ⁹ cells per ml or nearly 10 ⁷ cells per ml culture medium).
D) Culture on Microcarrier
In an attempt to overcome the shortcomings of traditional fixed-dependent culture processes, van Wczel (1967) introduced the concept of a microcarrier culture system. In such a system, cells are propagated on the surface of small solid particles suspended in growth medium by slow stirring. The cells attach to the microcarrier, grow gradually, and join the microcarrier surface. In fact, such large-scale cultivation systems have improved adhesion dependent cultivation from a single disc process to a unit process where the unit process is a combination of monolayer and suspension cultures. Thus, the production is increased by combining the surface areas essential for cells to grow to have the advantage of homogeneous suspension culture.
There are several advantages of microcarrier culture over most other fixed-dependent large scale culture methods. First, microcarrier cultures have a high volumetric ratio to surface area (variable by varying carrier concentrations), which provides high density cell production and the possibility of obtaining highly concentrated cell products. Cell productivity is increased to 1-2 × 10 ⁷ cells / ml when the cultures are propagated in irrigated reactors. Second, instead of using many small, low productivity vessels (eg, flasks or dishes), they are grown in one unit process vessel. This results in improved nutrient availability and significant savings in culture medium. In addition, by propagating in one reactor, it is possible to reduce the installation space, the number of operation steps required per cell, thereby reducing labor costs and the possibility of contamination. Third, it is possible to monitor and control environmental conditions (concentrations of pH, pO ₂ and media components, etc.) with a well-mixed homogeneous microcarrier suspension culture, leading to more regeneration of cell proliferation and regeneration of product recovery. have. Fourth, it is possible to take representative samples for microscopy, chemical testing, and calculations. Fifth, since the microcarrier is easily precipitated from the suspension, it can be easily carried out using a feed-batch process or recovering cells. Sixth, the microcarrier-dependent culture propagation method involves cell manipulation such as delivering cells without the use of proteolytic enzymes, coculture of cells, transplantation into animals, glass bottles, columns, and fluidization that can hold microcarriers. It can be used for irrigation of cultures using prepared needles or pupil fibers. Seventh, microcarrier culture can be relatively easily increased in its culture scale using conventional equipment used to culture microorganisms and animal cells in suspension.
E) Microencapsulation of Mammalian Cells
One particularly useful method for culturing mammalian cells is microencapsulation. Mammalian cells are retained within the semipermeable hydrogel membrane. Porous membranes form around the cells, exchanging nutrients, gases, metabolic products, and bulk media around the capsules. Several methods have been developed that are soft, rapid and non-toxic, and the resulting membrane is strong and porous enough to sustain the growth of cellular material during culture. This method is based on soluble alginate gelled in contact with a solution comprising calcium. Lim (1982, US Pat. No. 4,352,883) describes the concentration of cells in about 1% sodium alginate, pushed through small pores to form droplets, then freely breaking into about 1% calcium chloride solution. The droplet then enters a layer of polyamino acid that ionic bonds to the surface alginate. Finally, the alginate is liquefied again by treating the drops with a chelating agent to remove calcium ions. Another method is to drop the cells in the calcium solution dropwise into the alginate solution and create a pupil alginate sphere. A similar method involves dropping cells in a solution of chirosan dropwise with alginate to create a pupil sphere.
Microencapsulated cells are easily propagated in a stirred tank reactor so that beads of 150-1500 μm diameter can be easily maintained in the irrigated reactor using a fine mesh screen. The total medium volume for the capsule volume is maintained at 1: 2 to 1:10. When the cell density in the capsule is about 10 ⁸ , the effective cell density for the culture is 1-5 × 10 ⁷ .
The advantages of the microcapture process over other processes are that they can be protected from the harmful effects of shear stresses from sparging and agitation, and the beads can be easily maintained and scaled up for the purpose of using irrigation systems. It is easy and transplant beads can also be used.
The present invention includes cells that are fixed-dependent in nature. For example, when 293 cells are fixed-dependent and grow in suspension, the cells are adsorbed to each other to form agglomerates, and eventually, by nuclear culture conditions. When it reaches an unstable size, the cells in the inner nucleus of each mass suffocate. Thus, the efficient use of these cells to produce large amounts of adenoviruses requires an effective means of culturing fixed-dependent cells on a large scale.
F) irrigated attachment system
A irrigated attachment system is a suitable form of the present invention. Irrigation refers to a continuous flow at a constant rate through a cell or cell population, which means that the cells are retained in the culture unit, as opposed to a continuous-flow culture in which cells are washed with recovered culture medium. Since the concept of irrigation was introduced at the beginning of the 20th century, it was mainly used to maintain small manipulation tissue for extensive microscopy. It mimics the in vivo cellular environment in which cells are constantly supplied with blood, lymph, or other body fluids. Without irrigation, the cells in the culture undergo feeding and starvation and are unable to fully exploit their growth and metabolism.
Currently used irrigation cultures are a response to the challenge of cells growing at high density (0.1-5 × 10 ⁸ cells / ml). To increase the density above 2-4 × 10 ⁶ cells / mL, the medium must be continuously supplied with fresh medium to supplement nutrient deficiencies and remove toxic products. Irrigation allows better control of the culture environment (pH, pO ₂ , nutrient levels, etc.) and significantly increases the surface area utilization in the culture for cell attachment.
The development of irrigated nested needle reactors using a non-knitted needle matrix provides a means for maintaining irrigation culture at densities of needle volumes in excess of 10 ⁸ cells / ml (CelliGen ^™ , New Brunswick Scientific, Edison, NJ; Wang et al., 1992; Wang et al., 1993; Wang et al., 1994). In brief, such a reactor consists of an improved reactor to culture both fixed-dependent and non-fixed dependent cells. The reactor was made of overlapping needles with internal recirculation means. Suitably the fiber matrix carrier is placed in a basket in the reaction vessel. Holes in the bottom and top of the basket allow the media to flow into the basket. Specially designed impellers allow the medium to be recirculated in the space occupied by the fiber matrix to provide a uniform supply of nutrients and to remove waste. This simultaneously allows a small amount of total cellular material to be suspended in the medium. Combining the basket and the recycling means provides a bubble-free flow to the oxygenated medium through the fiber matrix. The fiber matrix is a non-woven fiber having pores with a diameter of 10 μm to 100 μm, providing an internal pore volume corresponding to 1-20 times the volume of individual cells.
Compared with other culture systems, this method offers several important advantages. In the case of the fiber matrix carrier, the cells are protected from mechanical stress such as stirring and bubble generation. Freely moving media through the basket provides cells with adequately regulated oxygen, pH, and nutrients. The product is continuously removed from the culture, and the product obtained is free of cells and produced in medium with low protein content to carry out the subsequent purification step. The unique design of such a reaction system also provides an easy way to scale up the reactor. Currently available up to 30ℓ. Some 100 L and 300 L are being developed and theoretically calculated can scale up to 1000 L reactors. This technique is described in WO 94/17178 (August 4, 1994, Freedman et al.), Which is hereby incorporated by reference.
Cellcube ^™ (Corning-Costar) module provides a large styrene surface for growth and fixation of substrate attached cells. Several side-by-side culture plates are combined to create an integrally collected sterile disposable device that creates a thin sealed lamina flow space between adjacent plates.
Cellcube ^™ modules have inlet and outlet ports facing each other diagonally to help regulate media flow. In the first few days of growth, the culture is generally satisfied with the medium maintained in the system after the initial inoculation. Initial inoculation and media irrigation start time depend on the cell density and cell growth rate in the inoculation medium. By measuring the concentration of nutrients in the circulating medium, the culture state can be well understood. In establishing the process, the nutritional composition is monitored at various irrigation rates to determine the most economical and productive operating parameters.
Higher than the density of cells in the system and the solution density (cells / ml) of a conventional culture system. The most commonly used basal medium is designed to support 1-2 × 10 ⁶ cells / ml / day. A typical Cellcube ^™ with an 85,000 cm 2 surface area contains about 6 l medium in the module. Cell density exceeds 10 ⁷ cells / ml in the culture vessel. At confluence, 2-4 reactor volumes of medium per day are required.
The time and parameters of production of the culture depend on the specific cell line type and use. Production requires a different medium for many cultures beyond that required for the growth phase of the culture. Transitioning from one phase to another requires multiple wash steps in conventional culture. However, the Cellcube ^™ system uses an irrigation system. One of the advantages of using such irrigation systems is to provide flexible transitions between the various operating phases. The irrigation system does not require conventional washing steps to remove serum components from the growth medium.
In an embodiment of the invention, cells transfected with AdCMVp53 were grown using the CellCube ^™ system. 293 cells were seeded in Cellcube ^™ according to the manufacturer's recommendations. Inoculated cell density is in the range of 1-1.5 × 10 ⁴ / cm 2. Cells were grown for 7 days at 37 ° C. under pH = 7.20, DO = 60% air saturation culture conditions. Medium irrigation rate is controlled according to the glucose concentration in the CellCube ^™ . One day before virus infection, the irrigation medium is changed from 10% FBS to 2% FBS. On day 8 the cells are infected with the virus, with a MOI of 5. Medium irrigation is stopped for 1 hour immediately after infection and resumed during the virus production phase. Cultures are obtained 45-48 hours after infection. Of course, such culture conditions are examples, and may be variously changed depending on nutrients and growth conditions required for a specific cell line. Such changes can be made without experimentation, and are well known to those skilled in the art.
G) serum-free suspension culture
In certain embodiments, adenovirus vectors for gene therapy are made in a fixed-dependent 293 cell culture (293A cells) as described above. Large-scale production of adenovirus vectors is limited by fixed-dependent 293A cells. Considerable efforts have been made to develop another production process that can be scaled up to increase the scale of adenovirus vector production and meet additional requirements. The methods include growing 293A cells in microcarrier culture and adapting 293A producing cells to suspension culture. In the above, the microcarrier culture technique has been described. This technique is to attach production cells on the microcarrier surface suspended in the culture medium with mechanical agitation. There is a limit to the scale of microcarrier culture because it requires cell attachment.
Prior to the present invention, there have been no reports of using 293 suspended cells in producing adenovirus vectors for use in gene therapy. In addition, the reported suspension 293 cells should have 5-10% FBS in the culture medium for optimal cell growth and virus production. There is considerable interest in the use of bovine-derived proteins in cell culture media because Bovine Spongiform Encephalopathy (BSE) has occurred in some countries. A precise, complex downstream purification process must be developed to remove contaminating proteins and any accidental viruses in the final product. Serum-free 293 suspension cultures have been developed and will be a major improved process for producing adenovirus vectors that can be used for gene therapy.
Virus production in spinner flasks and 3 l stirred tank reactors resulted in cell specific viral productivity of 293SF cells at 2.5 × 10 ⁴ vp / cell, equivalent to about 60-90% of 293A cells. However, due to the high retention cell concentration, viral productivity by volumetric measurement of 293SF cultures is essentially the same for 293A cell cultures. The inventors observed that the exchange of fresh medium at the time of virus infection significantly increased virus production. The inventors evaluated the limiting factors in the medium.
Using these findings, a scale-up, effective, and easily assessed process for producing adenovirus vectors can be obtained. Such an adaptation method is not limited to 293A cells but can be equally applied to other adenovirus vector producing cells.
3. Cell Obtained and Lysed Method
When adenoviruses are infected, the infected cells are lysed. Dissolution characteristics due to adenovirus infection allow the virus to be produced in two different ways. One is to obtain infected cells prior to cell lysis. The other is to obtain viral supernatant after completion of cell lysis by the virus produced. In the latter case, more incubation time is required to completely lyse the cells. The prolonged incubation time after viral infection raises concerns about the increased likelihood of developing replication-reactive adenovirus (RCA), particularly the first adenovirus vector (E1-deleted vector). Thus, a method of obtaining infected cells is selected prior to lysing the cells. Table 1 lists the most common methods used to lyse cells after cell harvest.
Method used for cell lysis Way Procedures Remarks Freeze-thaw Dry ice and 37 ℃ water tank repeat Easy to run in the laboratory. Great effect on cell lysis. No mass production. Not suitable for large scale manufacturing. Solid shear French PressHughes Press Poor experience with capital equipment investment virus inclusion Surfactant dissolution Non-ionic surfactants such as Tween, Triton, NP-40 Variety of Surfactant Selections for Laboratory and Mass Production Residual surfactants in the final product Hypotonic solution dissolution Water, citric acid buffer Low melting effect Liquid shear HomogenizerImpinging JetMicrofluidizer Capital equipment investment virus inclusion problem Mass production problem Ultrasonic crushing ultrasonic wave Capital equipment investment virus inclusion problem Noise pollution mass production problem
A) surfactant
The cell is bound to the membrane. To release cellular components it is necessary to open the cells. The most commonly used method is to dissolve the membrane with a surfactant. Surfactants are amphoteric molecules with nonpolar ends having fatty or aromatic properties and polar ends with or without charge. Surfactants are more soluble in water than lipids because they are hydrophilic than lipids. This makes it possible to disperse insoluble compounds in aqueous media, which are used to separate and purify proteins in their natural form.
Surfactants can be modified or non-modified. The former is anionic such as sodium dodecyl sulfate or cationic such as ethyl trimethyl ammonium bromide. These surfactants completely degrade the membrane and disrupt protein-protein interactions. Non-modifying surfactants can be divided into non-anionic surfactants such as Triton ^R X-100, bile salts such as cholate, and amphoteric surfactants such as CHAPS. Amphotericity includes both cation and anionic groups in the same molecule. Positive charges are neutralized by negative charges in the same or adjacent molecules.
Denatured surfactants, such as SDS, bind to monomeric proteins and drive the reaction to equilibrium until saturated. Thus, knowing the monomer free concentration may reveal the required surfactant concentration. SDS binding is coordinated. For example, binding of SDS to one molecule increases the likelihood that another molecule is bound to the protein, and the protein is transformed into a rod to make its length proportional to molecular weight.
Non-modifying surfactants such as Triton ^R X-100 do not bind to intrinsic shape or have a cooperative manner of binding. Such surfactants are robust, have large nonpolar moieties, and are unable to penetrate proteins that are soluble in water. They bind to the hydrophobic portion of the protein. Triton ^R X100 and other polyoxyethylene non-anionic surfactants are ineffective at breaking the interaction between protein and protein and artificially aggregate the protein. However, such surfactants disrupt protein-protein interactions, but are much softer to maintain their native form and function.
Surfactants can be removed in several ways. Surfactants present as monomers utilize dialysis. If the surfactant has already aggregated to form micelles, dialysis is ineffective because the micelles are too large to pass through dialysis. Ion exchange chromatography can be used to solve this problem. The broken protein solution is placed on an ion chromatography column and the column is washed with a surfactant free buffer. If equilibrium is achieved between the surfactant solution and the buffer, the surfactant is removed. Alternatively, the protein solution can be passed through a density difference. If the protein precipitates through a density difference, the surfactant escapes due to chemical potential.
One surfactant is insufficient to analyze the protein environment found in cells and to dissolve proteins. The protein is dissolved in one surfactant and placed in another surfactant suitable for protein analysis. The protein surfactant micelles formed in the first step are separated from the pure surfactant micelles. If an analytical surfactant is added in excess, the protein is found in micelles with these two types of surfactants. The process of separating the surfactant-protein micelles uses ion exchange or gel filtration chromatography, dialysis or buoyant density separation.
Triton ^R X-surfactants: This type of surfactant (Triton ^R X-100, X114, NP-40) has the same basic characteristics but different hydrophobic-hydrophilic characteristics. All of these release surfactants have a branched 8-carbon chain attached to the aromatic ring. This part contributes most of the hydrophobic character of the surfactant. Triton ^R X surfactant is used to dissolve the membrane protein under non-denaturing conditions. Surfactants for solubilizing proteins, if chosen, depend on the hydrophobic character of the protein to be dissolved. Hydrophobic proteins require hydrophobic surfactants to dissolve them effectively.
Triton ^R X-100 and NP-40 are very similar in structure and hydrophobicity and are interchangeable in most fields such as cell lysis, delipidated protein degradation, membrane protein and lipid lysis. Generally, 1 mg protein is dissolved using 2 mg surfactant or 10 mg surfactant is used for 1 mg of lipid membrane. Triton ^R X-114 is used to separate hydrophobic moieties from hydrophilic proteins.
Brij ^R surfactants: These are structurally similar to Triton ^R X surfactants, which vary in polyoxyethylene length attached to hydrophobic chains. However, unlike Triton ^R X surfactants, Brij ^R surfactants do not have aromatic rings and can vary in carbon chain length. It is difficult to remove Brij ^R surfactant from solution using dialysis, but it can be removed using a surfactant removal gel. Brij ^R 58 is closest to Triton ^R X100 in hydrophobic / hydrophilic character. Brij ^R- 35 is the most commonly used surfactant in HPLC procedures.
Dialysisable nonionic surfactants: η-octyl-β-D-glucoside (octylglucopyranoside) and η-octyl-β-D-thioglucoside (octylglucopyranoside, OTG) are readily available from solution. It is an unmodified nonionic surfactant that is dialyzed. These surfactants are used to dissolve membrane proteins and have low UV absorbance at 280 nm. Octylglucoside has a high CMC of 23-25 mM, which is used at a concentration of 1.1-1.2% when dissolving the membrane protein.
Octylglucoside is first synthesized to provide another octylglucoside. Octylglucoside has some inherent problems in the biological system because it is expensive to manufacture and hydrolyzed by β-glucosidase.
Tween ^R Surfactant: Tween ^R surfactant is an unmodified, nonionic surfactant. These are fatty acid polyoxyethylene sorbitan esters. Tween ^R 20 and Tween ^R 80 surfactants are used as blocking agents in biochemistry and are added to protein solutions to prevent nonspecific binding to hydrophobic materials such as plastics or nitrocellulose. They are used as blocking agents in ELISA and blotting. Generally these surfactants are used at concentrations of 0.01-1.0% to prevent nonspecific binding to hydrophobic materials.
Tween ^R 20 and other nonionic surfactants appear to remove some protein from the nitrocellulose surface. Tween ^R 80 is used to dissolve membrane proteins that provide nonspecific binding of proteins to multiwell plastic tissue culture plates and to reduce nonspecific binding of serum proteins and biotinylated Protein A to polystyrene plates in ELISA. .
The difference between the surfactants is in the fatty acid chain length. Tween ^R 80 is derived from oleic acid, a C ₁₈ chain, and Tween ^R 20 is derived from lauric acid, a C ₁₂ chain. Longer fatty acid chains result in Tween ^R 80 surfactants that are less hydrophilic than Tween ^R 20 surfactants. These two surfactants are well soluble in water.
While Tween ^R surfactants are difficult to remove from solution using dialysis, Tween ^R 20 can be removed using a surfactant removal gel. The polyoxyethylene chains in these surfactants cause them to be oxidized (peroxide formation), as are the Triton ^R X and Brij ^R surfactants.
Zwitter Surfactant: The Zwitter surfactant, CHAPS, is a sulfobetane derivative of choline acid. Zwitter surfactants are useful for dissolving membrane proteins when protein activity is important. Such surfactants are useful in a wide range of pH ranges 2-12 and are easily removed from solution by dialysis because of their high CMCs (8-10 mM). Such surfactants have low absorption at 280 nm, which is useful when protein monitoring is essential at this wavelength. CHAPS is comparable to BCA protein assays, which can be removed from solution using a surfactant removal gel. Proteins can be iodoized in the presence of CHAPS.
CHAPS can be used to dissolve native membrane proteins and receptors, and to maintain the functional performance of the proteins. Dissolution of cytochrome P-450 in Triton ^R X-100 or sodium cholate results in aggregation.
B) non-surfactant methods
Although not suitable, various non-surfactant methods can be used with other advantages of the present invention.
Freeze-thaw: This is a widely used technique for lysing cells in a smooth and effective manner. Cells are generally frozen quickly in dry ice / ethanol baths until completely frozen and transferred to 37 ° C. baths until completely thawed. This procedure is repeated several times to completely lyse the cells.
Ultrasonic Crushing: High frequency ultrasonic vibrators are useful for cell destruction. The method of destroying cells by ultrasound is not fully understood, but providing ultrasonic vibrations to the suspension results in high transition pressures. The main disadvantage of this technique is that it generates a significant amount of heat. To minimize the thermal effect, specially designed glass containers are used to hold the cell suspension. This design allows the suspension to circulate outside the vessel in the ultrasonic probe, where the flask is suspended on ice.
High pressure extrusion: This method is mainly used to destroy microbial cells. The French press uses 10.4 x 10 ⁷ Pa (16,000 psi) pressure to destroy the cells. Such a device consists of a stainless steel chamber opened out by a needle valve. Place the cell suspension in the chamber with the needle valve closed. After inverting the chamber, open the valve and press the piston to bleed off the air in the chamber. If the valve is in the closed position, the chamber is returned to its original position, mounted on a solid base, and the hydraulic press is applied to the piston. When a certain pressure is reached, the needle valve opens by friction, releasing pressure slightly, thereby expanding and rupturing the cells. While maintaining the pressure, the valve is left open and a small amount of ruptured cells are collected.
Solid Shearing Method: Mechanically shearing can be effected by friction using a Mickle shake that vigorously vibrates (300-3000 time / min) in the presence of 500 nm diameter glass beads. This method can destroy organs. A more controlled method is to use a Hughes press, where the piston forces most of the cells by friction or forces frozen cells through a 0.25 mm diameter slot in the pressure chamber. Use up to 5.5 × 10 ⁷ Pa (8000p.si) to dissolve the bacterial preparations.
Liquid Shearing Method: This method uses a high speed reciprocating or rotating blade to pass high speed or two fluids through a mixer, an up and down moving flanger and a homogenizer (homogenizer) using a ball, a microfluidizer or a small diameter tube. Use a collision jet that collides with the flow at high speed. The blades of the mixer have different inclinations, allowing for effective mixing. Homogenizers typically operate at high speeds within seconds to minimize local heat generation. While this technique is not suitable for microbial cells, very soft liquid shear is suitable for destroying animal cells.
Hypotonic / Faulty Solution Method: Cells are exposed to solutions with very low (hypotonic) or very high (failable) solute concentrations. Different solute concentrations produce osmotic pressure differences. In a hypotonic environment, water enters a cell, causing the cell to swell and rupture. In a hypertonic environment, water flows out of cells, causing them to shrink, resulting in rupture.
4. Concentration and Filtration Method
One feature of the invention utilizes a method for purifying adenovirus from cell lysates. Such methods include clarification, concentration, filtration and the like. The initial stage of the purification process purifies the cell lysate to remove large particles, particularly cellular components, from the cell lysate. Use strong filters or tangential transfer filtration to purify the dissolved material. In a suitable embodiment of the invention, the cell lysate is passed through a strong filter, the filter consisting of a column filled with relatively nonabsorbable material (eg, polyester resin, sand, diatomeceous soil, colloid, gel, etc.). In the case of tangential flow filtration (TFF), the lysate solution performs a reverse diffusion of the solute from the membrane surface to the solution across the membrane surface. Membranes are arranged in various types of filter devices, including open channel plates, frames, pupil fibers, tubes, and the like.
After clarification and prefiltration of the cell lysate, the resulting viral supernatant is first concentrated and the buffer is exchanged through filtration. Virus suspensions are concentrated by tangential transfer filtration through ultrafiltration membranes that block 100-300K molecular weight. Ultrafiltration is a pressure-modified convection process that uses semipermeable membranes to separate types by molecular weight, shape, charge, and the like. It separates solvents from solutes of various sizes, regardless of solute molecular weight size. Ultrafiltration is mild and effective, which can be used to concentrate and remove salts from solution. Ultrafiltration membranes generally have two distinct layers; It consists of an open substructure with progressively open pores, such as a thin layer (0.1-1.5 μm), a dense skin with a pore size of 10-400 μs, and a relatively large opening relative to the penetration side of the ultrafilter. Any kind that can pass through the holes of the skin passes freely through the membrane. In order to ensure maximum solute retention, the membrane is selected as a membrane that blocks molecular weight below the molecular weight of the species to be retained. When the macromolecules are concentrated, the membrane provides a filtrate that contains a large amount of the desired biological species and is free of retained material. The fine solute is removed by solvent and convection. As the concentration of retained solute is increased, the ultrafiltration rate is reduced.
Using ultrafilters, diafiltration, buffer exchange, and the like are ideal methods for removing and exchanging salts, sugars, non-aqueous solvents from the combined species, removing low molecular weight materials, and rapidly changing ionic and pH environments. Becomes Microsolute can be removed most effectively by adding a solute to the ultrafiltration solution at the same rate as the ultrafiltration rate. The fine species are washed away from the solution in a constant volume and the retained species is purified. The present invention utilizes diafiltration to exchange virus supernatant buffer prior to Benzonase ^R treatment.
5. Virus Infection
In one embodiment, the present invention utilizes adenovirus infection in cells to make therapeutically important vectors. In general, the virus is briefly exposed to the appropriate host cell under physiological conditions to allow the virus to enter the host cell. Although adenovirus is taken as an example, the present invention may use other viral vectors described below.
A) Adenovirus
Adenoviruses are suitably used in gene transfer vectors because of their medium DNA genome size, ease of manipulation, high titers, broad target cells, and high infectivity. Roughly 100-200 bp reversed terminal repeats (ITRs) are linked to the 36 kB viral genome, which contains cis-acting elements essential for viral DNA replication and packaging. By DNA replication initiation, it is divided into early (E) and late (L) portions of the genome containing different transcription units.
The E1 portions (E1A and E1B) encode proteins that correspond to transcriptional regulation and several cellular genes in the viral genome. When the E2 portions (E2A and E2B) are expressed, proteins for viral DNA replication are synthesized. These proteins are involved in DNA replication, late gene expression, and host cell blockade (Renan, 1990). After one primary transcript released by the main late promoter (MLP) is processed, the late gene (L1, L2, L3, L4, L5) product, including most of the viral capsid proteins, is expressed. MLP (located in units of 16.8 maps) is particularly effective in the late stages of infection. All mRNAs released by this promoter have a 5 'triple region leader (TL) sequence, which makes the mRNA suitable for translation.
In order for adenoviruses to be suitable for gene therapy, the carrying capacity must be maximized to include large DNA fragments. There is also a need to reduce toxic and immunological responses associated with specific adenovirus products. Removal of a significant portion of the adenovirus genome and the use of helper viruses or helper cells to provide the in trans-defective gene product allows for the insertion of a significant portion of heterologous DNA into the vector. In this way, the toxicity and immunogenicity of the adenovirus gene product can be reduced.
Large DNA substitutions are possible because all cis elements required for viral DNA replication are located across the linear viral genome at the reverse terminal repeat (ITR) (100-200 bp). Plasmids containing ITR's can be replicated in the presence of non-defective adenoviruses (Hay et al., 1984). Thus, inclusion of these elements in an adenovirus vector allows replication.
In addition, a package signal for capping the virus is located at 194-385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al., 1987). This signal is similar to the protein recognition site in the bacteriophage λ DNA, with specific sequences close to the sinus ends but outside of the adhesion end sequences mediate binding to the proteins required to insert DNA into the head structure. The E1 substitution vector of the adenovirus explains that 450bp (0-1.25 map units) at the left end of the viral genome is related to the package in 293 cells (Levrero et al., 1991).
It has already been confirmed that certain parts of the adenovirus genome are linked to the genome of mammalian cells, whereby the encoded genes are expressed. These cell lines have the ability to support replication of adenovirus vectors that lack adenovirus functions encoded in the cell line. Complementarity of replication-deficient adenovirus vectors has also been described using “helping” vectors (eg, wild type viruses or conditionally deleted mutants).
Replication-deficient adenovirus vectors can be complementary in trans by helper viruses. This observation alone is not sufficient to isolate replication-defective vectors, but the helper virus necessary to provide replication functionality may be present to contaminate any preparation. Thus, additional elements are needed to provide specificity for the replication and packaging of the replication-deletion vector. As provided herein, such elements are derived from the package function of adenoviruses.
Package signals of adenoviruses were found to be present at the left end of a typical adenovirus map (Tibbetts, 1977). Subsequent studies have shown that mutations that lack the E1A (194-358 bp) portion of the genome have poor growth, even if the cell line complements early function (E1A) (Hearing and Shenk, 1983). Complementary complementary adenovirus DNA (0-353 bp) to the right end of the mutation causes the virus to package normally. Mutation analysis also identified elements that depend on a short repeated position at the left end of the Ad5 genome. When it is at the end of the genome, only one copy of the repeat has been shown to be sufficient for an effective package, which is insufficient if it migrates inside the Ad5 DNA molecule (Hearing et al., 1987).
Mutated packaging signals can be used to create helper viruses that are packaged with various effects. Generally, mutations are point mutations or deletion mutations. When a helper virus with low package effect is grown in helper cells, compared to the wild type virus, the virus, although packaged, has a reduced rate and multiplies the virus. When such a helper virus is grown in cells with a virus comprising a wild-type package signal, the wild-type package signal is recognized prior to the mutated signal. In the case of providing a limited amount of package factor, the virus comprising the wild type signal is optionally packaged as compared to the helper virus. If the preference is quite large, a nearly homogeneous stock can be obtained.
B) retroviruses
In the present invention, adenovirus infection was used in cells to make therapeutically important vectors, but in the present invention, retroviruses may be used to infect cells for the purpose of making such vectors. Retroviruses are a group of single stranded RNA viruses that have the ability to convert these RNAs into double stranded DNA in infected cells by reverse transcription (Coffin, 1990). The resulting DNA is stably bound to cellular chromosomes to become a provirus, producing viral proteins directly. By binding to the chromosome in this way, the viral genome sequence is maintained in the cells and their descendants. There are three genes in the retroviral genome, gag, pol, and env, which respectively encode capsid proteins, polymerase enzymes, and envelope components. The sequence found upstream of the gag gene (called Y) serves as a signal in which the genome is packaged into a virion. Two long terminal repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. This includes strong promoter and enhancer sequences, which are necessary to bind to the host cell genome (Coffin, 1990).
To make a retroviral vector, a nucleic acid encoding a promoter is inserted at a specific sequence site in the viral genome to create a replication-defective virus. To make barions, package cell lines containing gag, pol, and env genes but without LTR and Y components are made (Mann et al., 1983). When a recombinant plasmid containing human cDNA with retroviral LTR and Y sequences was introduced into this cell line (e.g. using calcium phosphate precipitation), the Y sequence packaged the RNA transcript of the recombinant plasmid into viral particles, Secreted in culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). Media containing recombinant retroviruses are obtained, selectively concentrated and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, incorporation and stable expression require division of host cells (Paskind et al., 1975).
New methods have been recently developed to specifically target retroviral vectors only, which are based on chemical modification of retroviruses by chemical addition of calactose residues to the viral envelope. Such modifications can specifically infect cells, such as hepatocytes, through the asialoglycoprotein receptor.
Another method of targeting recombinant retroviruses is to use biotinylated antibodies against retroviral envelope proteins and antibodies against specific cellular receptors. Antibodies bind to biotin components using streptavidin (Roux et al., 1989). Antibodies to histocompatibility complex I and II antigens have been used to describe the in vitro infection of exogenous viruses with various human cells containing these surface antigens (Roux et al., 1989).
C) other viral vectors
Other viral vectors can be used for the expression structure described in the present invention. Vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), Herpes virus, etc. Vector derived from the virus is used. These viruses provide several features that can be used to transfer genes into various mammalian cells.
6. Manipulation of Virus Vectors
In certain embodiments, the present invention provides for engineering the viral vector. Such a method obtains viral particles produced therefrom using a vector structure comprising heterologous DNA encoding a desired gene and a means for expressing the same, and replicating the vector in a suitable host cell, and transferring the recombinant viral particles to the cell. It consists of infecting. Genes encode large amounts of the desired protein, which is a large-scale production in vitro. In addition, the gene can be a therapeutic gene that treats cancer cells, expressing immunoregulatory genes to combat viral infections or to restore gene function due to gene defects. In gene therapy vectors, genes become heterologous DNA, which includes DNA derived from sources other than the viral genome that provide the basis for the vector. Finally, the virus expresses the antigen to act as a live viral vaccine to produce antibodies to the desired antigen. Genes can be derived from prokaryotic or eukaryotic cell sources such as bacteria, viruses, yeasts, parasites, plants or animals. Heterologous DNA may be derived from one or more sources, for example, may be multiple gene structures or fusion proteins. Heterologous DNA may include regulatory sequences derived from one source and genes derived from another source.
A) Therapeutic Genes
p53 is recognized as a tumor suppressor gene (Montenarh, 1992). Significant levels of mutant p53 have been found in many cells transformed by several viruses, including chemical tumorigenesis, UV transmission, and SV40. The p53 gene is a target mainly used for mutation inactivation in various human tumors and is known as the most frequently mutated gene in cancers common in humans (Mercer, 1992). More than 50% of human NSCLC (Hollestein et al., 1991) were mutated in a wide range of other tumors.
The p53 gene encodes a 393-amino acid phosphate protein that complexes with large-T antigens and host proteins such as E1B. Proteins are also found in normal tissues and cells, but their concentrations are generally low compared to those found in transformed cells or tumor tissues. Interestingly, wild type p53 appears to play an important role in regulating cell growth and division. In some cases, wild-type p53 is overexpressed in human tumor cell lines to provide anti-proliferation. Thus, p53 acts as a negative regulator of cell growth (Weinberg, 1991), and either directly inhibits cells that are not growing or indirectly activates genes that inhibit such growth. Thus, the absence or inactivation of wild type p53 can result in transformation. However, some studies have shown that mutation p53 is essential for the full expression of the gene's ability to transform.
Wild type p53 has been recognized as an important growth regulator in many cell types. Missense mutations are the most common for the p53 gene, which is known to occur in at least 30 distinct codons, which sometimes create dominant alleles that modify the cell phenotype without reducing homozygosity. In addition, many of these dominant negative alleles are resistant to organisms and are transferred to germline. Various mutant alleles appear to be at least strongly functional, invasive, dominant negative allele ranges (Weinberg, 1991).
Casey and colleagues report that transfection of wild-type p53-encoding DNA into two human breast cancer cell lines reverts growth inhibition regulation in these cells (Casey et al., 1991). Similar results were obtained when transfecting human lung cancer cell lines with non-mutated wild type p53 (Takahasi et al., 1992). p53 is dominant for mutant genes and can be selected for proliferation when the mutant genes are transfected into cells. Normal expression of transfected p53 is not deleterious to normal cells with endogenous wild type p53. Thus, such a structure can provide normal cells without other side effects. Treatment of p53-related cancers with wild type p53 expression constructs reduces the number of malignant tumor cells or decreases their growth rate. In addition, recent studies have shown that some p53 wild-type tumors are sensitive to exogenous p53 expression effects.
Major metastasis of eukaryotic cells is stimulated by cyclin dependent kinase or CDK. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the G ₁ phase. The activity of this enzyme phosphorylates Rb in later G _1- . CDK4 activity is regulated by subunit D- ^cycline and inhibitory subunit p16 ^INK4 , which can modulate Rb phosphorylation because it has the biochemical characteristics of a protein that specifically binds to and inhibits CDK4. (Scrrano et al., 1993; Serrano et al., 1995). Since the p16 ^INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene increases CDK4 activity, resulting in excessive phosphorylation of the Rb protein. p16 is known to modulate CDK6 function.
p16 ^INK4 belongs to the newly classified CDK- inhibitory protein, which includes a ^B p16, p21 ^{WAF1.CIP1.SDII,} p27 ^KIP1. The p16 ^INK4 gene leads to 9p21, a chromosome portion that is largely missing in many tumor types. Homologous deletions and mutations of the p16 ^INK4 gene are frequent in human tumor cell lines. This indicates that the p16 ^INK4 gene is a tumor suppressor gene. However, this interpretation has changed since we observed that the frequency of p16 ^INK4 gene alteration was lower in non-negative tumors than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian et. al., 1994; Kamb et al., 1994a; Kamb et al., 1994b; Mori et al., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al., 1994; Arap et al , 1995). Restoration of wild type p16 ^INK4 function by transfection with a plasmid expression vector reduces colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).
C-CAM is expressed in practically all epithelial cells (Odin and Obrink, 1987). C-CAM, with a surface molecular weight of 105 kD, was originally isolated from the plasma membrane of rat hepatocytes by reacting with specific antibodies that neutralize cell aggregation (Obrink, 1991). According to a recent study, structurally C-CAM belongs to the immunoglobulin (Ig) superfamily, whose sequence is highly homologous to the carcinoimbroinin antigen (CEA) (Lin and Guidotti, 1989). Using a baculovirus expression system, Cheung et al. (1993a; 1993b and 1993c) demonstrated that the initial Ig domain of C-CAM is important for cell adsorption activity.
Cell adsorption molecules or CAMs are known to be involved in the molecular interaction of complex processes that regulate organ development and cell differentiation (Edelman, 1985). Recent studies suggest that aberrant expression of CAMs is associated with the development of several neoplastic tumors. For example, several types of neoplasia are associated with progression, primarily due to decreased expression of E-cadherin, which is expressed in epithelial cells. (Edelman and Crossin, 1991; Frixen et al., 1991; Bussemakers et al., 1992; Matsura et al., 1992; Umbas et al., 1992). In addition, Giancotti and Ruoslahti (1990), when the expression of α ₅ β ₁ integrin increased by gene transfer may reduce the tumorigenic capacity of Chinese hamster Ovari cells in vivo. C-CAM appears to inhibit tumor growth in vitro and in vivo.
Other tumor suppressors used according to the invention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, BRCA1, VHL, FCC, MMAC1, MCC , p16, p21, p57, C-CAM, p27, BRCA2. Inducing apoptosis such as Bax, Bak, Bcl-X ₅₇ Bik, Bid, Harakiri, Ad E1B, Bad, ICE-CED3 protease can also be used in accordance with the present invention.
According to the invention various enzyme genes are of interest. Such enzymes include cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-1-phosphate uridiltransferase, phenylalanine hydroxylase, glucoserbrosidase, sphingomyelinase, α -L-iduronidase, glocose-6-phosphate dehydrogenase, HSV thymidine kinase, human thymidine kinase and the like.
Another group of genes that can be used in the vectors described herein are hormones. Hormones include growth hormone, proactin, placental lactogen, progesterone cystic-stimulating hormone, chronic gonadotropin, thymic-stimulating hormone, leptin, adenocroticotropine (ACTH), angiotensin I, II, β- Related to endropin, β melanoma stimulating hormone (β-MSH), cholecytokine, endothelin I, galanine, gastrointestinal inhibitory peptide (GIP), glucagon, insulin, reportropin, neuropisin, somatostatin, calcitonin, calcitonin gene Peptide (CGRP), β-calcitonin gene related peptide, hypercalcemia of malignant tumor factor (1-40), parathyroid hormone-related protein (107-139) (PTH-rP), parathyroid hormone related protein (107-111) (PTH-rP), glucagon-like peptide (GLP-1), pancreatine, pancreatic peptide, peptide YY, PHM, secretin, vascular active visceral peptide (VIP), oxytocin, vasopressin (AVP), vasotocin, enkephalin Amides, metropinamide, alpha monocyte stimulating hormone (alpha-MSH), sil Diuretic factor (5-28) (ANF), amylin, amyloid P component (SAP-I), corticotropin releasing hormone (CRH), growth hormone releasing factor (GHRH), progesterone releasing hormone (LHRH) , Neuropeptide Y, substance K (neurokinin A), substance P, tyrotropin releasing hormone (TRH) and the like.
Other kinds of genes that can be inserted into the vectors of the present invention include interleukins and cytokines. Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, GM-CSF, G-CSF.
Diseases currently available for viral vectors include, but are not limited to, adenosine diminase deficiency, human blood coagulation factor IX deficiency in hemophilia B, and bladder fibrosis (which involves bladder fibrosis transmembrane and substitution of receptor genes). Vectors embodied in the present invention can be treated by delivering a gene encoding an angiogenesis inhibitor or a cell process inhibitor, such as hyperproliferative diseases or restenosis, such as rheumatoid arthritis. Prodrug activators such as the HSV-TK gene are delivered to treat hyperproliferative diseases including cancer.
B) antisense structures
Oncogenes such as ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl are suitable targets. However, for therapeutic benefit, such oncozin is expressed as an antisense nucleic acid and inhibits oncogene expression. "Antisense nucleic acid" refers to an oligonucleotide that is complementary to DNA and RNA sequences encoding oncogene. When introduced into target cells, antisense oligonucleotides specifically bind to their target nucleic acids and interfere with transcription, RNA processing, transport or translation. Providing oligonucleotides to double stranded (ds) DNA forms triple helices, while targeting RNA forms double helices.
Antisense structures allow for binding to the exon-intron boundaries of promoters, other reference moieties, exons, introns or genes. Antisense RNA constructs or DNA encoding such antisense RNAs can inhibit gene transcription or translation or both in host cells, including humans, in vitro or in vivo. Nucleic acid sequences consisting of “complementary nucleotides” can be base paired according to standard Watson-Crick complementary rules. That is, large purines form base pairs with less pyrimidine, guanine complexes with cytosine (G: C), adenine base pairs with thymine in DNA (A: T), and uracil and base pairs in RNA. (A: U).
As used herein, "complementary" or "antisense sequence" means that there is very little mismatched to the actual complementary nucleic acid sequence throughout its length. For example, a nucleic acid sequence of length 15 refers to having complementary sequences at 13 or 14 positions in which one or two are mismatched. Originally, a “fully complementary” nucleic acid sequence is one that is not mismatched to a nucleic acid sequence that is completely complementary throughout its length.
If all or part of the gene sequence is used in the antisense construct, only any sequence with statistically 17 bases is generated in the human genome, which is sufficient to specify a unique target sequence. Shorter oligomers are easier to make and better accessible in vivo, but many other factors are involved in determining hybridization specificity. The binding affinity and sequence specificity that the oligonucleotides bind to their complementary targets increases with increasing length. Oligonucleotides having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs may be used. By examining the constructs in vitro to determine whether the function of an endogenous gene is affected or the expression of a relevant gene with complementary sequences, the antisense nucleic acid given to target the corresponding host cell gene is determined to be effective. Can be.
In certain embodiments, other elements may be included in the antisense structure, for example, may include C-5 propine pyrimidine. Oligonucleotides comprising C-5 propine analogs of uridine and cytidine bind with high affinity to RNA and appear to be potent antisense inhibitors of gene expression (Wagner et al., 1993).
Another example of targeted antisense transport can utilize targeted ribozymes. "Ribozyme" refers to an RNA-based enzyme that targets oncogene DNA and RNA and can cleave specific nucleotide sequences. Ribozymes can be directly targeted to a cell in the form of an RNA oligo-nucleic acid bound ribozyme sequence or introduced into the cell in an expression construct encoding the desired ribozyme RNA. Ribozymes can be used and applied in the same manner as described for antisense nucleic acids.
C) antigens for the vaccine
Other therapeutic genes include genes that encode antigens such as viral antigens, bacterial antigens, fungal antigens or parasitic antigens. Viruses include picornavirus, coronavirus, togavirus, flaviviru, rhabdovirus, paramyxovirus, and orthomyxovirus ), Bunyavirus, arenvirus, arevirus, retrovirus, retrovirus, papovavirus, parvovirus, herpesvirus, poxvirus ( poxviruses, hepadnaviruses, sponge foam viruses, and the like. Suitable viral targets include influenza, herpes simplex virus 1, 2, measles, smallpox, polio, HIV, and the like. The etiology includes tripanozoma, tapeworms, roundworms, and parasites. In addition, tumor markers such as fetal antigens or prostate specific antigens can be targeted in this manner. Suitable examples include HIV env protein and hepatitis B surface antigen. Administration of the vector for the purpose of vaccination according to the present invention requires a vector-associated antigen that is non-immunogenic, since it is possible to express the transgene for a long time if a strong immune response is desired. Appropriately, a person's vaccination should be done once a year or once every two years, providing immunologically long-term protection against infectious agents.
D) adjusting part
In order for the viral vector to effectively express the transcript encoding the therapeutic gene, the polynucleotide encoding the therapeutic gene is given under formomotor and polyadenylation signal transcriptional control. A "promoter" is a DNA sequence that is recognized by the synthetic mechanism of a host cell or is a DNA sequence introduced into a synthetic mechanism necessary to initiate specific transcription of a gene. Polyadenylation signal refers to a DNA sequence in a synthetic mechanism that is recognized or introduced by the synthetic mechanism of a host cell, which is needed to add a series of nucleotides directly to the ends of the mRNA transcript for proper processing and for translation. It is necessary for transcription outside the nucleus into the cytoplasm. "Under transcriptional control" means that the promoter is in the correct position relative to the polynucleotide to regulate RNA polymerase initiation and polynucleotide expression.
Herein, the promoter means a transcriptional control module gathered around the start site of RNA polymerase II. Much of how the promoter is organized comes from analyzing several viral promoters, including HSV thymidine kinase (tk), the SV40 early transcription unit. As can be seen in this study, augmented by a recent study, the promoter consists of modules that function separately, each consisting of about 7-20 bp DNA, one for transcriptional activators or protein inhibitors. It includes more than two cognitive sites.
At least one module in each promoter functions at the starting site for RNA synthesis. The best known for this is the TATA box, and some TATA-free promoters, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late gene, have been found to have a separate element anchored at their starting site. Help.
Additional promoter elements regulate the frequency of transcription initiation. In general, they are located 30-110 bp upstream of the starting site, and recently a promoter of saccharide was shown to contain functional elements downstream of the starting site. Since the space between the promoter elements is fluid, the function of the promoter is preserved if the elements are reversed or can move. In the case of the tk promoter, the spacing between promoter elements can be increased to 50 bp, but beyond this the activity is reduced. Depending on the promoter, the individual elements either co-ordinate or function independently to activate transcription.
If the polynucleotide can be expressed in the target cell, the particular promoter used to regulate the expression of the therapeutic gene does not necessarily have to be used. Thus, when targeting human cells, it is desirable to position the polynucleotide coding moiety adjacent thereto under the control of a promoter that can be expressed in human cells. Such promoters generally include human or viral promoters. Promoters are provided in Table 2.
Promoter Immunoglobulin heavy chain Immunoglobulin light chain T-cell receptor HLA DQ αDQ β β-interferon Interleukin-2 Interleukin-2 receptor MHC Class II 5 MHC Class II HLA-DRα β-actin Muscle Creatine Kinase Prealbumin (transstratin) Elastase I Metallothionine Collagenase Albumin gene α-iron protein ι-globin β-globin c-fos c-HA-ras insulin Neuronal Adsorption Molecules (NCAM) α _{I-Antitrypsin}H2B (TH2B) Histone Rat or Type I Collagen Glucose Regulatory Protein (GRP94 GRP78) Rat growth hormone Human Serum Amyloid A (SAA) Troponin (TNI) Platelet-Induced Growth Factors Duchenne Muscle Problems SV40 Polyoma Retrovirus Papilloma virus B hepatitis virus Human immunodeficiency virus Cytomegalovirus Gibbon Ape Leukemia Virus
Promoter can be characterized as an inducible promoter. Inducible promoters are inactive or very low activity promoters, except when inducer materials are present. Examples of promoters included in the present invention include MT II, MMTV, collagenase, stromelysin, SV40, murine MX gene, α-2-macroglobulin, MHC I gene h-2kb, HSP70, prolipin, tumor necrosis Factors, including but not limited to thymic stimulating hormone α gene. The related inducers are shown in Table 3. Any inducible promoter can be used in the present invention, and such promoters are all within the scope of the claimed invention.
element Inducer MT II Probol Ester (TPA) Heavy Metals MMTV (rat breast tumor virus) Glycocorticoids β-interferon poly (rI) Xpoly (rc) Adenovirus 5 E2 Ela c-jun Probol ester (TPA), H ₂ O ₂Collagenase Probol Ester (TPA) Stromielcin Probol Ester (TPA), IL-1 SV40 Probol Ester (TPA) Rat MX gene Interferon, Newcastle Disease Virus GRP78 gene A23187 α-2-macroglobulin IL-6 Vimentin serum MHC Class I Gene H-2kB Interferon HSP70 Ela, SV40 Large T antigen Proliperin Probol Ester-TPA Tumor necrosis factor FMA Thymic stimulating hormone α gene Thymus hormone
In various embodiments, the human cytomegalovirus (CMV) immediate gene promoter, the SV40 early promoter, and the Raus Salcoma virus long terminal repeats are used to express significant levels of the desired polynucleotides. Other viral or mammalian cell or bacterial phase promoters known in the art can be used to express the polynucleotides provided that the expression level is sufficient to produce a growth inhibitory effect.
Promoters with known properties can be used to optimize the expression levels and patterns of polynucleotides after transfection. For example, promoters with activity in specific cells, for example, tyrosinase (melanoma), alpha-blue protein and albumin (liver tumor), CC10 (lung tumor), prostate specific antigen (prostate tumor) are therapeutic genes. Allows for tissue specific expression.
Enhancers were perceived as genetic material to increase transcription from promoters in separate locations on the same DNA molecule. This ability to function at significant distances was unprecedented in prokaryotic transcription control studies. In subsequent studies, DNA portions with enhancer activity were organized similarly to promoters. That is, it consists of many individual elements, each of which is bound to one or more transcription proteins.
The fundamental difference between enhancers and promoters is in operation. The enhancer portion stimulates transcription at a distance, but does not necessarily need to be a promoter portion or a component thereof. Promoters, on the other hand, have one or more elements that initiate RNA synthesis at specific sites and in specific directions, but enhancers do not have this feature. Promoters and enhancers often appear to have very similar modular organization, overlapping or adjacent.
Thus, as per the Eukaryotic Promoter Data Base (EPDB), the expression of specific structures is induced. The T3, T7, SP6 cytoplasmic expression system may be used and may be another embodiment. If the appropriate bacteriophage polymerase is provided as part of the transport complex or additional inheritance is provided as an expression vector, it may support cytoplasmic transcription from certain bacteriophage promoters.
If cDNA inserts are used, it is desirable to include polyadenylation signals that can affect proper polyadenylation of gene transcripts. The characteristics of the polyadenylation signal are not critical to the practice of the present invention and any sequence can be used. These polyadenylation signals from the SV40, bovine growth hormone, and herpes simplex virus thymidine kinase genes have been shown to function in a variety of target cells.
7. Gene Delivery Method
The helper cell lines of the present invention are made and various gene structures (such as DNA) are transported into cells for the production of recombinant adenovirus vectors for use therewith. One way of accomplishing this is through viral transduction using infectious viral particles, for example by transforming with the adenovirus vector of the invention. Alternatively, retroviruses or bovine papilloma viruses can be used, all of which permanently transform host cells with the desired genes. In other situations, the nucleic acid to be delivered is included in an uninfected or infectious viral particle. Delivery of such genetic material should be in a non-viral way.
Several non-viral methods can be used to deliver expression constructs to cultured mammalian cells. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979), ultrasonography (Fechheimer et al., 1987), high speed Gene bombardment using microinjection (Yang et al., 1990), receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).
Once the construct is delivered to the cell, the nucleic acid encoding the therapeutic gene is left standing and expressed at another site. In certain embodiments, the nucleic acid encoding a therapeutic gene is stably bound to the genome of the cell. Such binding may be at the same position and orientation via homologous recombination (gene substitution) or at random non-specific positions (gene propagation). In another embodiment, the nucleic acid can be stably maintained in the cell as an episomal DNA fragment separately. Such nucleic acid fragments or "episomes" encode sequences sufficient to replicate and maintain independently or identically to host cell processes. The way in which the expression construct is delivered to the cell and the nucleic acid is maintained in the cell depends on the type of expression construct used.
In one embodiment of the invention, the expression construct simply consists of naked recombinant DNA or plasmid. The constructs can be delivered using any of the methods mentioned above that can cross the cell membrane physically or chemically. It is particularly suitable for performing in vitro, but can also be used in vivo. Dubensky et al., (1984) have successfully injected polymavirus DNA in the form of CaPO ₄ precipitates into the liver and spleen of adult rats and newborn rats, demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also injected CaPO ₄ precipitated plasmids directly into the peritoneum to express transfected genes. DNA encoding CAM is delivered in vivo in a similar manner to express CAM.
Another way to deliver naked DNA into cells is to spray particles. In this method, high-speed DNA-coated microprojectiles depend on their ability to penetrate the cell membrane and enter the cell without killing the cell (Klein et al., 1987). Several devices have been developed to accelerate small particles. One such device emits a high voltage to generate a current, which is the driving force (Yang et al., 1990). The microprojectils used consist of biologically inert materials such as tungsten or gold beads.
In another embodiment of the invention, the expression construct can be entrapped in liposomes. Liposomes have a vesicular structure with a phospholipid bilayer and an internal water soluble medium. Multilamellar liposomes have several lipid layers separated by aqueous media. Suspension of the phospholipid in an aqueous solution spontaneously forms a multilayer lamellae. Lipid components are arranged on their own, forming closed structures, capturing water, and dissolving solutes between lipid bilayers (Ghosh and Bachhawat, 1991).
Liposomal-mediated nucleic acid transport and expression of foreign DNA in vitro are very successful. Using the β-lactamase gene, Wong et al., (1980) can perform external DNA expression in liposome-mediated transported and cultured chick embryos, HeLa, liver tumor cells. Nicolau et al., (1987) successfully performed liposome-mediated gene delivery after intravenous injection. Various methods are also included using the "lipopeptin" technique.
In certain embodiments of the invention, liposomes may be combined with hematoglutinin virus (HVJ). This has been shown to fuse cell membranes and facilitate the entry of DNA trapped in liposomes into cells (Kaneda et al., 1989). In another embodiment, liposomes are complexed with or used with nuclear bihistone chromosomal protein (HMG-1) (Kato et al., 1991). In another embodiment liposomes are complexed with or used with HVJ and HMG-1. Since such expression constructs have been successfully used for the delivery and expression of nucleic acids in vitro and in vivo, they can be used in the present invention.
Another expression construct used to carry nucleic acids encoding therapeutic genes into cells is receptor-mediated transport media. They have the advantage of being able to selectively accept macromolecules by receptor-mediated phagocytosis in almost all eukaryotic cells. Because various receptors are distributed according to cell morphology, transport is very specific (Wu and Wu, 1993).
Receptor-mediated gene target mediators generally consist of two components; Cell receptor-specific ligands and DNA binding substances. Several ligands are used to deliver receptor-mediated genes. The most known ligands are asialolosomucoids (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1990). Recently, synthetic neoglycoproteins that recognize the same receptors as ASOR have been used as gene transfer carriers (Ferkol et al., 1993; Perales et al., 1994), and epidermal growth factor (EGF) is applied to the side cancer cells. It was used to deliver (Myers, EPO 0273085).
In another embodiment, the transport vehicle consists of a ligand and a liposome. For example, Nicolau et al. (1987) found that the use of lactosyl-ceramide, galactose-terminal asialganglioside bound to liposomes increases insulin gene uptake by hepatocytes. Thus, using various receptor-ligand systems with or without liposomes, nucleic acids encoding therapeutic genes can be specifically transported to cells such as prostate, epithelial or tumor cells. For example, human prostate-specific antigens (Watt et al., 1986) can be used as receptors to carry nucleic acids to prostate tissue.
8. Removal of Nucleic Acid Contaminants
The present invention used nucleases to remove impurity nucleic acids. Nucleases include Benzonase ^r , Pulmozyme ^R , or other DNases or RNases commonly used in the art.
Enzymes like Benzonase ^R degrade nucleic acids but have no proteolytic activity. By rapidly hydrolyzing nucleic acids, Benzonase ^R is the ideal enzyme to reduce the rate of cell lysis. Adsorption interferes with separation due to agglomeration, particle size changes or changes in particle charge, so that little product is recovered with a given purification process. Benzonase ^R reduces nucleic acid loading during purification, thus reducing interference and improving yield.
Like all endonucleases, Benzonase ^R hydrolyzes phosphodiester bonds between specific nucleotides. When fully digested, all free nucleic acids present in solution are oligonucleotides of 2 to 4 bases in length.
9. Refining Technology
The present invention uses several different purification methods to purify the adenovirus vectors of the present invention. Such techniques include those based on precipitation and chromatography as described below.
A) Density Difference Centrifugation
There are two methods for density difference centrifugation, the rate zonal technique and the isopycnic technique, both of which quantitatively separate all components in the particle mixture. It can also be used to measure the density of voids or to measure sedimentation constants.
Particle separation by the velocity zone technique is to separate according to size and settling rate. This technique allows the sample solution to be carefully placed on top of the liquid density difference implemented to separate particles of maximum density that exceed the density of the dense particles. The sample is then centrifuged until it is separated to the desired level, e.g., centrifuged for a time such that the particles can shift in concentration differences to form separate zones or bands separated by the relative velocity of the particles. . Because the technique is time dependent, centrifugation must be stopped before any separated area forms pellets at the bottom of the tube. This method is used to isolate enzymes, hormones, RNA-DNA hybrids, ribosomal subunits, organelles, analyze the size distribution of polysome samples, and fractionate lipoproteins.
The sample is placed on top of a continuous density difference leading to the full density range of the particles to be separated. Therefore, the maximum density of the slope should always be greater than the density of the dense particles. During centrifugation, the particles precipitate until the particle's buoyant density and gradient density are equal (P _p = P _m in Equation 2.12). Regardless of how long the centrifugation lasted, no further precipitation occurs at this point because the particles are suspended in a material buffer having a density greater than the density they retain.
In contrast to the velocity zone technique, isotropic centrifugation is a balanced method whereby each particle forms a band at these inherent suspended densities. If not all of the components in the particle mixture are required materials, the unwanted components in the mixture settle to the bottom of the centrifuge tube, while the desired particles can select the slope range to settle at their respective equal density positions. . This technique combines velocity zones and equal density methods.
Equal density centrifugation depends on the suspended density of the particles and is independent of the size, shape and time of the particles. Very similar density (eg p = 1.3 g cm ^-3 in sugar solution) soluble proteins cannot be separated in this way, whereas subcellular organs (Golgi in sucrose solution is p = 1.11 g cm ^-3 , Mitochondria are p = 1.19 g cm ^-3 and peroxysomes are p = 1.23 g cm ^-3 ).
As another method of dropping the particle mixture to be separated at the preformed gradient, the sample is first mixed with the gradient medium to provide a homogeneous solution density, and the gradient is itself formed during centrifugation by precipitation balance. In this method (commonly referred to as the equilibrium equal density method), heavy metals (eg, caseinium or rubidium), sucrose, colloidal silica or metrizaamide salts are used.
Sample (DNA) is mixed homogeneously with cesium chloride concentrate. Centrifugation of the concentrated cesium chloride solution precipitates CsCl molecules, creating a concentration gradient and forming a density difference. Initially homogeneously distributed sample molecules (DNA) in the tube reach sites where their floating and solution densities are equal (e.g., equal density sites), rising or settling until zones are formed. This technique has the disadvantage of centrifuging for a very long time to achieve equilibrium (eg 36 to 48 hours). However, they are commonly used in analytical centrifugation to determine the suspended density of particles, to determine the base composition of double stranded DNA, or to separate linear DNA from circular DNA.
The technique used by Leselson and Stahl is a technique used to elucidate the DNA replication mechanism in Escherichia coli and increases the density difference between different types of DNA by adding isotopes ( ¹⁵ N) during biosynthesis or dyes such as heavy metal ions or ethidium bromide. Can be improved by combining. Viruses are isolated and purified using isometric density gradients and human plasma lipoproteins are analyzed.
B) Chromatography
In certain embodiments of the invention, it is desirable to make purified adenoviruses. Purification techniques are those known in the art. These techniques are to separate the cellular environment to separate adenoviruses from other components in the mixture. When the adenovirus particles are separated from other components, the adenoviruses are purified using chromatography and electrophoretic techniques for complete pure separation. Particularly suitable analytical methods for preparing pure adenovirus particles of the invention include ion-exchange chromatography, size extrusion chromatography, polyacrylamide gel electrophoresis and the like. A particularly effective purification method that can be used in the present invention is HPLC.
One aspect of the present invention relates to the purification of adenovirus particles. As used herein, the term "tablet" refers to a composition that can be separated from other components, where the adenovirus particles are purified to a degree comparable to those available in nature. Thus, purified adenovirus particles refer only to adenovirus components that do not have a naturally occurring environment.
Generally, "purified" refers to adenovirus particles aliquoted to remove a variety of other components, wherein the composition retains the biologically expressed biological activity. By “actually purified” is meant that particles, proteins, or peptides of the composition form the main component of the composition, which consists of about 50% or more of the composition of the composition.
Based on the description of the present invention, those skilled in the art will recognize various methods of quantifying the degree of purification of a protein or peptide. These methods include using SDS / PAGE analysis to determine the specific activity of an active aliquot or to measure the amount of polypeptide in the aliquot. A suitable method of assessing the purity of an aliquot calculates the specific activity of the aliquot, compares the specific activity with the calculated activity of the native extract, and calculates the purity, which is referred to as "several times purity". The actual unit used to represent the amount of activity depends upon the particular assay selected for purification and whether the expressed protein or peptide has detectable activity.
There is no general rule that adenoviruses should be provided in their purest form. Also, in certain embodiments, products with slightly less actual purity may be used. Partial purification can be accomplished using fewer purification steps or other forms in the same purification process. For example, it will be appreciated that cation-exchange column chromatography performed using HPLC apparatus is several orders of magnitude greater than the same technique using low pressure chromatography systems. Relatively low purity methods are beneficial for recovering total protein or maintaining the activity of the expressed protein.
Of course, chromatographic techniques and purification methods known in the art may be used to purify the protein expressed by the adenovirus vector of the present invention. Exemplary purification techniques used to purify adenovirus particles include ion exchange chromatography and high performance drug chromatography, which are described in more detail below.
Ion-Exchange Chromatography: The basic principle of ion-exchange chromatography is that the affinity of the exchange material depends on the electrical properties of the material and the relative affinity of the other charged substrate in the solvent. The bound material can be eluted by changing the pH, changing the charge of the material or by adding a competing material such as a salt. Because different materials have different electrical properties, the conditions for releasing each bound molecular species vary. In general, for good separation, the options that can be chosen are continuous ionic strength gradient elution or gradual elution (not only pH gradients are used, because it is difficult to make a pH difference without increasing ionic strength). ). In the case of anion exchangers, the pH or ionic strength is gradually increased or only the ionic strength is increased. In the case of cation exchangers, pH and ionic strength are increased. It is advisable to select the dissolution process through trial and error and with regard to stability. For example, for unstable materials it is important to keep the pH constant at all times.
Ion exchangers are solids with chemically bonded charges in which ions are electrically coupled, and can exchange these ions with ions in an aqueous solution. Ion exchange is used for column chromatography to separate molecules according to charge; Indeed, other molecular features are also important because chromatographic behavior is sensitive to charge density, charge distribution and molecule size.
A molecule with the desired charge of ion exchange chromatography reversibly binds to the ion exchanger and changes the ionic environment to bind or elute the molecule. Separation in the ion exchanger consists of two steps in total; First, the material to be separated is bound to the exchanger under conditions that can provide a stable, firm bond; The column is then eluted with the components and compositions of the buffer that can compete with materials bound to different pHs, ionic strengths, or other binding sites or binding sites.
Ion exchangers are typically three-dimensional matrices or networks comprising charged groups covalently attached. If the group has a negative charge, the cation is exchanged, which becomes a cation exchanger. A common group used for cation exchangers is SO ₃ ⁻ . In the case that H ⁺ ions are bound to the exchange may exchange the two Ca ²⁺ as in the acid form, which in the H ⁺ Na ⁺ or two H ^+. The sulfonic acid group is called a strong acid cation exchanger. Other commonly used groups are hydroxy phenol and carboxy phenol, both of which are weak acid cation exchange groups. If the charged group is positive, for example a tetravalent amino group, it becomes a strong base anion exchange group. The most commonly used reverse base anion exchanger is an aromatic or aliphatic amino group.
The matrix is made of various materials. The most commonly used materials are dextran, cellulose, agarose, styrene and polyvinylbenzene interpolymers, wherein the divinylbenzenes are all crosslinked to the polyester strands and have charged groups. Table 4 provides many ion exchanger compositions.
The total capacity of the ion exchanger is evaluated by its ability to accept exchangeable groups at 1 mg dry molecular weight. This number is provided by the manufacturer, and in the case of excess capacity, the completion is very important because the ions do not bind and pass through the column.
matrix exchange Function Trade name Dextran Strong cation weak cation strong anion weak anion Sulfopropylcarboxymethyldiethyl- (2-hydroxypropyl) -aminoethyldiethylaminoethyl SP-SephadexCM-SephadexQAE-SephadexDEAE-Sephadex Cellulose Cationic cation anion anion anion anion Carboxymethylphosphodiethylaminoethylpolyethyleneiminebenzoylate-naphthoylate, diethylaminoethylp-aminobenzyl CM-CelluloseP-celDEAE-cellulosePEI-CelluloseDEAE (BND) -cellulosePAB-cellulose Styrene-divinyl-benzene Strong cation strong anion strong cation + strong anion Sulfonic acid sulfonic acid + tetramethylammonium AG 50AG 1AG 501 Acryl Phenoxyxoxyamine Weak cationic strong cationic weak anion Carboxylic sulfonic acid tetravalent amino Bio-Rex 70 Bio-Rex 40AG-3
Available doses refer to doses under certain experimental conditions (eg, pH, ionic strength). For example, the degree to which the ion exchanger is charged depends on the pH (the effect of pH is less on strong ion exchangers). Another factor is the ionic strength because small ions around the charged group compete with the same molecule for these groups. This competition is quite effective when the molecule is a macromolecule, which means that if the diffusion constant of small ions is larger, more encounters are made. Clearly, competition increases as the concentration of buffer increases.
The porosity of the matrix is also an important factor because it is inside and outside of the charged matrix, and the matrix also plays a role in filtering molecules. Large molecules cannot pass through the pores, and as the molecular size increases, the capacity decreases. The porosity of the polyester resin is determined by the amount of crosslinking of divinylbenzene. The porosity decreases when the amount of divinylbenzene is increased. In the case of the Dowex and AG series, the number after X represents the divinylbenzene ratio, for example Dowex 50-X8 says that the divinylbenzene is 8%.
Ion exchangers vary in particle size, ie mesh size. Fine mesh refers to a large volume to surface area ratio, thus increasing capacity and reducing exchange time in a given exchanger volume. On the other hand, fine mesh has a slow moving speed, which increases the diffusion spread. The use of very thin particles having a diameter of 10 μm and the use of high pressure to maintain proper flow is called high-performance or high pressure liquid chromatography or simply HPLC.
Other properties, such as charge, capacity, porosity, mesh, etc., may be chosen that are suitable for collecting and exchanging different exchangers. The following examples illustrate how to determine the shape of the column material and how to select binding and elution conditions.
There are several choices when using ion exchange chromatography. First of all, it is selected whether the exchanger is an anion or a cation. The choice is simple if the material to be bound to the column is a single charge (+ or-). However, since most substances (eg proteins, viruses) have both negative and positive charges, the overall charge depends on the pH. In such cases, the main factor is the stability of the material at various pH values. Most proteins have a stable pH range (eg, a pH range that does not denature), where they have a positive or negative charge. An anion exchanger is used if the protein is stable at a pH value above the isoelectric point, and a cation exchanger is used if it is stable below the isoelectric point.
The strong or weak exchanger is selected in consideration of the pH effect on charge and stability. For example, when chromatography weakly ionized materials that require very low or very high pH for ionization, strong ion exchangers are used because they function over the entire pH range. Weak ion exchangers are suitable when the material is unstable because strong ion exchangers sometimes modify the molecules by modifying them. The pH at which the material is stable is consistent with a very narrow range of pH at which certain weak exchangers are charged. Weak ion exchangers are also suitable for separating molecules with large charges from small charges because weakly charged ions do not bind. Weak exchangers have also been shown to separate materials better when the charge difference is very small. If the macromolecule has a very strong charge, it is impossible to elute from the strong exchanger, and a weak exchanger is appropriate. Weak exchanges are generally more useful than strong exchangers.
Sephadex and Bio-gel exchangers are particularly beneficial for macromolecules that are unstable at low ionic strength. Even when the matrix is large in polarity, the density of the ionizable groups can be several times greater than that of cellulose ion exchangers because of the insolubility of the matrix due to crosslinking in these materials. The increased charge density increases the affinity so that adsorption can occur even at higher ionic strengths. On the other hand, such exchangers have some molecular filtration properties, and sometimes molecular weight differences remove the distribution due to the charge difference, resulting in better separation due to molecular filtration effects.
Small molecules are best separated on matrices with small holes (high cross-linking) because of the large available capacity, whereas large molecules require large hole sizes. However, except for the Sephadex form, most ion exchangers do not provide an opportunity to match molecular weight and porosity.
Cellulose ion exchangers have proven to be most effective in purifying large molecules such as proteins and polynucleotides. This is because the matrix is fibrous and all functional groups are available on the largest molecules on the surface. In most cases, however, bead types such as DEAE-Sephacel and DEAE-Biogel P are more useful because they have better flow rates and the molecular sieving effect helps in separation.
Choosing a mesh size is always a difficult problem. Smaller mesh sizes improve dissolution but reduce flow rate, which increases zone spreading and lowers resolution. Therefore, the appropriate mesh size should be determined by experiment.
Since the buffer itself consists of ions, they can also be exchanged and the pH equilibrium is affected. To avoid this problem, the role of the buffer should be used. Anion exchangers and cation buffers, anion buffers and cation exchangers are used. Since ionic strength is a factor in binding, the buffer should choose a buffer with high buffering capacity, but the ionic strength should not be too large. In addition, to obtain the best resolution, the ionic conditions (so-called starting conditions) used to provide the sample to the column are close to those used to elute the column.
High performance liquid chromatography (HPLC) is characterized by a rapid resolution with significant resolution peaks. This is done using very fine particles and high pressure to maintain a proper flow rate. Can be separated within minutes or up to an hour. The particles are also very small and very tight, so that the void becomes a very small part of the bed volume, even with a small amount of sample. Also, the concentration of the sample does not need to be high because the band is so narrow that the sample is rarely diluted.
10. Pharmaceutical Compositions and Formulations
When purified using the method described above, the virus particles of the present invention can be administered in vitro, ex vivo, in vivo and the like. Therefore, it is desirable to prepare the complex in the form of a pharmaceutical composition suitable for the desired use. Generally, it is essentially a pharmaceutical composition that is free of pyrogens and free of other impurities that can be harmful to humans or animals. It is also desirable to use appropriate salts and buffers to stabilize the complex and allow the target cells to receive the complex.
The water-soluble composition of the present invention is composed of an expression construct effective amount and a nucleic acid, which are dissolved or dispersed in a pharmaceutically acceptable carrier or water-soluble medium. Such a composition is called an inoculum. By "pharmaceutical or pharmacologically acceptable" is meant the whole molecule and composition which, when administered to an animal or human, does not create side effects, allergies or other unwanted reactions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic solutions, absorption delaying agents, and the like. Such media and materials for pharmaceutically active substances are known. Except insofar as any conventional media or substance does not adapt with the active ingredient, they may be used in therapeutic compositions. Auxiliary activity statements can be bound to the composition.
As a free base or pharmacologically acceptable salt, the active compound solution is used by appropriately mixing water with a surfactant such as hydroxypropylcellulose. Dispersions can be made with grillcerol, liquid polyethylene glycols, mixtures thereof and oils. Under ordinary conditions for storage and use, these preparations contain a preservative to inhibit microbial growth.
Viral particles of the invention include traditional pharmaceutical preparations that can be used in therapeutic regimens, including administration to humans. Therapeutic compositions according to the invention are administered via any conventional route so long as target cells are available via the route of administration utilized. Such routes of administration include oral, nasal, buccal, rectal, vaginal or topical routes. It may also be administered via eye, dermis, subcutaneous, muscle, peritoneal or intravenous injection. Such compositions are administered in conjunction with a pharmaceutically acceptable composition comprising primarily a physiologically acceptable carrier, buffer, other excipient. For use in tumors, injection directly into the tumor, injection into the excised tumor bed, site (eg via lymph) or systemic administration may be considered. It is also preferable to carry out continuous irrigation for several hours or days using a catheter at a disease site such as a tumor or a tumor site.
Therapeutic compositions of the invention are preferably administered in the form of injectable compositions in liquid solutions or suspensions, and may also be prepared in solid form, which may be in solution, suspension or liquid prior to injection. Such preparations can also be emulsified. Typical compositions for this purpose consist of about 100 mg human serum albumin per ml phosphate buffer salt. Other pharmaceutically acceptable carriers may be used, such as aqueous solutions, salts, preservatives, buffers and the like, non-toxic excipients and the like. Non-aqueous solvents include polypropylene glycol, polyethylene glycol, vegetable oils, injectable organic esters such as ethyl oleate, and the like. Aqueous carriers include extracellular intestinal carriers such as water, alcohol / aqueous solutions, salt solutions, sodium chloride, Ringer's dextrose and the like. Intravenous carriers include fluids and nutrient replenishers. Preservatives include antimicrobials, antioxidants, chelating agents, inert gases, and the like. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted using known parameters.
There is an additional preparation for oral administration. Oral formulations include pharmaceutically usable levels of typical excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. The composition may be in the form of solutions, suspensions, tablets, pills, capsules, sustained release preparations, powders and the like. If the route is topical, it may be in the form of a cream, ointment, plaster, spray, or the like.
(i) inhibit tumor cell proliferation; (ii) kill or remove tumor cells; (iii) vaccination; (vi) The effective amount of the therapeutic agent substance is determined according to the desired purpose, such as gene delivery for long-term expression of the therapeutic gene. "Unit dose" refers to physically discrete units suitable for use in a subject, each unit comprising a predetermined amount of the dentifrice composition calculated for administration to produce the desired response. The amount to be administered depends on the number and duration of the treatment, the individual to be treated, the condition of the individual and the desired outcome. In the case of adenoviruses, multiple gene therapy regimes can be expected.
In certain embodiments of the invention, adenovirus vectors encoding tumor suppressor genes are used to treat cancer patients. Typical amounts of an adenovirus vector for use in cancer gene therapy with 10 ³ -10 ¹⁵ PFU / dose (10 ^3, 10 ^4, 10 ^5, 10 ^6, 10 ^7, 10 ^8, 10 ^9, 10 ^10, 10 ^11, 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ ), where the dose can be divided by several injections to other sites within the filling tumor. The treatment regime involves several processes of administering the gene transfer vector for 3-10 weeks. It may be beneficial to administer it for many hours, ranging from months to years.
In another embodiment of the present invention, adenovirus vectors encoding therapeutic genes can be used to vaccinate humans or other mammals. In general, in the case of vaccination, the amount of virus required to achieve the desired effect is administered to humans or animals to express the transgene for a long time and to obtain a strong host immune response. Two additional booster injections after the first injection can induce a long-term immune response. Typical dosages range from 10 ⁶ to 10 ¹⁵ PFU, depending on the desired result. Small amounts of antigen can induce potent cell-mediated immune responses, while large amounts of antigens induce antibody-mediated immune responses. The exact amount of therapeutic composition depends on the judgment of each practitioner and varies from individual to individual.
11.Example
The following examples illustrate suitable embodiments of the present invention. Those skilled in the art will be representative of the techniques found in the examples found by the inventors to perform optimal functions in carrying out the invention. However, one of ordinary skill in the art will recognize that many variations can be made to the specific embodiments described based on the teachings of the present invention, which can yield similar results without departing from the scope of the present invention.
Example 1
Materials and methods
A) cells
293 cells (human epidermal embryonic kidney cells) were obtained from the Master Cell Bank for research.
B) badge
Dulbecco's modified Eagle's medium (DMEM, 4.5 g / L glucose) + 10% fetal calf serum (FBS) was used for cell growth. On virus growth, the concentration of FBS in DMEM was lowered to 2%.
C) virus
Cytomegalovirus (CMV) A replication-responsive human type 5 adenovirus, AdCMVp53, was developed that expresses human wild-type p53 protein under the control of an early early promoter.
D) Celligen Bioreactor
Virus supernatants were prepared by microcarrier culture using a 5 L (3.5 L working volume) Celligen Bioreactor (New Brunswick Scientific, Co. Inc.). Microcarriers (SoloHill) coated on 13 g / l glass were used to incubate the cells in the bioreactor.
E) Producing viral supernatant in Celligen bioreactor
293 cells obtained from Master Cell Bank (MCB) were thawed and extended to Cellfactories (Nunc). Cells join at about 85-90% level. Cells were seeded in the bioreactor at a concentration of 1 × 10 ⁵ cells / ml. The cells are agitated intermittently to attach to the microcarrier. Stirring was started continuously at a speed of 30 rpm about 6-8 hours after cell inoculation. Cells were incubated for 7 days with pH = 7.20, dissolved oxygen (DO) = 60%, air saturation, and temperature fixed at 37 ° C. On day 8, cells are infected with AdCMVp53 with a MOI value of 5. 50 hours after virus infection, the agitation rate was increased from 30 rpm to 150 rpm to allow cell lysis and release the virus into the supernatant. Virus supernatants were obtained 74 hours after infection. Viral supernatant is filtered for further concentration and filtration.
F) Cellcube ^TM Bioreactor System
AdCMVp53 virus was produced using the Cellcube ^™ Bioreactor System (Corning-Costar). It consists of a disposable cell culture module, an oxygen generator, a medium circulation pump, and an irrigation medium pump. The cell culture module used has a 21,550 cm 2 (1mer) culture surface area.
G) Virus production in Cellcube ^TM
Thaw 293 cells obtained from the Master cell Bank (MCB) and extend them from Cellfactories (Nunc). Cells join at about 85-90% level. Cells were seeded in Cellcube ^™ as recommended by the manufacturer. Inoculated cell density is 1-1.5 × 10 ⁴ / cm 2. Cells were grown for 7 days at pH = 7.20, DO-60% air saturation, 37 ° C. Cell irrigation rate was adjusted according to glucose concentration in Cellcube ^™ . One day before virus infection, the irrigation medium was changed from DMEM + 10% FBS to DMEM + 2% FBS. On day 8, cells are infected with AdCMVp53, with a MOI value of 5. The medium irrigation is stopped for 1 hour after infection and then again for the rest of the virus production phase. Cells were obtained 45-48 hours after infection.
H) dissolution solution
Cells are lysed at the end of the virus production phase in Cellcube ^™ using 1% (v / v) Tween-20 (Fisher Chemicals) in 20 mM Tris + 0.25 M NaCl + 1 mM MgCl ₂ , pH = 7.50 warm buffer.
I) Purification and Filtration
Virus supernatants obtained from Celligen bioreactors and virus solutions obtained from Cellcube ^™ are first clarified using a strong filter (Preflow, Gelman Sciences) and then filtered through a 0.8 / 0.22 μm filter (SuporCap 100, Gelman Sciences).
J) Concentration / Diafiltration
Using a tangential flow filter (TFF), the virus supernatant obtained from Celligen Bioreactor and the virus solution obtained from Cellcube ^™ are concentrated and the buffer is exchanged. Concentration and diafiltration are performed using a 300K molecular weight blocking (NMWC) PelliconII mini cassette (Millipore). The viral solution is first concentrated 10 times. The 4 volume buffer is then exchanged for 20 mM Tris + 1.0 M NaCl + 1 mM MgCl ₂ , pH = 9.00 buffer using a constant volume diafiltration method.
Similar concentrations and diafiltration were performed for column purified viruses. Pellicon II mini cassettes (100K NMWC) are used instead of 300K NMWC. Diafiltration is performed using 20 mM Tris + 0.25 M NaCl + 1 mM MgCl ₂ , pH = 9.00 buffer or Dulbecco's Phosphate Buffered Salt (DPBS).
K) benzonase treatment
The concentrated / diafiltered virus solution is treated with Benzonase ^™ (American International Chemicals) at a concentration of 100 u / ml at room temperature to reduce the concentration of impurity nucleic acids in the virus solution.
L) CsCl gradient ultracentrifugation
The unfiltered virus solution is purified by double CsCl gradient ocular centrifugation using a Beckman ultracentrifuge (XL-90) SW40 rotor. First, a 7 ml stock virus solution is placed on top of the same amount of CsCl decanter (which consists of 2.5 ml 1.25 g / ml and 2.5 ml 1.40 g / ml CsCl solution). CsCl gradient is centrifuged at 35,000 rpm for 1 hour at room temperature. Virus bands can be recovered between the slopes. The recovered virus was further purified through isodensity CsCl gradient. Mix the virus solution with at least 1.5 times the volume of 1.33 g / ml CsCl solution. The CsCl solution was centrifuged at 35,000 rpm for at least 18 hours at room temperature. The lower band consisting of the native virus was recovered. Virus was directly dialyzed against 20 mM Tris + 1 mM MgCl ₂ , pH = 7.50 buffer to remove CsCl. Dialysis virus is stored at -70 ° C for later use.
M) Ion Exchange Chromatography (IEC) Purification
The benzonase treated virus solution was purified using IEC. For purification, a strong anion resin Toyopearl SuperQ 650M (Tosohaas) was used. The first developed method used a FPLC system (Pharmacia) using an XK16 column (Pharmacia). Further large volume studies used the BioPilot system (Pharmacia) using XK16 columns (Pharmacia). In brief, the column is filled with resin, sterilized with 1N NaOH, then filled with Buffer B, and then conditioned with Buffer A. Buffers A and B consisted of 20mM Tris + 0.25M NaCl + 1mM MgCl ₂ , pH = 9.00 and 20mM Tris + 2M NaCl + 1mM MgCl ₂ , pH = 9.00, respectively. The virus solution sample was placed on a conditioned column and the column was washed with buffer A until UV absorption reached the basal level. Purified virus is eluted from the column using a 10-fold column volume with a linear NaCl gradient.
N) HPLC analysis
HPLC analysis procedures have been developed to assess virus production and purification efficiency. Tris (hydroxymethyl) aminomethane (tris) obtained from Fisher Biotech (Cat # BP154-1; Fair Lawn, New Jersey, U.S.A.); Sodium chloride (NaCl) was used by Sigma (Cat # S-7653, St. Louis, MO, U.S.A.), which was used directly by further purification fish. HPLC analysis using Gold Workstation Software (126 binary pump and 168 diode array detector) on Beckman's analytical gradient system equipped with an anion exchange column, TosoHaas (7.5 cm × 7.5 mm ID, 10 µm particle size, Cat # 18257) It was. A 1 ml Resouce Q (Pharmacia) anion-exchange column was used to evaluate the method developed by Huyghe et al., Using the HEPES buffer system. This method was only used in bioreactor systems.
The buffers currently used in HPLC systems are as follows: Buffer A is 10 mM Tris buffer, pH 9.0. Buffer B is 1.5M NaCl + Buffer A, pH 9.0. The buffer is filtered using a 0.22 μm bottle top filter from Corning (Cat # 25970-33). All samples are filtered through 0.8 / 0.22 μm Aerodise PF (Gelman Sciences (Cat # 4187)) prior to parent injection.
Samples are injected into the HPLC column at a volume of 60-100 μl. After injection, the column (TosoHaas) is washed with 20% B at 0.75 ml / min rate for 3 minutes. After 6 minutes, concentration differences begin to appear, ie B increases from 20% to 50%. This slope is 50% to 100% after 3 minutes and 100% for the next 6 minutes. The salt concentration returns stepwise back to 20% after 4 minutes and then maintains 20% B for 6 minutes. Holding time of Adp53 is 9.5 ± 0.3, A ₂₆₀ / A ₂₈₀ = 1.26 ± 0.03. After each chromatography was finished, the column was washed with 100 μl 0.15 M NaOH, followed by gradient.
Example 2
Virus production and the impact of media on the drenched Cellcube ^TM Purification
In the case of irrigation cell culture systems such as Cellcube ^™ , medium irrigation rate plays an important role in the yield and quality of the product. Two different medium irrigation strategies were used. One is to maintain glucose concentration in the Cellcube ^™ at ≧ ² g / l (fast irrigation). The other, the glucose concentration is maintained at > 1 g / l (low speed medium irrigation).
When two different irrigation rates were used, no significant change in culture parameters such as pH and DO was observed. Equivalent crude virus (prior to purification) was produced after obtaining with 1% Tween-20 lysis solution as shown in Table 5. However, it can be seen that there is a significant difference in the HPLC profile of the viral solution with high and low irrigation.
Effect of Glucose Concentration on Medium in Virus Production Glucose concentration (g / L)≥2.0≥1.0 Virus Production (PFU)4 × 10 ¹² 4.9 × 10 ¹²
As can be seen in FIG. 1, a slow medium irrigation yielded a virus peak (hold time 9.39 min) which was very well separated from the virus solution. It was found that after one step ion exchange chromatography purification of virus solutions produced under slow medium irrigation, viruses with optimal purity and biological activity could be obtained. On the other hand, the virus peak was not separated at 9.39 minutes from the virus solution obtained under high-speed irrigation. This means that under fast medium irrigation, contaminants with the same eluate as the virus are produced. Although the characteristics of the contaminants are not clear, the contaminants appear to be associated with increased extracellular matrix protein production under high-speed medium irrigation from the producing cells. As can be seen in the following examples, the separation characteristics found in HPLC make the IEC purification process difficult. As a result, the medium irrigation rate used during cell growth and virus production phase on Cellcube ^™ has a significant impact on IEC purification of the virus. Slow medium irrigation is appropriate. The use of low-speed medium irrigation facilitates the purification of the original product as well as lowers the medium consumption and is effective in terms of cost.
Example 3
Cell acquisition and lysis method
Based on existing experiments, the inventors first evaluated the freeze-thaw method. Cells were obtained from Cellcube ^™ 45-48 hours after infection. First, Cellcube ^™ is separated from the culture system and the medium used is drained. The 50 mM EDTA solution is then pumped into the Cube to separate the cells from the culture surface. The cell suspension obtained was then centrifuged at 1,500 rpm (Beckman GS-6KR) for 10 minutes. The resulting cell pellet is resuspended in Dulbecco's Phosphate Buffer (DPBS). The cell suspension releases the virus from the cells by repeating the freeze / thaw process five times in a 37 ° C. water bath and dry ice container. The obtained crude cell lysate (CCL) was analyzed by HPLC.
2 shows the HPLC profile. No virus peak was observed at 9.32 minutes. Instead, two peaks were produced at 9.11 and 9.78 minutes. This profile means that other contaminants with similar elution times as viruses are present in the CCL, which interferes with virus purification. As a result, it can be seen that the purification efficiency is very low when the CCL is purified by IEC using FPLC.
In addition to being low in purification efficiency, the amount of product loss during the cell harvesting process is significant, as can be seen in Table 6. About 20% of the product is lost in the discarded EDTA solution. In addition, about 24% of the unpurified viral product was present in the spent media. Thus, there are only about 56% unpurified viral product in CCI. In addition, the freeze-thaw process is the limiting factor in scaling up to the most changing process. There is a need to develop more efficient cell lysis processes with less loss of product.
Virus loss during EDTA obtaining of cells from Cellcube ^™ waste Unrefined productTotal product (PFU)Badge usedEDTA SolutionCell lysateVolume (ml)2800200082- Titer (PFU / mL)2.6 × 10 ⁸ 3 × 10 ⁸ 2 × 10 ¹⁰ - Total Virus (PFU)7.2 × 10 ¹¹ 6 × 10 ¹¹ 1.64 × 10 ¹² 3 × 10 ¹²ratio24%20%56%
Data obtained from 1 mer Cellcube
Evaluation of Non-ionic Surfactants for Cell Lysis SurfactantsConcentration (w / v)chemical substanceRemarks Thesit1% 0.5% 0.1%Dodecylpoly (ethylene glycol ether) _n n = 9-10Much precipitation NP-401% 0.5% 0.1%Ethylphenolpoly (ethylene-glycoether) _n n = 9-11Much precipitation Tween-201% 0.5% 0.1%Poly (oxyethylene) _n -sorbitan-monolaurate n = 20Small precipitation Brij-581% 0.5% 0.1%Cetylpoly (ethyleneglycoether) _n n = 20Cloudy solution Triton X-1001% 0.5% 0.1%Oxylphenol poly (ethylene glycol ether) _n n = 10Much precipitation
Surfactants were used to effect cell lysis to release intracellular organs. As a result, the inventors evaluated a method of dissolving the surfactant to release the adenovirus. Table 7 lists five different non-ionic surfactants during cell lysis. Using 50 mM EDTA, cells were obtained from Cellcube ^™ 48 hours after infection. Cell pellets were suspended at different concentrations of the different surfactants shown in Table 7.
Cell lysis was performed at room temperature or ice for 30 minutes. Centrifugation to remove precipitates and cell debris yields a clear lysis solution. The dissolution solution was treated with benzonaise and then analyzed by HPLC. 3 shows the HPLC profile of the dissolution solution with different surfactants. Thesit and NP-40 work similarly to the Triton X-100. The lysis solution with 1% Tween-20 had the best viral resolution and the lowest viral resolution observed with Brij-58. The most effective cell lysis occurs at 1% (w / v) surfactant concentration. Dissolution temperature is not an important factor for virus resolution under the tested surfactant concentration. To simplify the process, dissolution at room temperature is recommended. Virus obtained from Cellcube ^™ and cell lysis were obtained using a lysis solution consisting of 1% Tween-20 in a 20 mM Tris + 0.25M NaCl + 1mM MgCl ₂ pH = 7.50 solution.
Example 4
Effect of Enrichment and Diafiltration on Virus Recovery
The virus solution of the lysis step was clarified and filtered before concentration / diafiltration. The effect of concentration / diafiltration was evaluated on TFF membranes with different NMWCs, including 100K, 300K, 500K, 1000K. When using 300K NMWC membranes, maximum media flow can be obtained with minimal virus loss in the filtrate. Use of larger NMWC membranes results in higher media flow, but increases virus loss in the filtrate, while using less NMWC membranes results in insufficient media flow. The virus solution is first concentrated 10-fold and then subjected to a 4-fold sample volume diafiltration on 20 mM Tris + 0.25 M NaCl + 1 mM MgCl ₂ , pH = 9.00 buffer using a constant volume method. The pressure difference through the membrane during the concentration / diafiltration process is maintained at ≤ 5 psi. As can be seen from Table 8, it can be seen that virus recovery levels are consistently high during enrichment / diafiltration.
Concentration / Diafiltration of Unpurified Virus SolutionTiter (PFU / mL)Volume (ml)Total Virus (PFU)collectionRun # 1Run # 2Run # 1Run # 2Run # 1Run # 2Run # 1Run # 2 Ex conc./diafl.2.6 × 10 ⁹ 2 × 10 ⁹ 190020004.9 × 10 ¹² 4 × 10 ¹² After conc./diafl.2.5 × 10 ¹⁰ 1.7 × 10 ¹⁰ 2002005 × 10 ¹² 3.4 × 10 ¹² 102%85% density 9.510 factor Filtrate5 × 10 ⁵ 1 × 10 ⁶ 300030001.5 × 10 ⁹ 3 × 10 ⁹
Example 5
Effect of salt addition during benzonase treatment
After concentration / diafiltration the virus solution is treated with benzonase (nuclease) to reduce the impurity nucleic acid concentration in the virus solution. The effects of reducing the nucleic acid concentration at different concentrations of 50, 100, 200, 300 units / ml were evaluated. In order to simplify the process, it was treated overnight at room temperature. It can be seen that after benzonase treatment, contaminating nucleic acids that can hybridize to human genomic DNA are significantly reduced.
As can be seen from Table 9, it can be seen that the concentration before and after the benzonase treatment was significantly reduced. Viral solutions were analyzed via HPLC before and after benzonase treatment. As can be seen in Figures 4A and 4B, it can be seen that the peak of the contaminant nucleic acid was drastically reduced after the benzonase treatment. This is consistent with nucleic acid hybrid test results. Because of the effect, the unpurified virus solution is treated with benzonase at a concentration of 100 u / ml.
Reduction of contaminant nucleic acid concentrations in viral solutions (treatment conditions: benzonaise concentration of 100 u / ml, temperature of room temperature, overnight overnight)Before treatmentAfter treatmentdecrease Contaminant Nucleic Acid Concentration200 µg / ml10ng / ml2 × 10 ⁴ -fold
There was a significant change in HPLC profile before and after benzonase treatment. No viral peak isolated at 9.33 hours after benzonase treatment was detected. At the same time, a major peak with 260 nm high absorption at 9.54 minutes was observed. According to the titer test results, benzonase treatment does not have a bad effect on virus titer, and the virus remains intact and infectious even after benzonase treatment. This is because the cellular nucleic acid released during the cell lysis step interacts with the virus, aggregates with the virus or is adsorbed to the virus surface during the benzonase treatment.
To minimize the interaction of the nucleic acid with the virus during the benzonase treatment, different concentrations of NaCl are added to the virus solution prior to the benzonase treatment. There was no significant change in the HPLC profile obtained by treatment with benzonase in the presence of 1M NaCl in the virus solution. Figure 5 shows the HPLC profile of the viral solution after benzonase treatment in the presence of 1M NaCl. As can be seen in FIG. 4, there was still a viral peak at 9.35 minutes after benzonase treatment. This result shows that 1M NaCl disrupts the interaction of the nucleic acid with the virus during the benzonase treatment and allows for further purification of the virus from contaminating nucleic acid.
Example 6
Ion Exchange Chromatography Purification
Anion exchangers were evaluated for adenovirus purification when there was a negative charge on the surface of the adenovirus at physiological pH. A strong anion exchanger Toyoperal Super Q 650M was used to develop the purification method. The NaCl concentration and the pH effect of the dropping buffer (buffer A) upon virus purification were assessed using the FPLC system.
A) Method Development
In the case of ion exchange chromatography, the pH of the buffer is the most important variable and has a significant impact on the purification effect. Regarding medium pH, conductivity during virus production, the inventors set buffer A to 20 mM Tris + 1 mM MgCl ₂ + 0.2 M NaCl, pH = 7.50. An XK16 column filled with Toyopearl SuperQ 650M at 5 cm height was conditioned with Buffer A.
The virus supernatant is treated with benzonase in a Celligen bioreactor and 5 ml of concentrated / diafiltered sample is added dropwise to the column. After washing the column, elution was performed with Tris + 1 mM MgCl ₂ + 2M NaCl, pH = 7.50, with a linear gradient of 10 times or more.
6 shows an elution profile. Three peaks can be observed during elution without satisfactory separation. Based on a baseline study conducted with 293 cell conditioned medium (no virus), the first two peaks were associated with the virus. In order to improve the separation effect, the effect of buffer pH was evaluated. The other conditions are kept constant while increasing the buffer pH to 9.00. As can be seen in FIG. 7, it can be seen that a significant improvement compared to the case of pH 7.50 buffer. Aliquots # 3, # 4, # 8 were analyzed using HPLC.
As can be seen in Figure 8, most of the virus was found in # 4, the virus was not detected in # 3 and # 8. # 8 contains mainly contaminating nucleic acid. However, there seems to be less purification yet. The overlap between # 3 and # 4 still indicates that there is contaminant at # 4.
Based on the chromatography of FIG. 7, it can be seen that increasing the concentration of salt in Buffer A can improve virus purification. As a result, the contaminants present at # 3, just before the virus peaks, flow through the friction. NaCl concentration in Buffer A is increased to 0.3 M, but other conditions remain constant. Figure 9 shows the elution profile in the buffer solution A with a salt concentration of 0.3M NaCl.
The purification effect was drastically improved. As expected, the contamination peaks seen in FIG. 7 disappear when the salt concentration is increased. Samples were taken from the crude virus supernatant and passed through to analyze peaks # 1 and # 2 via HPLC. No virus was detected in the flow through friction. HPLC analysis of peak # 1 shows one virus peak. These HPLC peaks are equivalent to those obtained from double CsCl gradient purified virus. The peaks observed at 3.14 minutes and 3.61 parts in the peak purified by the CsCl gradient are glycerol related peaks. The A260 / A280 ratio of the purified virus is 1.27 ± 0.03. This is similar to the results reported by Huyghe et al. (1996), as well as similar values of the double CsCl purified virus. Peak # 2 consists mainly of contaminating nucleic acid. Based on the purification results, the inventors proposed the following method for IEC purification of adenovirus supernatants taken from bioreactors.
Buffer A: 20 mM Tris + 1 mM MgCl ₂ + 0.3 M NaCl, pH = 9.00
Buffer B: 20 mM Tris + 1 mM MgCl ₂ + 2M NaCl, pH = 9.00
Elution: 10-fold column volume, linear gradient
B) how to increase capacity
After developing the method, the same purification method was used to scale up the purification method using an XK50 column (5 cm I.D., 10-fold scale up) instead of the XK16 column (1.6 cm I.D.). Similar elution profiles were obtained in the XK50 column as can be seen in FIG. 11. Virus aliquots were analyzed on HPLC, which showed virus purity equivalent to that obtained on XK16 column.
During the scale up study, it is convenient to use conductivity to quantify the salt concentration of Buffer A. The appropriate conductivity range of buffer A is 25 ± 2 mS / cm in Ceylon (21 ° C). Samples generated during purification and the double CsCl purified virus were analyzed together on SDS-PAGE.
As can be seen in Figure 12, most major adenovirus structural proteins were detected on SDS-PAGE. IEC purified virus showed similar staining to that of the double CsCl purified virus. Bovine serum albumin (BSA) concentrations were significantly reduced during purification. The BSA concentration in the purified virus was lower than that detected by the Western blot test of FIG. 13.
The extent to which contaminating nucleic acid concentrations are reduced during the purification process is measured using nucleic acid slot blots. ³² P labeled human genomic DNA was used as a hybrid probe because 293 cells are human embryonic kidney cells. Table 10 shows nucleic acid concentrations at different stages of purification. The nucleic acid concentration in the final purified virus solution was 60 pg / ml, a 3.6 × 10 ⁶ fold reduction compared to the concentration of the initial virus supernatant. Virus titers and infectivity of the total particles from the purified virus were evaluated and the results compared to those of the dual CsCl purification procedure in Table 9. Virus recovery and particle / PFU ratio were very similar in both purification methods. The titer of column purified virus solution can be further increased by performing concentration.
Removal of Contaminating Nucleic Acids During Purification Purification stepsContaminant Nucleic Acid Concentration Bioreactor Virus Supernatant220 µg / ml Concentrated / filtration supernatant190 μg / ml Supernatant after Benzonase treatment (O / N, RT, 100u / ml)10ng / ml Column Purified Virus210 pg / ml Purified virus after enrichment / filtration60 pg / ml CsCl purified virus800 pg / ml
Example 7
Other purification methods
In addition to the strong anion exchange chromatography method, the purity of the AdCMVp53 virus was assessed using other modes of chromatography (eg, size extrusion chromatography, hydrophobic interaction chromatography, cation exchange chromatography, metal ion affinity chromatography). When compared to that of Toyopearl Super Q, all purification methods were rather ineffective and the product recovery was low. Therefore, Toyopearl Super Q resin is recommended when purifying AdCMVp53. However, in some modifications of the process, other tetravalent ammonia chemicals using strong anion exchange may be suitable when attempting to purify AdCMVp53.
Example 8
Purification of Unpurified AdCMVp53 Virus from Cellcube ^TM
Two different production methods have been developed to make the AdCMVp53 virus. One is to use microcarriers in stirred tank reactors. The other is to use Cellcube ^™ bioreactor. As described above, the purification method was developed using unpurified viral supernatant generated in a stirred tank bioreactor. The same media, cells, and viruses were used to produce viruses in bioreactors and Cellcube ^™ , but the surface area on which cells adsorb was different.
In the case of bioreactors, cells were grown on glass-coated microcarriers and, in the case of Cellcube ^™ , on proprietary treated polyester culture surfaces. On the other hand, the cellcube ^TM was constantly irrigated, and the bioreactor did not carry out the medium irrigation. In the case of Cellcube ^™ , the crude virus product is obtained in the virus infected cell type, whereas in the bioreactor the virus supernatant type is obtained.
The crude purified cell lysate (CCL) produced after five freeze-thaw cycles of virus infected cells was purified by IEC using the method described above. Unlike virus supernatants obtained from bioreactors, purification was not satisfactory for CCL material obtained from Cellcube ^™ . 14 shows a chromatogram. These results indicate that virus solutions obtained from Cellcube ^™ by freeze-thawing are not purified by the IEC method.
Other purification methods including hydrophobic interaction, metal chelate chromatography for virus purification in CCL were also examined. Unfortunately, there was no improvement in tablets in these methods. Given the difficulty in purifying viruses in CCL and the drawbacks associated with freeze-thaw in the production process, the inventors decided to investigate other lysis methods.
A) Purification of unpurified virus solution in lysis buffer
As can be seen in Examples 1 and 3, HPLC analysis was used to screen different surfactant dissolution methods. Based on HPLC results, a 1% Tween-20 20 mM Tris + 0.25 M NaCl + 1 mM MgCl ₂ , pH = 7.50 solution was used as lysis buffer. Instead of obtaining infected cells by the end of the virus production phase, the medium used was removed and lysis buffer was fed to Cellcube ^™ . After 30 minutes of incubation, the cells are lysed and the virus is released into the lysis buffer.
After clarification and filtration, the viral concentrate is concentrated / diafilterated and treated with benzonase to reduce the contaminating nucleic acid concentration. The treated virus solution was purified by the method described above using Toyopearl SuperQ resin. A separation result similar to that obtained in the virus supernatant obtained from the bioreactor was obtained. 15 shows an elution profile. However, analysis of virus aliquots on HPLC detects peaks other than virus peaks. The results are shown in Figure 16A.
To further purify the virus, the collected virus aliquots were purified again in the same manner. As can be seen in FIG. 16B, the purity of the virus aliquot after the second purification was significantly improved. It was evaluated whether metal chelate chromatography was suitable as the second purification method. As seen in the second IEC, virus purity is similarly improved. However, the IEC was chosen as the second purification method because of its simplicity.
As described in Example 2, the media irrigation used during cell growth and virus production has a significant impact on the HPLC separation profile resulting in Tween-20 unpurified virus yield. In the case of the unpurified virus solution produced under high speed irrigation, two ion exchange columns are required to achieve the required virus purity.
Based on the significant improvement in separation in HPLC for virus solutions produced under slow medium irrigation, purification through one ion exchange column will yield the required viral purity. 17 shows the elution profile of the crude virus solution produced under slow medium irrigation. High virus peaks were obtained during elution. HPLC analysis of virus aliquots showed that after one ion exchange chromatography, the viral purity is similar to that of the CsCl gradient. 18 shows the result of HPLC analysis.
Purified virus was analyzed by Western blot for SDS-PAGE and BSA and subjected to nucleic acid slot blot to determine contaminating nucleic acid concentration. The analysis results are shown in FIGS. 19A, 19B, and 19C, respectively. All analyzes showed that the column purified virus had a purity comparable to that of the purified virus by double CsCl gradients. Table 11 shows virus titers and recovery rates before and after column purification. For comparison, the general virus recovery obtained by double CsCl gradient purification was included. Both methods had similar virus recovery.
Comparison of ICE and Dual CsCl Gradient Ultracentrifugation Purification of AdCMVp53 from Cellcube ^TM Titer (PFU / mL)A260 / A280Particle / PFUcollection IEC1 × 10 ¹⁰ 1.273663% Ultracentrifugation2 × 10 ¹⁰ 1.263860%
A) Resin Capacity Study
The dose of Toyopearl Super Q resin was evaluated to purify the virus solution obtained with Tween 20 produced under low irrigation. Fill the XK50 column with 100 ml resin. Different amounts of unpurified virus solution were purified through the column using the method described herein.
Virus destruction and purification effects were examined on HPLC. 20 shows the results of HPLC analysis. If the sample / column volume ratio is greater than 2: 1, the purity of the viral aliquot is reduced. Contaminants elute with the virus. If the loading factor is 3: 1 or higher, the destruction of the virus can be observed in the flow process. Thus, the working dropping capacity of the resin allows the sample / column volume ratio to be 1: 1.
B) Concentration / Diafiltration after Purification
The reason for concentration / diafiltration after column purification is to increase virus titer, which can be exchanged with virus buffer specific to the virus product, if necessary. The purification step uses a 300K NMWC TFF membrane. Since no purified protein-like or nucleic acid contaminants exist in the purified virus, very large buffer flows can be obtained without a rapid drop in pressure through the membrane.
Changing the buffer to 20mM Tris + 1mM MgCl ₂ + 0.15M NaCl, pH = 7.50 recovers almost 100% virus during this step. Purified virus can be converted to DPBS during the enrichment / diafiltration step. The concentration factor is determined by the virus titer desired for the final product and the titer of the virus solution eluting from the column. This fluidity helps to maintain the concentration of the final purified virus product.
C) Evaluation of Defective Viruses on IEC Purified AdCMVp53
Since the package efficiency of adenovirus in production cells is less than 100%, some defective adenovirus is present in the unpurified virus solution. Since the defective virus does not capture DNA in the virus capsid, the density difference can be used to isolate the native virus using CsCl gradient ultracentrifugation. Since both viruses have similar surface chemistries, it may be difficult to isolate defective viruses from native viruses using ion exchange chromatography. The presence of excess deletion virus will affect the purity of the purified product.
To assess the proportion of missing virus particles present, the purified concentrated virus is subjected to isotensive CsCl ultracentrifugation. As can be seen in FIG. 21, a faint band can be observed on the native virus band after centrifugation. Two bands are recovered and dialyzed against 20 mM Tris + 1 mM MgCl ₂ , pH = 7.50 buffer to remove CsCl. Dialysis virus was analyzed by HPLC and the results are shown in FIG. 22. Both viruses have similar holding times. However, the defective virus has a lower A260 / A280 ratio than that of the native virus. This means that the defective virus has less viral DNA.
If glycerol is added to the virus (10% v / v) before freezing to −70 ° C., peaks can be seen between 3.02 and 3.48 minutes. Defective virus rate is less than 1% of total virus. As such, the percentage of defective virus affects the infectious virus (PFU) for the total particles in the purified virus product. Two viruses were analyzed by SDS-PAGE (FIG. 19A). Compared with native virus, the defective virus lacked the DNA associated core protein at 24 and 48.5 KD. This result is consistent with the presence of DNA in the defective virus.
D) Production and Purification of AdCMVp53 Virus
Based on the above development results, the inventors provided the production and purification sequence of AdCMVp53, as can be seen in FIG. Based on 1 mer Cellcube ^™ , a virus recovery step with corresponding virus production is included. The final virus recovery rate is 70 ± 10%. This is about three times greater than the virus recovery reported by Huyghe et al. (1996), using metal chelate chromatography purification to purify the p53 protein encoding the DEAE ion exchanger and adenovirus. The final purified virus product in 1 mer Cellcube ^™ is about 3 × 10 ¹² PFU. The final yield was comparable when compared to the current production method using double CsCl gradient ultracentrifugation for purification.
E) Scale up
4mer Cellcube ^TM were running up-scale study of successful systems to evaluate the virus produced from the current 16 mer Cellcube ^TM system. The resulting crude virus solution is purified, concentrated and diafiltered using a Pellicon cassette. Determine the quality and recovery of the virus. After benzonase treatment, the unpurified virus solution is purified using 20 cm and 30 cm BioProcess columns, 4mer and 16mer, respectively.
Example 9
Improved Ad-p53 Production in Serum-Free Suspension Cultures
Adaptation of 293 Cells
Continuously lowering the FBS concentration in the T-flask allows 293 cells to adapt to commercial IS293 serum free medium (Irvine Scientific; Santa Ana, Calif.). Thaw frozen cells in one vial PDWB, place in 10% FBS DMEM medium in a T-flask, and cells adapt to serum-free IS 293 medium in the T-flask by continuously lowering the FBS concentration in the medium. After six passages to the T-75 flask, the FBS% was estimated to be about 0.019%. The cells are further passaged two or more times to the T flask before transfer to the spinner flask.
293 cells serum-free adapted to T flasks are adapted to suspension culture.
The serum-free adaptive cells in T flasks are transferred to serum-free 250 ml spinner suspension culture (100 ml working volume) for suspension culture. Viability is reduced during cell culture, and large cell masses are observed. After two or more passages in the T-flask, it is adapted to suspension culture again. In the second trial, heparin was added to the medium at a concentration of 100 mg / l to prevent cell aggregation and the initial cell density increased to 1.18E + 5 vc / ml. During cell culture, cell density is slightly increased and cell viability is maintained. The doubling time of the cells was significantly reduced after and during passage of the cells in the spinner flask at least seven times, at the end of the seven passages the doubling time was approximately 1.3 days, which resulted in doubling of cells under 10% FBS in adherent cell culture. It is comparable to the time (1.2 days). In serum-free IS293 medium with heparin added, almost all cells are present individually without forming a cell population in suspension culture (Table 12).
Serum-free suspension culture: adapted to suspension Passage numberFlask No.Average doubling time (days) 11 Reduced viability 13 3.4 14 3.2 15 Heparin addition1234Viability Reduction4.75.03.1 1612345.54.84.34.3 1712342.93.52.41.7 1812343.513.16.13.8 1912342.52.62.32.5 2012341.3 (97% Viability) 1.5 (99% Viability) 1.8 (92% Viability) 1.3 (96% Viability)
Virus Production and Cell Growth with Serum-Free Suspension Cultures in Spinner Flasks
To test Ad5-CMVp53 vector production in serum-free suspension cultures, the cells adapted to serum-free suspension cultures were grown in 100 ml serum-free IS293 medium supplemented with 250% spinner flasks with 0.1% Pluronic F-68 and heparin. Let's do it. Cells are infected at 1.36E + 06 viable cells / ml on day 3 (MOI is 5). Supernatants are analyzed daily by HPLC to calculate virus particles / ml after infection. No virus was detected in samples older than 3 days. On day 3 it is 2.2E + 09 vps / ml and on day 6 it is 2.6 +/- 0.6E + 07 pfu / ml. The pfu per cell was found to be 19, which was about 46 times less than when adsorption cultures were in media supplemented with serum. As a reference, cell growth was checked without infection.
Serum-free suspension culture: virus production and cell growthStandard without virus infectionVirus infection without medium exchangeMedium exchange and virus infection Initial Density (vc / mL)2.1 × 10 ⁵ 2.1 × 10 ⁵ 2.1 × 10 ⁵Cell density at infection (vc / mL)9.1 × 10 ⁵ 1.4 × 10 ⁶ 1.5 × 10 ⁶Virus production per volume (pfu / mL) 6 days P.I.NA2.7 × 10 ⁷ 2.8 × 10 ⁸Virus Production Per Volume (HPLC vps / mL) 6 days P.I.NANA1.3 × 10 ¹⁰Virus production per cell (HPLC vps / cell)NANA1.3 × 10 ⁴
293 Cell Bank Preparation Adapted to Serum-Free Suspensions
As described above, cells produce an Ad-p53 vector and are propagated in serum-free IS293 medium supplemented with 0.1% F-68 and 100 mg / l heparin in a spinner flask to 1.0E + 07 (live cells / ml / vial). Cell banks adapted to serum-free suspensions). To collect the cells, the cells were centrifuged when in a moderate log state, viability was found to be greater than 90%, resuspended in serum-free medium supplemented with IS293, and centrifuged again to wash the cells. The cells are again suspended in cold IS293 with 0.1% F-68, 100 mg / l heparin, 10% DMSO, 0.1% methylcellulose to 1E + 07 viable cells / ml. The cell suspension is transferred to sterile cold vials, sealed and frozen overnight at -70 ° C. The vial is transferred to a liquid nitrogen reservoir. Mycoplasmism test was negative.
To revive frozen cells, one vial is thawed in 50 ml IS293 medium supplemented with T-150, 0.1% F-68, 100 mg / l heparin. The culture is then passaged twice in a 250 ml spinner flask. In another study, one vial was thawed in a 250 ml spinner flask with 100 ml serum free supplemented IS293 medium. Pass it twice in a serum-free spinner flask. In both studies the cells grew well.
Medium Exchange and Virus Production of Serum-Free Suspension Cultures in Spinner Flasks
In the case of suspension-free culture in spinner flasks, in the case of existing serum-free virus production, virus productivity per cell was so low that serum-free suspension culture was not performed. This is likely due to nutrient depletion and the production of inhibitory byproducts. To exchange the medium used with fresh serum-free IS293 medium, cells were centrifuged on day 3 and resuspended in fresh IS-293 medium supplemented with F-68 and heparin (100 mg / L) to yield cell density. 1.20E + 06 vc / ml and cells are infected at 5 MOI with Ad5-DMVp53 vector. Extracellular HPLC vps / ml at day 3 was 7.7E + 09 vps / ml, 1.18E + 10 vps / ml at day 4, 1.2E + 10 vps / ml at day 5 and 1.3E + 10 vpa at day 6 / Ml and on day 6 the pfu is 2.75 +/- 0.86E + 08 tvps / ml. The pfu ratio for HPLC virus particles is about 47. In addition, the cells are allowed to settle by centrifugation and the cells are lysed using the same type of surfactant lysis buffer as used in CellCube yield. Cellular HPLC vps / ml is 1.6E + 10 vps / ml on Day 2, 6.8E + 09 vps / ml on Day 3, 2.2E + 09 vpa / ml on Day 4 and 2.24E + 09 vps on Day 5 / ML and 2.24E + 09 vps / mL on day 6.
Replacing the medium used with fresh serum-free IS 293 medium significantly increases Ad-p53 vector production. Substituting medium increased extracellular HPLC virus particle production by about 3.6 times more than the previous level on day 3 and increased 10 times more extracellular pfu titer production on day 6 compared to the previous level. Ad-p53 vector production per cell was found to be about 1.33E + 04 HPLC vps.
Two days after infection, intracellular HPLC virus particle peaks appeared and particle counts decreased. Again extracellular viral particles are gradually increased by 6 days of harvest. Almost all Ad-p53 vectors are produced two days after infection, localized in the cell, and then the virus is released out of the cell. Nearly half of the virus is released into extracellular buffer two to three days after infection, and the rate of release decreases over time.
All cells infected with the Ad-p53 vector lost viability at 6 days post infection, whereas uninfected cells survived 97%. In the presence of infection, the pH is not changed when the medium is changed and the pH is 6.04, 5.97 when the medium is changed. In the absence of infection, the pH is 7.00 (Table 12).
Virus production and cell culture in stirred tanks when media exchange and gas are provided
To increase Ad-p53 vector productivity, the environment was further controlled using a 5 L CelliGen bioreactor. In the case of a 5 LCelliGen bioreactor, pH, dissolved oxygen and temperature are controlled. Oxygen and carbon dioxide are connected to an oxygen supply solenoid valve and adjust the pH. Use the sea propeller to mix better with less shearing environment. Air is continuously supplied during operation to keep the lift positive within the bioreactor.
To inoculate the bioreactor, the cell vials are thawed in a 250 ml spinner flask in 100 ml serum free medium and the cells are extended in a 250 or 500 ml spinner flask. 800 ml cell inoculum grown in a 500 ml flask was mixed with 2700 ml freshly prepared medium in a 10 L box and transferred to the CelliGen bioreactor at gas pressure.
The initial working volume of CelliGen Bioreactor is approximately 3.5 liters of culture. The initial stirring speed of the propeller is 80 rpm, the temperature starts at 37 ° C., the pH is 7.1, the pH is 7.0 after infection and the DO is about 40% throughout.
Initial cell density is 4.3E + 5 vc / ml (97% viability), after 4 days, when the cell density reaches 2.7E + 6 vc / ml (93% viability), the cells are reproduced in fresh medium and CelliGen Transfer to Bioreactor. After medium exchange, the cell density is 2.1E + 6 vc / ml and the cells are infected at 10 MOI. Drop the DO below 40%. To maintain DO above 40%, approximately 500 ml cultures were withdrawn from CelliGen to lower oxygen demand by cell culture and the upper propella blades located close to the gas and liquid yarns, increasing surface regeneration rate. Improve oxygen delivery. Then keep it above 40% until the operation is finished.
In the case of pH adjustment, the cell culture is acidified with CO ₂ gas and the cell culture is made alkaline with 1N NaHCO ₃ . Initial pH is set to 7.10. The initial pH of the culture is 7.41. Consuming approximately 280 mL 1N NaHCO ₃ solution stabilizes the pH of the cell culture to 7.1. After infecting the cell culture with the virus, the pH is lowered to 7.0 and the CO ₂ gas supply line is cut off to reduce NaHCO ₃ consumption. Using too much NaHCO ₃ solution to adjust the pH will increase the volume of the cell culture more than necessary. Consume 70 mL 1N NaHCO ₃ solution and the pH is mostly between 7.0 and 7.1. The temperature is maintained at 35 ° C to 37 ° C.
After infection, cell viability slowly decreases until day 6 post-infection. On the day of harvest, there are no viable cells. The virus production volume of the CelliGen bioreactor is 5.1E + 10 HPLC vps / ml as compared to the spinner flask (1.3E + 10 vps / ml). Adjusting the environment of the CelliGen bioreactor increases Ad-p53 vector production by about four-fold compared to that produced in spinner flasks with medium change. This is because at the time of infection the cell density increases from 1.2E + 6 to 2.1E + 6 vc / ml and the virus production per cell increases from 1.3E + 4 to 2.5E + 4 vps / ml. 2.5E4 vps / ml is comparable to 3..5E + 4 vps / cell for serum supplemented adsorbed cell cultures.
Virus Production and Cell Culture in a Stirred-Spread Bioreactor
In the first study, cells were successfully grown in agitated virus for virus production, and oxygen, CO ₂ , was fed to the top of the bioreactor. However, this method is limited in scaling up because gas transport is not effective. Thus, in a second study, to investigate cell growth and Ad-p53 production in a sprayed bioreactor to realize a scale up of serum-free suspension culture, serum-free IS293 medium (F-68) was used with pure oxygen and CO ₂ gas bubbles. 0.1%) and heparin (100 mg / L supplement).
Pure oxygen bubbles are supplied through the liquid medium to supply the dissolved oxygen to the cells, and the pure oxygen supply is controlled through the solenoid valve to maintain the dissolved oxygen at 40% or more. In order to supply pure oxygen, a stainless steel sintered oxygen diffuser with a 0.22 μm aperture is used to effectively supply oxygen with minimal damage to the cells. The CO ₂ gas was bubbled into the liquid medium using the same diffuser and tube fed with oxygen, maintaining the pH at about 7.0. For pH adjustment, Na ₂ CO ₃ solution (106 g / l) is fed to the bioreactor. Air is supplied to the upper part of the bioreactor to maintain the internal pressure of the bioreactor. The other bioreactor shapes are the same as those used in the first study.
Inoculate cells from frozen vials. One frozen cell (1.0E + 7vc) is thawed in 50 ml medium in a T-150 flask and passaged three times in 200 ml medium in a 500 ml spinner flask. 400 ml inoculated cells grown in two 500 ml spinner flasks are mixed with IS293 medium supplemented with F-68 and heparin in a 10 L container to make a 3.5 L cell suspension, which is transferred to a 5 LCelliGen bioreactor.
The initial density of the bioreactor is 3.0E + 4 vc / ml. Initial cell density is less than that of the first study. Four 500 ml spinner flasks were used for inoculation in the first study. Although the initial cell density is low, at 7 days in the spray environment, the cell density grows to 1.8E + 6 vc / ml and the survival is about 98%. During the 7 day survival, glucose concentration decreased from 5.4 g / l to 3.0 g / l and lactate concentration increased from 0.3 g / l to 1.8 g / l.
On day 7, once the cell density was 1.8E + 6 vc / ml, the cells in the bioreactor were allowed to settle by centrifugation, and in a 10 L vessel with 3.5 L fresh serum-free IS293 medium (F-68 and heparin supplemented). Resuspend. 293 cells were infected with 1.25E + 11 pfu Ad-p53 and transferred to CelliGen bioreactor. In the bioreactor, the cell viability is 100%, but the cell density is only 7.2E + 5 vc / ml. There was cell loss during the medium exchange operation. Virus titers in the medium were 2.5E + 10HPLC vps / ml on day 2 obtained; On day 3 2.0E + 10HPLC vps / ml; On day 4 2.8E + 10HPLC vps / ml; 3.5E + 10HPLC vps / ml on Day 5; On day 6 it is 3.9E + 10 HPLC vpa / ml. The first bioreactor study to provide gas was 5.1E + 10 HPLC vps / ml. Lowering the virus concentration in the second run results in a lower cell density during infection. 2.1E + 6 vc / ml was used in the first trial when compared to 7.2E + 5 vc / ml in the second trial. In fact, Ad-p53 production per second sprayed CelliGenqkdldhfldprxjdptj cells was found to be 5.4E + 4 vps / cell, which is more peak production than ever obtained. Production per cell in the first serum-free CelliGen bioreactor without spraying and productivity in serum supplemented T-flasks were 2.5E + 4 vps / cell and 3.5E + 4 vps / cell, respectively.
After viral infection, the viability of the cells is reduced from 100% to 13% on day 6 of harvest. Glucose concentration decreased from 5.0 g / l to 2.1 g / l and lactate increased from 0.3 g / l to 2.9 g / l during 6 days post infection. 20 ml Na ₂ CO ₃ (106 g / l) solution was used during the entire operation.
Experimental results show that Ad-p53 can be produced technically and economically in spray stirred reactors. Spraying and stirring reactors were also allowed to scale up and unit operations thereof.
All compositions and methods described herein can be used without experimentation based on the teachings of the present invention. While the compositions and methods of the present invention have been described through appropriate embodiments, those skilled in the art can make changes to the compositions, methods, steps, etc. described herein without departing from the gist of the present invention. More specifically, certain chemical and physiologically relevant substances may be substituted for the materials described herein, with similar results. All such substitutions and alterations are within the scope of the present invention.
references
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权利要求:
Claims (69)
[1" claim-type="Currently amended] In a method for producing adenovirus;
a) growing host cells in medium under low irrigation,
b) infecting the host cell with an adenovirus,
c) obtaining and lysing host cells to obtain crude cell lysate,
d) concentrating the crude cell lysate,
e) exchange the unpurified cell lysate buffer,
f) reducing the concentration of contaminating nucleic acid in the unpurified cell lysate.
[2" claim-type="Currently amended] The method of claim 1, further comprising separating the adenovirus particles from the cell lysate using chromatography.
[3" claim-type="Currently amended] The method of claim 1, wherein the glucose concentration in the medium is maintained at 0.7 to 1.7 g / l.
[4" claim-type="Currently amended] The method of claim 1, wherein the method of exchanging the buffer solution comprises diafiltration.
[5" claim-type="Currently amended] The method of claim 1 wherein the adenovirus consists of adenovirus vectors that encode exogenous gene constructs.
[6" claim-type="Currently amended] 6. The method of claim 5, wherein the gene structure is operably linked to a promoter.
[7" claim-type="Currently amended] 7. The method of claim 6, wherein the promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late promoter, foriloma F9-1, tyrosinase.
[8" claim-type="Currently amended] The method of claim 1 wherein the adenovirus is a replication-responsive adenovirus.
[9" claim-type="Currently amended] 9. The method of claim 8, wherein the adenovirus lacks a portion of the E1-1 portion.
[10" claim-type="Currently amended] 10. The method of claim 9, wherein the adenovirus is deficient in some of the E1A or E1B moieties.
[11" claim-type="Currently amended] The method of claim 1, wherein the host cell is capable of complementing replication.
[12" claim-type="Currently amended] The method of claim 1, wherein the host cell is 293 cells.
[13" claim-type="Currently amended] 6. The method of claim 5, wherein the exogenous gene structure encodes a therapeutic gene.
[14" claim-type="Currently amended] The method of claim 1, wherein the therapeutic gene is antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fms, antisense jun, antisensetrk, antisense ret, antisense gsp, antisense hst, antisense bcl, antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zacl, scFV ras, DCC, NF- 1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, Characterized by encoding IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF G-CSF, thymidine kinase, p53 Way.
[15" claim-type="Currently amended] The method of claim 14, wherein the therapy gene encodes p53.
[16" claim-type="Currently amended] The method of claim 1, wherein the cells are obtained and lysed ex situ using hypotonic solution, hypertonic solution, freeze-thaw, sonication, impingement jet, microfluidization or surfactant.
[17" claim-type="Currently amended] The method of claim 1, wherein the cells are obtained and lysed in situ using a storage solution, a hypertonic solution, and a surfactant.
[18" claim-type="Currently amended] 18. The method of claim 17, wherein the cells are obtained and lysed using a surfactant.
[19" claim-type="Currently amended] 19. The method of claim 18, wherein the surfactant is Thesit ^R , NP-40 ^R , Tween-20 ^R , Brij-58 ^R , Triton X ^R- 100, octyl glucoside.
[20" claim-type="Currently amended] The method of claim 1, wherein the infected cells are autolyzed to lyse.
[21" claim-type="Currently amended] The method of claim 1, wherein the cell lysate is treated with Benzonase ^R or Pulmozyme ^R.
[22" claim-type="Currently amended] The method of claim 2, wherein the separation process is performed using one chromatography.
[23" claim-type="Currently amended] The method of claim 22, wherein the chromatography step is ion exchange chromatography.
[24" claim-type="Currently amended] The method of claim 23, wherein the ion exchange chromatography is anion exchange chromatography.
[25" claim-type="Currently amended] The method of claim 24, wherein the anion exchange chromatography uses DEAE, TMAE, QAE, PEI.
[26" claim-type="Currently amended] The method of claim 24, wherein the anion exchange chromatography uses Toyopearl Super Q 650M, MonoQ, Source Q, Fractogel TMAE.
[27" claim-type="Currently amended] The method of claim 24, wherein the ion exchange chromatography is carried out in the range of pH 7.0 to 10.0.
[28" claim-type="Currently amended] The method of claim 1, further comprising the step of concentrating using membrane filtration.
[29" claim-type="Currently amended] 29. The method of claim 28, wherein the filtration is tangential transfer filtration.
[30" claim-type="Currently amended] 29. The method of claim 28, wherein the filtration uses 100 to 300 K NMWC, regenerated cellulose, polyether sulfone membranes.
[31" claim-type="Currently amended] a) growing host cells in medium under low irrigation,
b) infecting the host cell with an adenovirus,
c) obtaining and lysing host cells to obtain crude cell lysate,
d) concentrating the crude cell lysate,
e) exchange the unpurified cell lysate buffer,
f) Adenovirus, characterized in that produced according to the process consisting of reducing the concentration of contaminating nucleic acid in unpurified cell lysate
[32" claim-type="Currently amended] 32. The adenovirus according to claim 31, wherein the adenovirus consists of adenovirus vectors encoding exogenous gene constructs.
[33" claim-type="Currently amended] 32. The adenovirus according to claim 31, wherein the gene structure is operably linked to a promoter.
[34" claim-type="Currently amended] 32. The adenovirus of claim 31, wherein the adenovirus is a replication-responsive adenovirus.
[35" claim-type="Currently amended] 35. The adenovirus of claim 34, wherein the adenovirus lacks a portion of the E1-1 portion.
[36" claim-type="Currently amended] 32. The adenovirus of claim 31, wherein the adenovirus lacks some of the E1A or E1B moieties.
[37" claim-type="Currently amended] 32. The adenovirus of claim 31, wherein the host cell has the ability to complement replication.
[38" claim-type="Currently amended] 32. The adenovirus according to claim 31, wherein the host cell is 293 cells.
[39" claim-type="Currently amended] 32. The adenovirus of claim 31, wherein the exogenous gene structure encodes a therapeutic gene.
[40" claim-type="Currently amended] The method of claim 39, wherein the therapeutic gene is antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fms, antisense jun, antisensetrk, antisense ret, antisense gsp, antisense hst, antisense bcl, antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zacl, scFV ras, DCC, NF- 1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, Characterized by encoding IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF G-CSF, thymidine kinase, p53 Adenovirus.
[41" claim-type="Currently amended] 41. The adenovirus of claim 40, wherein the therapy gene encodes p53.
[42" claim-type="Currently amended] 34. The adenovirus of claim 33, wherein the promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late promoter, foriloma F9-1, tyrosinase.
[43" claim-type="Currently amended] A method for purifying adenovirus;
a) growing host cells,
b) infecting the host cell with an adenovirus,
c) obtaining a host cell and contacting with a surfactant to lyse the cell to obtain a cell lysate,
d) concentrating the crude cell lysate,
e) exchange the unpurified cell lysate buffer,
f) reducing the concentration of contaminating nucleic acid in the unpurified cell lysate.
[44" claim-type="Currently amended] 44. The method of claim 43, further comprising separating adenovirus particles from cell lysate using chromatography.
[45" claim-type="Currently amended] 44. The method of claim 43, wherein the glucose concentration in the medium in which the host cells are grown is maintained at 0.7 to 1.7 g / l.
[46" claim-type="Currently amended] 44. The method of claim 43, wherein the method of exchanging buffers comprises diafiltration.
[47" claim-type="Currently amended] 45. The method of claim 43, wherein the surfactant is Thesit ^R , NP-40 ^R , Tween-20 ^R , Brij-58 ^R , Triton X ^R- 100, octyl glucoside.
[48" claim-type="Currently amended] 48. The method of claim 47, wherein the surfactant concentration present in the dissolution solution is about 1% (w / v).
[49" claim-type="Currently amended] 44. The method of claim 43, wherein the separation is carried out using one chromatography.
[50" claim-type="Currently amended] 45. The method of claim 44, wherein the chromatography step is ion exchange chromatography.
[51" claim-type="Currently amended] a) growing host cells,
b) infecting the host cell with an adenovirus,
c) obtaining a host cell and contacting with a surfactant to lyse the cell to obtain a cell lysate,
d) concentrating the crude cell lysate,
e) exchange the unpurified cell lysate buffer,
f) Adenoviruses produced by a process comprising reducing the concentration of contaminating nucleic acid in unpurified cell lysate.
[52" claim-type="Currently amended] A method for purifying adenovirus;
a) host cells are grown in serum-free medium,
b) infecting the host cell with an adenovirus,
c) obtaining host cells and lysing the cells to obtain crude cell lysate,
d) concentrating the crude cell lysate,
e) exchange the unpurified cell lysate buffer,
f) reducing the concentration of contaminating nucleic acid in the unpurified cell lysate.
[53" claim-type="Currently amended] 53. The method of claim 52, wherein the host cell is adapted for growth in serum free medium.
[54" claim-type="Currently amended] 53. The method of claim 52, wherein the cells grow as in cell suspension culture.
[55" claim-type="Currently amended] 53. The method of claim 52, wherein the cells grow as in fixed-dependent cultures.
[56" claim-type="Currently amended] 54. The method of claim 53, wherein adapting for growth to the serum-free medium comprises slowly decreasing the fetal bovine serum content in the growth medium.
[57" claim-type="Currently amended] 54. The method of claim 53, wherein the serum free medium has a fetal calf serum content of 0.03% v / v or less.
[58" claim-type="Currently amended] 53. The method of claim 52, further comprising separating adenovirus particles from the lysate using chromatography.
[59" claim-type="Currently amended] 53. The method of claim 52, wherein lysis is via self lysis of infected cells.
[60" claim-type="Currently amended] 53. The method of claim 52, wherein the method of exchanging buffer is performed by diafiltration.
[61" claim-type="Currently amended] 53. The method of claim 52, wherein the surfactant is Thesit ^R , NP-40 ^R , Tween-20 ^R , Brij-58 ^R , Triton X ^R- 100, octyl glucoside.
[62" claim-type="Currently amended] 53. The method of claim 52, wherein the surfactant concentration present in the dissolution solution is about 1% (w / v).
[63" claim-type="Currently amended] 53. The method of claim 52, wherein the separating is carried out using one chromatography.
[64" claim-type="Currently amended] 53. The method of claim 52, wherein the chromatography is performed using ion exchange chromatography.
[65" claim-type="Currently amended] a) host cells are grown in serum-free medium,
b) infecting the host cell with an adenovirus,
c) obtaining host cells and lysing the cells to obtain crude cell lysate,
d) concentrating the crude cell lysate,
e) exchange the unpurified cell lysate buffer,
f) Adenovirus, characterized in that it is made through a process consisting of reducing the concentration of contaminating nucleic acid in unpurified cell lysate.
[66" claim-type="Currently amended] 293 host cells, adapted to grow in serum-free medium.
[67" claim-type="Currently amended] 67. The cell of claim 66, wherein the cell is adapted to grow in suspension culture.
[68" claim-type="Currently amended] 67. The cell of claim 66 wherein the cell has been deposited as an IT293SF cell in an ATCC.
[69" claim-type="Currently amended] 67. The cell of claim 66, wherein the method of adapting to growth in serum-free medium comprises gradually reducing the fetal calf serum content in the growth medium.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1996-11-20|Priority to US3132996P

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1996-11-20|Priority to US60/031,329

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1997-11-20|Application filed by 파커 데이비드 엘., 인트로겐 테라페티스, 인코퍼레이티드

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2000-09-15|Publication of KR20000057160A

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2005-07-26|Application granted

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2005-07-26|Publication of KR100503701B1

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2007-04-25|First worldwide family litigation filed

优先权:

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申请号 | 申请日 | 专利标题

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US3132996P| true| 1996-11-20|1996-11-20|

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US60/031,329|1996-11-20|