STABILIZED LIQUID PHARMACEUTICAL FORMULATIONS CONTAINING ANTI-PCSK9 ANTIBODIES

专利摘要:
Stabilized Formulations Containing Anti-pcsk9 Antibodies The present invention provides pharmaceutical formulations comprising a human antibody that specifically binds to human subtilisin/kexin type 9 proprotein convertase (pcsk9). the formulations may contain, in addition to an anti-pcsk9 antibody, at least one amino acid, at least one sugar, or at least one non-ionic surfactant. the pharmaceutical formulations of the present invention exhibit a substantial degree of antibody stability after storage for several months.
公开号:BR112014001712B1
申请号:R112014001712-3
申请日:2012-07-27
公开日:2021-09-08
发明作者:Scott Walsh；Daniel Dix
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

FIELD
[0001] The present invention relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of pharmaceutical formulations comprising a human antibody that specifically binds to human proprotein subtilisin/kexin type 9 convertase (PCSK9). SEQUENCE LISTING
[0002] A ST.25 compliant text file of a string listing is filed concurrently with the present application. The contents of the text file are here, incorporated by reference. A paper copy of the string listing, which is identical in content to the ST.25 compliant text file, is included as part of this application. FOUNDATION
[0003] Therapeutic macromolecules (eg, antibodies), must be formulated in a way that not only makes the molecules suitable for administration to patients, but also maintains their stability during storage and subsequent use. For example, therapeutic antibodies in liquid solution are prone to degradation, aggregation or undesirable chemical modification unless the solution is properly formulated. The stability of an antibody in the liquid formulation depends not only on the types of excipients used in the formulation, but also on the amounts and proportions of the excipients relative to each other. In addition, other considerations aside from stability must be taken into account when preparing a liquid antibody formulation. Examples of such additional considerations include the solution viscosity and antibody concentration that can be accommodated by a given formulation, and the visual quality or appeal of the formulation. Thus, when formulating a therapeutic antibody, great care must be taken to arrive at a formulation that remains stable, contains an adequate concentration of antibody, and has an adequate viscosity, as well as other properties that allow the formulation to be conveniently administered to patients.
[0004] Antibodies to human proprotein convertase subtilisin/kexin protein type 9 (PCSK9) are an example of a therapeutically relevant macromolecule that requires proper formulation. Anti-PCSK9 antibodies are clinically useful for treating or preventing diseases, such as hypercholesterolemia and other dyslipidemias, and other conditions. Exemplary anti-PCSK9 antibodies are described, inter alia, in WO 2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, US Patent Nos. 7,572,618, WO 2010/077854, EU. 2010/0166768, and US. 2011/0065902.
[0005] Although anti-PCSK9 antibodies are known, there is a need in the prior art for new pharmaceutical formulations comprising anti-PCSK9 antibodies that are sufficiently stable and suitable for administration to patients. SUMMARY
[0006] The present invention satisfies the above-mentioned need by providing pharmaceutical formulations comprising a human antibody that specifically binds to human proprotein convertase subtilisin/kexin protein type 9 (PCSK9).
[0007] In one aspect, there is provided a liquid pharmaceutical formulation, which comprises: (i) a human antibody that specifically binds to human proprotein convertase subtilisin/kexin protein type 9 (PCSK9); (ii) a buffer; (iii) an organic co-solvent; (iv) a stabilizer; and optionally, (v) a viscosity reducer.
In one embodiment, the antibody is provided at a concentration of approximately 50 ± 7.5 mg/ml to approximately 200 ± 30 mg/ml. In another embodiment, the antibody is provided at a concentration of approximately 50 mg/ml ± 7.5 mg/ml. In another embodiment, the antibody is provided at a concentration of approximately 100 mg/ml ± 15 mg/ml. In another embodiment, the antibody is provided at a concentration of approximately 150 mg/ml ± 22.5 mg/ml. In another embodiment, the antibody is provided at a concentration of approximately 175 mg/ml ± 26.25 mg/ml. In another embodiment, the antibody is provided at a concentration of approximately 200 mg/ml ± 30 mg/ml.
[0009] In one embodiment, the antibody comprises any one or more of an amino acid sequence SEQ ID NO: 1 - 8. In one embodiment, the antibody comprises (a) a heavy chain variable region (HCVR) comprising determining regions of heavy chain complementarity 1, 2 and 3 (HCDR1-HCDR2-HCDR3) each comprising a sequence of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and (b) a variable region of light chain (LCVR) comprising light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3) each comprising a sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively . In a specific embodiment, the antibody comprises an HCVR and an LCVR, each of which comprises the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 5, respectively.
[00010] In one embodiment, the pH of the liquid formulation is approximately pH 6.0 ± 0.5, pH 6.0 ± 0.4, pH 6.0 ± 0.3, pH 6.0 ± 0 .2, pH 6.0 ± 0.1, pH 6.0 ± 0.05, pH 6.0 ± 0.01, or pH 6.0. In a specific embodiment, the pH of the liquid formulation is approximately pH 6.0 ± 0.3. In one embodiment, the liquid pharmaceutical buffer comprises one or more buffers, which have an effective buffering range of approximately 5.5 to approximately pH 7.4, or a pKa of approximately 6.0.
[00011] In one embodiment, the buffer is histidine. In one embodiment, histidine is at a concentration of 5 mM ± 0.75 mM to 50 mM ± 7.5 mM. In one embodiment, histidine is at a concentration of 10 mM ± 1.5 mM, or approximately 10 mM. In one embodiment, histidine is at a concentration of 20 mM ± 3 mM or approximately 20 mM. In one embodiment, histidine is at a concentration of 40 nM ± 6 mM or approximately 40 nM.
[00012] In one embodiment, the organic co-solvent is a nonionic polymer containing a polyoxyethylene portion. In some embodiments, the organic co-solvent is any one or more of polysorbate 20, poloxamer 188, and polyethylene glycol 3350. In a specific embodiment, the organic co-solvent is polysorbate 20.
[00013] In one embodiment, the organic co-solvent is at a concentration of approximately 0.005% ± 0.00075% to about 1% ± 0.15% "weight by volume" or "w/v", wherein, by example, 0.1 g/ml = 10% and 0.01 g/ml = 1%. In one embodiment, the organic co-solvent is polysorbate 20, which is at a concentration of about 0.2% ± 0.03% w/v. In another embodiment, the organic co-solvent is polysorbate 20, which is at a concentration of 0.01% ± 0.0015% w/v or about 0.01% w/v.
[00014] In one modality, the stabilizer is a sugar. In one embodiment, the sugar is selected from the group consisting of sucrose, mannitol and trehalose. In a specific modality, the stabilizer is sucrose.
[00015] In one modality, the stabilizer is at a concentration of, 1% ± 0.15% w/v to 20% ± 3% w/v. In a specific embodiment, the stabilizer is sucrose at a concentration of 5% ± 0.75% w/v or about 5% w/v. In another specific modality, the stabilizer is sucrose at a concentration of 10% ± 1.5% w/v or about 10% w/v. In another specific modality, the stabilizer is sucrose at a concentration of 12% ± 1.8% w/v or about 12% w/v.
[00016] In one embodiment, the viscosity reducer is a salt selected from the group consisting of arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride and acetate sodium. In one embodiment, the viscosity reducer is L-arginine hydrochloride.
[00017] In one embodiment, the viscosity reducer is at a concentration of about 10 mM ± 1.5 mM to 150 mM ± 22.5 mM. In one embodiment, the viscosity reducer is L-arginine hydrochloride at a concentration of 50 mM ± 7.5 mM or about 50 mM. In one embodiment, the viscosity reducer is L-arginine hydrochloride at a concentration of 40 mM ± 6 mM or about 40 mM.
[00018] In one embodiment, the viscosity of the liquid or reconstituted lyophilized pharmaceutical formulation at 25oC is less than or equal to about 15 cPoise ± 10%. In one embodiment, the viscosity at 25°C is between 1.0 cPoise ± 10% and 18 cPoise ± 10%. In one embodiment, the viscosity at 25°C is 1.6 cPoise ± 10%, 1.7 cPoise ± 10%, 3.3 cPoise ± 10%, 3.5 cPoise ± 10%, 4.8 cPoise ± 10%, 6.0 cPoise ± 10%, ± 7.0 cPoise 10%, 7.1 cPoise ± 10%, 7.2 cPoise ± 10%, 7.9 cPoise ± 10%, 8.9 cPoise ± 10%, 10, 0 cPoise ± 10%, 10.6 cPoise ± 10%, 11.4 cPoise ± 10%, 11.6 ± 10% cPoise , 11.8 cPoise ± 10%, 12.4 cPoise ± 10%, 13.9 cPoise ± 10%, 14.0 cPoise ± 10%, 15.5 cPoise ± 10%, or 17.9 cPoise ± 10%.
[00019] In one modality, the osmolality of the liquid pharmaceutical formulation is between 100 ± 15 mOsm/kg and 460 ± 69 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 103 ± 15 mOsm/kg, or about 103 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 195 ± 29 mOsm/kg, or about 195 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 220 ± 33 mOsm/kg, or about 220 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 330 ± 50 mOsm/kg, or about 330 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 435 ± 65 mOsm/kg, or about 435 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 440 ± 66 mOsm/kg, or about 440 mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceutical formulation is 458 ± 69 mOsm/kg, or about 458 mOsm/kg.
[00020] In one embodiment, at least 96% or at least 97% of the non-aggregated and undegraded form of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation after three months of storage of the liquid pharmaceutical formulation at -80° C as determined by size exclusion chromatography. In one embodiment, at least 56% of the non-basic and non-acid form (i.e., major peak or major charge form) of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation after three months of storage of the formulation. pharmaceutical liquid at -80°C as determined by ion exchange chromatography.
[00021] In one embodiment, at least 96% or at least 97% of the non-aggregated and undegraded form of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation after three months of storage of the liquid pharmaceutical formulation at -30° C as determined by size exclusion chromatography. In one embodiment, at least 56% of the anti-PCSK9 antibody main loading form is recovered from the liquid pharmaceutical formulation after three months of storage of the liquid pharmaceutical formulation at -30°C, as determined by ion exchange chromatography.
[00022] In one embodiment, at least 96% or at least 97% of the non-aggregated and undegraded form of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation after three months of storage of the liquid pharmaceutical formulation at -20° C as determined by size exclusion chromatography. In one embodiment, at least 56% of the anti-PCSK9 antibody main loading form is recovered from the liquid pharmaceutical formulation after three months of storage of the liquid pharmaceutical formulation at -20°C, as determined by ion exchange chromatography.
[00023] In one embodiment, at least 96% of the non-aggregated and undegraded form of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation, after six months of storage of the liquid pharmaceutical formulation, at 5°C, as determined by size exclusion chromatography. In one embodiment, at least 58% or 59% of the anti-PCSK9 antibody main loading form is recovered from the liquid pharmaceutical formulation, after three months of storage of the liquid pharmaceutical formulation, at 5°C, as determined. by ion exchange chromatography.
[00024] In one embodiment, at least 94% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation, after six months of storage of the liquid pharmaceutical formulation, at 25°C, as determined by size exclusion chromatography. In one embodiment, at least 45% or 47% of the non-basic and non-acidic form of the anti-PCSK9 antibody are recovered from the liquid pharmaceutical formulation after six months of storage of the liquid pharmaceutical formulation at 25°C as determined by ion exchange chromatography.
[00025] In one embodiment, at least 91% or 92% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody are recovered from the liquid pharmaceutical formulation, after 28 days of storage of the liquid pharmaceutical formulation at 45°C, as determined by size exclusion chromatography. In one embodiment, at least 35% or 37% of the non-basic and non-acidic form of the anti-PCSK9 antibody is recovered from the liquid pharmaceutical formulation after 28 days of storage of the liquid pharmaceutical formulation at 45°C, as determined by chromatography of ion exchange.
[00026] In one aspect, there is provided a liquid pharmaceutical formulation, which comprises: (i) from 50 ± 7.5 mg/ml to 175 ± 26 mg/ml of a human antibody that specifically binds to human PCSK9 , (ii) from 0 mM to 40 ± 6 mM histidine, (iii) from 0% to 0.2% ± 0.03% (w/v) polysorbate 20, (iv) from 0% to 12 % ± 1.8% (w/v) sucrose, and (v) from 0 mM to 50 ± 7.5 mM arginine, under a pH of about 5.3 to about 6.7 . The anti-PCSK9 antibody of that aspect comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), such that the HCVR/LCVR combination comprises heavy and light chain complementarity determining regions (HCDR1-HCDR2 - HCDR3/ LCDR1-LCDR2-LCDR3), which comprise the amino acid sequences of SEQ ID NOs: 2 - 3 - 4/SEQ ID NOs: 6 - 7 - 8, respectively. In a particular embodiment, the anti-PCSK9 antibody comprises a heavy chain variable region (HCVR) and light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 5, respectively (henceforth onwards "mAb-316P").
[00027] In one embodiment of this aspect, the liquid formulation comprises (i) 50 ± 7.5 mg/ml mAb-316P; (ii) 10 ± 1.5 mM histidine; (iii) 0.1% ± 0.015% (w/v) of polysorbate 20; and (iv) 6% ± 0.9% (w/v) sucrose, under a pH of 6.0 ± 0.3. In one embodiment of this particular formulation, the viscosity is about 1.7 cPoise. In one embodiment of this particular formulation, the osmolality is 220 ± 44 mOsm/kg.
[00028] In another embodiment, the liquid formulation comprises (i) 100 ± 20 mg/ml mAb-316P, (ii) 20 ± 4 mM histidine, (iii) 0.2% ± 0.04% (w/ v) of polysorbate 20; and (iv) 12% ± 2.4% (w/v) sucrose, under a pH of 6.0 ± 0.3. In one embodiment of this particular formulation, the viscosity is about 3.5 cPoise. In one embodiment of this particular formulation, the osmolality is 440 ± 88 mOsm/kg.
[00029] In another embodiment, the liquid formulation comprises (i) 150 ± 22.5 mg/ml mAb-316P; (ii) 10 ± 1.5 mM histidine; (iii) 0.2% ± 0.03% or 0.01% ± 0.0015% (w/v) of polysorbate 20; and (iv) 10% ± 1.5% (w/v) sucrose, under a pH of 6.0 ± 0.3. In one embodiment of this particular formulation, the viscosity is about 6 cPoise. In one embodiment of this particular formulation, the osmolality is 435 ± 65.25 mOsm/kg. In one embodiment of this particular formulation, after storage of the formulation at 45oC for 28 days, >92% of the antibody is native and >35% of the antibody is in the main charge form. In one embodiment of this particular formulation, after storage of the formulation at 25oC for six months, >94% of the antibody is native and >45% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storage of the formulation at 5°C for six months, >96% of the antibody is native and >58% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storage of the formulation at -20oC for twelve months, >97% of the antibody is native and >56% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storage of the formulation at -30oC for twelve months, >97% of the antibody is native and >56% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storage of the formulation at -80oC for twelve months, >97% of the antibody is native and >56% of the antibody is in the major charge form.
[00030] In some embodiments of this particular formulation, >85% of the antibody retains its biological potency after 28 days at 45oC, >82% after 28 days at 37oC, and/or >98% after 28 days at 25oC. In some modalities of this particular formulation, >85% of the antibody retains its biological potency after six months at -20oC, >70% after six months at -30oC, and/or >79% after six months at -80oC. In some embodiments of this particular formulation, >81% of the antibody retains its biological potency after eight freeze-thaw cycles, and/or >84% of the antibody retains its biological activity after 120 minutes of agitation.
[00031] In another embodiment of this aspect, the liquid formulation comprises (i) 175 ± 26.25 mg/mL of mAb-316P; (ii) 10 ± 1.5 mM histidine; (iii) 0.01% ± 0.0015% (w/v) of polysorbate 20; (iv) 5% ± 0.75% (w/v) sucrose; and (v) 50 ± 7.5 mM arginine, at a pH of 6.0 ± 0.3. In one embodiment of this particular formulation, the viscosity is about 10.6 cPoise. In one embodiment of this particular formulation, the osmolality is 330 ± 50 mOsm/kg. In one embodiment of this particular formulation, after storage of the formulation at 45°C for 28 days, >91% of the antibody is native and >38% of the antibody is in the main charge form. In one embodiment of this particular formulation, after storing the formulation at 25°C for six months, >94% of the antibody is native and >47% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storage of the formulation at 5°C for six months, >96% of the antibody is native and >59% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storing the formulation at -20°C for three months, >96% of the antibody is native and >56% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storing the formulation at -30°C for three months, >96% of the antibody is native and >56% of the antibody is in the major charge form. In one embodiment of this particular formulation, after storing the formulation at -80°C for three months, >96% of the antibody is native and >56% of the antibody is in the major charge form.
[00032] In one aspect, a liquid pharmaceutical formulation of any of the foregoing aspects is provided in a container. In one embodiment, the container is a polycarbonate bottle. In another embodiment, the container is a glass vial. In one embodiment, the glass vial is a type 1 borosilicate glass vial with a fluorocarbon coated butyl rubber stopper. In another embodiment, the container is a microinfuser. In another embodiment, the container is a syringe. In a specific embodiment, the syringe comprises a plunger coated with fluorocarbons. In a specific embodiment, the syringe is a 1 ml long glass syringe containing less than about 500 parts per billion of tungsten equipped with a 27-G needle, a fluorocarbon-coated butyl rubber stopper, and a cap with non-cytotoxic latex-free rubber tip. In a more specific embodiment, the syringe is a NUOVA WIPO 1 mL long glass syringe equipped with a 27-G thin wall needle, a 4023/50 rubber stopper coated with FLUROTEC, and an FM rubber end cap. 27. In another specific modality, the syringe is a 1 ml or 3 ml plastic syringe equipped with a 27-G needle. In a more specific modality, the plastic syringe is distributed by BECTON DICKINSON.
[00033] In one aspect, there is provided a pharmaceutical formulation comprising (a) 175 mg/ml ± 26.25 mg/ml of an anti-PCSK9 antibody, (b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3; (c) 0.01% w/v ± 0.0015% polysorbate 20; (d) 5% w/v ± 0.75% sucrose, and (e) 50 mM ± 7.5 mM arginine, wherein (a) the antibody comprises an HCVD of SEQ ID NO: 1 and an LCVD of SEQ ID NO: 5; (b) more than 90% of the antibodies in the formulation have a molecular weight of 155 kDa ± 1 kDa; (c) more than 50% of the antibodies in the formulation have an isoelectric point of about 8.5, and (d) 75% to 90% of the antibodies in the formulation are fucosylated.
[00034] In one embodiment, the pharmaceutical formulation consists of (a) 175 mg/ml ± 26.25 mg/ml of the anti-PCSK9 antibody from the immediately preceding paragraph; (b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3; (c) 0.01% w/v ± 0.0015% polysorbate 20; (d) 5% w/v ± 0.75% sucrose; and (e) 50 mM ± 7.5 mM arginine, in water.
[00035] In one aspect, there is provided a pharmaceutical formulation comprising (a) 150 mg/ml ± 22.5 mg/ml of an anti-PCSK9 antibody; (b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3; (c) 0.2% w/v ± 0.03% polysorbate 20, and (d) 10% w/v ± 1.5% sucrose, wherein (i) the antibody comprises an HCVD of SEQ ID NO : 1 and an LCVD of SEQ ID NO: 5; (ii) more than 90% of the antibodies in the formulation have a molecular weight of 155 kDa ± 1 kDa, (iii) more than 50% of the antibodies in the formulation have an isoelectric point of about 8.5, and (iv) from 75% to 90% of the antibodies in the formulation are fucosylated.
[00036] In one embodiment, the pharmaceutical formulation consists of (a) 150 mg/ml ± 22.5 mg/ml of the anti-PCSK9 antibody from the immediately preceding paragraph, b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3, c) 0.2% w/v ± 0.03% polysorbate 20, and (d) 10% w/v ± 1.5% sucrose, in water.
[00037] In one aspect, there is provided a pharmaceutical formulation comprising (a) 150 mg/ml ± 22.5 mg/ml of an anti-PCSK9 antibody, (b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3, (c) 0.01% w/v ± 0.0015% polysorbate 20, and (d) 10% w/v ± 1.5% sucrose, wherein (i) the antibody comprises an HCVD of SEQ ID NO: 1 and an LCVD of SEQ ID NO: 5, (ii) more than 90% of the antibodies have a molecular weight of 155 kDa ± 1 kDa, (iii) more than 50% of the antibodies in the formulation have a spot isoelectric of about 8.5, and (iv) from 75% to 90% of the antibodies in the formulation are fucosylated.
[00038] In one embodiment, the pharmaceutical formulation consists of (a) 150 mg/ml ± 22.5 mg/ml of the anti-PCSK9 antibody from the immediately preceding paragraph, b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3, (c) 0.01% w/v ± 0.0015% polysorbate 20, and (d) 10% w/v ± 1.5% sucrose, in water.
[00039] In one aspect, there is provided a pharmaceutical formulation comprising (a) 100 mg/ml ± 15 mg/ml of an anti-PCSK9 antibody, (b) 20 mM ± 3 mM histidine, pH 6 ± 0.3, (c) 0.2% w/v ± 0.03% polysorbate 20, and (d) 12% w/v ± 1.8% sucrose, wherein (i) the antibody comprises an HCVD of SEQ ID NO : 1 and an LCVD of SEQ ID NO: 5, (ii) more than 90% of the antibodies in the formulation have a molecular weight of 155 kDa ± 1 kDa, (iii) more than 50% of the antibodies in the formulation have an isoelectric point of about 8.5, and (iv) from 75% to 90% of the antibodies in the formulation are fucosylated.
[00040] In one embodiment, the pharmaceutical formulation consists of (a) 100 mg/ml ± 15 mg/ml of the anti-PCSK9 antibody from the immediately preceding paragraph, b) 20 mM ± 3 mM histidine, pH 6 ± 0.3 , (c) 0.2% w/v ± 0.03% polysorbate 20, and (d) 12% w/v ± 1.8% sucrose, in water.
[00041] In one aspect, there is provided a pharmaceutical formulation comprising (a) 50 mg/ml ± 7.5 mg/ml of an antibody, (b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3 , (c) 0.1% w/v ± 0.015% polysorbate 20, and (d) 6% w/v ± 0.9% sucrose, wherein (i) the antibody comprises an HCVD of SEQ ID NO: 1 and an LCVD of SEQ ID NO: 5, (ii) more than 90% of the antibodies in the formulation have a molecular weight of 155 kDa ± 1 kDa, (iii) more than 50% of the antibodies in the formulation have an isoelectric point of about from 8.5, and (iv) from 75% to 90% of the antibodies in the formulation are fucosylated.
[00042] In one embodiment, the pharmaceutical formulation consists of (a) 50 mg/ml ± 7.5 mg/ml of the anti-PCSK9 antibody from the immediately preceding paragraph, (b) 10 mM ± 1.5 mM histidine, pH 6 ± 0.3, (c) 0.1% w/v ± 0.015% polysorbate 20, and (d) 6% w/v ± 0.9% sucrose, in water.
[00043] In one aspect, there is provided a method for preparing a freeze-dried composition comprising an anti-PCSK9 antibody and less than 0.3% in water. The method comprises the steps of (a) combining in a glass vial water, an anti-PCSK9 antibody, histidine, sucrose and polysorbate 20, (b), then keeping the combination at about 5oC for approximately 60 minutes, ( c) then lower the temperature, at a rate of about 0.5oC per minute, (d) then hold the combination at about -45oC for approximately 120 minutes, (e) then reduce the atmospheric pressure at about 13.33 Pa (100 mTorr), (f) then increase the temperature at a rate of about 0.5oC per minute, (g) then keep the blend at about -25oC for about 78 hours (h) then increase the temperature at a rate of 0.2oC per minute, (i) then keep the combination at about 35oC for about 6 hours, (j) then decrease the temperature by a rate of about 0.5oC, (k) and then keep the blend at about 25oC for about 60 minutes, before storage.
[00044] In one embodiment, the method additionally comprises the steps of (I) filling the glass vial containing the combination of step (k) with nitrogen gas, and (m) capping the vial under about 80% of atmospheric pressure. In one embodiment, the composition is brought to 2-8°C, after step (i), (j) or (k) and before the vial stoppering step.
[00045] In some embodiments, in step (a) the anti-PCSK9 antibody is at 50 mg/ml ± 7.5 mg/ml, the histidine is at 10 mM ± 1.5 mM (pH 6.0), the polysorbate 20 is in 0.1% ± 0.015%, and sucrose is in 6% ± 0.9%. In one embodiment, the anti-PCSK9 antibody comprises an HCDR1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO: 4, an LCDR1 of SEQ ID NO: 6, an LCDR2 of ID SEQ NO: 7, and an LCDR3 of SEQ ID NO: 8. mAb-316P. In one embodiment, the anti-PCSK9 antibody comprises an HCVD of SEQ ID NO: 1 and an LCVD of SEQ ID NO: 5. In one embodiment, (i) the antibody comprises an HCVD of SEQ ID NO: 1 and a LCVD of SEQ ID NO: 5, (ii) more than 90% of the antibodies in the combination have a molecular weight of 155 kDa ± 1 kDa, (iii) more than 50% of the antibodies in the combination have an isoelectric point of about 8.5, and (iv) from 75% to 90% of the antibodies in the combination are fucosylated.
[00046] In one aspect, there is provided a freeze-dried pharmaceutical composition comprising an anti-PCSK9 antibody and less than 0.3% in water, which is produced according to the method of the preceding aspect.
[00047] In one aspect, there is provided a pharmaceutical composition, which comprises the freeze-dried pharmaceutical composition of the preceding aspect resuspended in water. In one embodiment, the pharmaceutical composition consists of 50 mg/ml ± 7.5 mg/ml of anti-PCSK9 antibody, 10 mM ± 1.5 mM histidine (pH 6.0), 0.1% ± 0.015% polysorbate 20, and 6% ± 0.9% sucrose, in water. In one embodiment, the pharmaceutical composition consists of 100 mg/ml ± 15 mg/ml of anti-PCSK9 antibody, 20 mM ± 3 mM histidine (pH 6.0), 0.2% ± 0.03% polysorbate 20 and 12% ± 1.8% sucrose, in water. In another embodiment, the pharmaceutical composition consists of 150 mg/ml ± 22.5 mg/ml of anti-PCSK9 antibody, 30 mM ± 4.5 mM histidine (pH 6.0), 0.3% ± 0.045% polysorbate 20 and 18% ± 2.7% sucrose, in water. In yet another embodiment, the pharmaceutical composition consists of 175 mg/ml ± 26.25 mg/ml of anti-PCSK9 antibody, 35 mM ± 5.25 mM histidine (pH 6.0), 0.35% ± 0. 0525% polysorbate 20 and 21% ± 3.15% sucrose, in water.
[00048] In some embodiments, the anti-PCSK9 antibody comprises an HCVD of SEQ ID NO: 1 and an LCVD of SEQ ID NO: 5, and (b) more than 90% of the antibodies in the composition have a molecular weight of 155 kDa ± 1 kDa, (c) greater than 50% of the antibodies in the composition have an isoelectric point of approximately 8.5 and (d) 75% to 90% of the antibodies in the composition are fucosylated.
[00049] In one aspect, there is provided a pharmaceutical composition of any of the foregoing aspects, wherein the composition is contained in a container. In one embodiment, the container is a vial, which in some embodiments is a glass vial. In another embodiment, the container is a syringe. In some embodiments, the syringe is a low tungsten glass syringe. In one embodiment, the syringe is a 1 mL NUOVA OMPI long glass syringe equipped with a 27-G thin wall needle, a FLUROTEC coated 4023/50 rubber stopper, and an FM 27 rubber tip cap.
[00050] In one aspect, there is provided a kit comprising a pharmaceutical composition of any of the foregoing aspects, a container, and instructions. In one embodiment, the container is a filled syringe. In a particular embodiment, the syringe is a 1 mL NUOVA OMPI long glass syringe equipped with a 27-G thin wall needle, a FLUROTEC coated 4023/50 rubber stopper, and an FM 27 rubber end cap.
[00051] Other embodiments of the present invention become apparent from a review of the following detailed description. DETAILED DESCRIPTION
[00052] Before describing the present invention, it should be understood that this invention is not limited to particular methods and experimental conditions described, as such, methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
[00053] Unless otherwise defined, all technical and scientific terms used in this report have the same meaning as commonly understood by one skilled in the art to which this invention belongs. As used in this report, the term "approximately", when used in reference to a particular recited numerical value or range of values, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "approximately 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
[00054] Although any methods and materials similar or equivalent to those described in this report may be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated into this report by reference to describe in their entirety. PHARMACEUTICAL FORMULATIONS
[00055] As used in this report, the term "pharmaceutical formulation" means a combination of at least one active ingredient (eg, a small molecule, macromolecule, compound, etc., which is capable of exerting a biological effect on a human animal or non-human), and at least one inactive ingredient, which, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term "formulation" as used in this report means "pharmaceutical formulation" unless specifically indicated otherwise. The present invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. In accordance with certain embodiments of the present invention, the therapeutic polypeptide is an antibody, or antigen-binding fragment, that specifically binds to the human proprotein subtilisin/kexin protein type 9 convertase (PCSK9). More specifically, the present invention includes pharmaceutical formulations comprising: (i) a human antibody that specifically binds to human PCSK9 (ii) a histidine buffer; (iii) an organic co-solvent which is a non-ionic surfactant, (iv) a thermal stabilizer which is a carbohydrate; and optionally (v) a viscosity reducer, which is a salt. Specific exemplary components and formulations included within the scope of the present invention are described in detail below. ANTIBODIES THAT SPECIFICALLY BIND TO PCSK9
[00056] The pharmaceutical formulations of the present invention may comprise a human antibody, or an antigen-binding fragment thereof, that specifically binds to human PCSK9. As used herein, the term "PCSK9" means a human proprotein convertase belonging to the proteinase K subfamily of the secretory subtylase family. Evidence suggests that PCSK9 increases plasma LDL levels by binding to the low-density lipoprotein particle receptor and promoting its degradation. An exemplary human PCSK9 amino acid sequence is described in SEQ ID NO: 9. Antibodies to human PCSK9 are described in patent application publications US 2010/0166768, EU 2011/0065902, and WO 2010/077854.
[00057] The term "antibody", as used in this report, is generally intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds as well. as multimers thereof, (eg, IgM), however, immunoglobulin molecules consisting of only heavy chains (ie, lacking light chains) are also encompassed in the definition of the term "antibody". Each heavy chain comprises a heavy chain variable region (abbreviated in this report as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated in this report as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[00058] Unless specifically indicated otherwise, the term "antibody" as used herein is intended to encompass complete antibody molecules as well as antigen-binding fragments. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply, "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody which retain the ability to specifically bind to human PCSK9 or an epitope thereof.
[00059] An "isolated antibody" as used in this report is intended to refer to an antibody that is substantially free of other antibodies that have different antigenic specificities (eg, an isolated antibody that specifically binds to human PCSK9 , is substantially free of antibodies that specifically bind to antigens other than human PCSK9).
[00060] The term "specifically binds", or similar, means that an antibody or antigen-binding fragment thereof, a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by a dissociation constant of at least about 1 x 10-6 M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasma resonance, and the like. An isolated antibody that specifically binds to human PCSK9 may, however, be cross-reactive with other antigens, such as PCSK9 molecules from other species (orthologs). In the context of the present invention, multispecific (e.g., bispecific) antibodies that bind to human PCSK9, as well as one or more additional antigens, are considered to "specifically bind" to human PCSK9. Furthermore, an isolated antibody can be substantially free of other cellular material or chemicals.
[00061] Examples of anti-human PCSK9 antibodies that may be included in the pharmaceutical formulations of the present invention are presented in patent application publications EU 2010/0166768, EU 2011/0065902, and WO 2010/077854, the disclosures of which are incorporated as reference in its entirety.
[00062] According to certain embodiments of the present invention, the anti-human mAb-316P PCSK9 antibody is a human IgG1 that comprises a heavy chain variable region that is of the IGHV3-23 subtype and a light chain variable region that is of the subtype IGKV4-1 (see Barbie and Lefranc, The Human Immunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ) Segments, Exp. Clin. Immunogenet., 1998; 15:171-183, and Scaviner, D. et al., Protein Displays of the Human Immunoglobulin Heavy, Kappa and Lambda Variable and Joining Regions, Exp. Clin. Immunogenet., 1999; 16:234-240).
[00063] In some embodiments, the anti-human mAb-316P PCSK9 antibody comprises at least one amino acid substitution, which results in a charge change on an exposed surface of the antibody relative to the germline sequence IGKV4-1 . The IGKV4-1 germline sequence, and amino acid position assignment numbers presented herein are compatible with the International Immunogenetics (IMGT) information system, as described in Lefranc, M.-P., et al., IMGT®, the International ImMunoGeneTics® information system, Nucl. Acids Res., 37, D1006-D1012 (2009). In some embodiments, the exposed surface comprises a complementarity determining region (CDR). In some embodiments, amino acid substitution(s) are selected from the group consisting of a basic amino acid replaced by an uncharged polar amino acid in CDR1 (eg, at position 32) of IGKV4-1. Unique permutations in the charge distribution of an antibody, especially at an environmental interface (such as, for example, at a CDR), would be expected to create unpredictable conditions to maintain or promote the stability of the antibody in solution.
[00064] In some embodiments, the anti-human mAb-316P PCSK9 antibody comprises at least one amino acid substitution, which creates a charge change within a framework region of an antibody variable region relative to the antibody sequence. germline IGHV3-23 or the germline sequence IGKV4-1. In some embodiments, the amino acid substitution(s) are selected from the group consisting of (a) a hydrophobic amino acid replaced by a polar amino acid in framework region 3 (FR3) (e.g., at position 77) of IGHV3-23, and (b) a polar amino acid substituted by a basic amino acid in framework region 2 (FR2) (eg, at position 51) of IGKV4-1. Changes in the ability of the peptide chain to fold, especially within a framework region, which affects the CDR interface with the solvent, would be expected to create unpredictable conditions to maintain or promote the stability of the antibody in solution.
[00065] According to certain embodiments of the present invention, the anti-human PCSK9 antibody, or antigen-binding fragment thereof, comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2. of SEQ ID NO: 3, and an HCDR3 of SEQ ID NO: 4. In certain embodiments, the anti-human PCSK9 antibody, or antigen binding fragment thereof, comprises an HCVD of SEQ ID NO: 1.
[00066] According to certain embodiments of the present invention, the anti-human PCSK9 antibody, or antigen-binding fragment thereof, comprises a light chain complementarity determining region (kappa) (LCDR) 1 of SEQ ID NO: 6 , an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8. In certain embodiments, the anti-human PCSK9 antibody, or antigen-binding fragment thereof, comprises an LCVD of SEQ ID NO: 5.
[00067] The exemplary non-limiting antibody used in the Examples in this report is referred to as "mAb-316P". This antibody is also referred to in EU 7,608,693 as H4H098P. mAb-316P (H4H098P) comprises an amino acid sequence pair HCVR/LCVR having domains SEQ ID Nos: 1/5, and HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3 represented by SEQ ID Nos: 2 - 3 - 4/ SEQ ID Nos: 6 - 7 - 8.
[00068] The amount of antibody, or antigen-binding fragment thereof, contained in the pharmaceutical formulations of the present invention may vary depending on the specific desired properties of the formulations, as well as the particular circumstances and purposes for which the formulations are intended to be used. In certain embodiments, pharmaceutical formulations are liquid formulations which may contain from 50 ± 7.5 mg/ml to 250 ± 37.5 mg/ml of antibody; 60 ± 9 mg/ml to 240 ± 36 mg/ml antibody, 70 ± 10.5 mg/ml to 230 ± 34.5 mg/ml antibody; 80 ± 12 mg/ml to 220 ± 33 mg/ml antibody; 90 ± 13.5 mg/ml to 210 ± 31.5 mg/ml antibody; 100 ± 15 mg/ml to 200 ± 30 mg/ml antibody; 110 ± 16.5 mg/ml to 190 ± 28.5 mg/ml antibody; 120 ± 18 mg/ml to 180 ± 27 mg/ml antibody; 130 ± 19.5 mg/ml to 170 ± 25.5 mg/ml antibody; 140 ± 21 mg/ml to 160 ± 24 mg/ml antibody; 150 ± 22.5 mg/ml antibody; or 175 ± 26.25 mg/ml. For example, the formulations of the present invention can comprise about 50 mg/ml; about 60 mg/ml; about 65 mg/ml; about 70 mg/ml; about 75 mg/ml; about 80 mg/ml; about 85 mg/ml; about 90 mg/ml; about 95 mg/ml; about 100 mg/ml; about 105 mg/ml; about 110 mg/ml; about 115 mg/ml; about 120mg/ml; about 125 mg/ml; about 130 mg/ml; about 135mg/ml; about 140 mg/ml; about 145 mg/ml; about 150mg/ml; about 155 mg/ml; about 160 mg/ml; about 165mg/ml; about 170 mg/ml; about 175 mg/ml; about 180 mg/ml; about 185 mg/ml; about 190 mg/ml; about 195 mg/ml; about 200 mg/ml; about 205 mg/ml, about 210 mg/ml; about 215 mg/ml; about 220 mg/ml; about 225 mg/ml; about 230 mg/ml; about 235 mg/ml; about 240 mg/ml; about 245 mg/ml; or about 250 mg/ml of an antibody or antigen-binding fragment, which specifically binds to human PCSK9. EXCIPIENTS AND pH
[00069] The pharmaceutical formulations of the present invention comprise one or more excipients. The term "excipient" as used in this report means any non-therapeutic agent added to the formulation to provide a desired consistency, viscosity or stabilizing effect.
[00070] In certain embodiments, the pharmaceutical formulation of the invention comprises at least one organic co-solvent in a type and in an amount that stabilizes the human PCSK9 antibody under conditions of rough handling or agitation, such as, for example, centrifugation. In some embodiments, what is meant by "stabilizes" is the prevention of the formation of more than 3% of aggregated antibody of the total amount of antibody (on a molar basis) during the course of gross manipulation. In some embodiments, rough manipulation is vortexing a solution containing the antibody and organic co-solvent for about 60 minutes or approximately 120 minutes.
[00071] In certain embodiments, the organic co-solvent is a non-ionic surfactant, such as an alkyl poly(ethylene oxide). Specific nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates, such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85 ; poloxamers such as poloxamer 181, poloxamer 188, poloxamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as Tween 20, sorbitan monolaurate and polyoxyethylene sorbitan monolaurate. Poloxamer 188 is also known as PLURO-NIC F68.
[00072] The amount of nonionic surfactant contained in the pharmaceutical formulations of the present invention may vary depending on the specific desired properties of the formulations, as well as the particular circumstances and purposes for which the formulations are intended to be used. In certain embodiments, the formulations may contain from 0.01% ± 0.0015% to 0.2% ± 0.03% surfactant. For example, the formulations of the present invention may comprise about 0.0085%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05% , about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about of 0.20%, about 0.21%, about 0.22%, or about 0.23% polysorbate 20 or poloxamer 188.
[00073] The pharmaceutical formulations of the present invention may also comprise one or more stabilizers in a type and in an amount that stabilizes the human PCSK9 antibody under thermal stress conditions. In some embodiments, what is meant by "stabilizes" is to maintain greater than about 91% of the antibody in a native conformation when the solution containing the antibody and thermal stabilizer is kept at about 45°C for up to about 28 days. In some embodiments, what is meant by "stabilizes" is where less than about 6% of the antibody is aggregated when the solution containing the antibody and thermal stabilizer is kept at about 45°C for up to about 28 days. As used in this report, "native" means the major form of the antibody by size exclusion, which is generally an intact monomer of the antibody.
[00074] In certain embodiments, the thermal stabilizer is a sugar or sugar alcohol selected from sucrose, trehalose and mannitol, or any combination thereof, the amount of which contained in the formulation may vary depending on the specific circumstances and intended purposes for the formulation to be used. In certain embodiments, the formulations may contain about 3% to about 14% sugar or sugar alcohol, about 4% to about 13% sugar or sugar alcohol, about 5% to about 12% sugar or sugar alcohol, about 6% to about 11% sugar or sugar alcohol, about 7% to about 10% sugar or sugar alcohol, about 8% to about 9% sugar or sugar alcohol, about 4% to about 6% sugar or sugar alcohol; about 5% to about 7% sugar or sugar alcohol, about 9% to about 11% sugar or sugar alcohol; or about 11% to about 13% sugar or sugar alcohol. For example, the pharmaceutical formulations of the present invention may comprise 4% ± 0.6%, 5% ± 0.75%, 6% ± 0.9% and 7% ± 1.05% and 8% ± 1.2 %, 9% ± 1.35%, 10% ± 1.5%, 11% ± 1.65%, 12% ± 1.8%, 13% ± 1.95%, or about 14% ± 2. 1% sugar or sugar alcohol (eg sucrose, trehalose or mannitol).
[00075] The pharmaceutical formulations of the present invention may also comprise a buffer or buffering system that serves to maintain a stable pH and help stabilize the human PCSK9 antibody. In some embodiments, what is meant by "stabilizes" is where less than 4.5% ± 0.5% or less than 6.0 ± 0.5% of the antibody is aggregated when the solution containing the antibody and buffer it is kept around 45oC for up to 28 days. In some embodiments, what is meant by "stabilizes" is where less than 3% ± 0.5% or less than 2.6% ± 0.5% of the antibody is aggregated when the solution containing the antibody and buffer is kept at about 37oC for up to about 28 days. In some embodiments, what is meant by "stabilizes" is where at least 91% ± 0.5% or at least 92% ± 0.5% of the antibody is in its native conformation as determined by size exclusion chromatography , when the solution containing the antibody and the buffer is kept at about 45oC for up to about 28 days. In some embodiments, what is meant by "stabilizes" is where at least 94% ± 0.5% or at least 95% ± 0.5% of the antibody is in its native conformation as determined by size exclusion chromatography , when the solution containing the antibody and the buffer is kept at about 37oC for up to about 28 days. By "native" or "native conformation" what is meant is the fraction of antibody that is not aggregated or degraded. This is generally determined by an assay that measures the relative size of the antibody entity, such as a size exclusion chromatography assay. Non-aggregated, non-degraded antibody elutes under a fraction that is equivalent to the native antibody, and is generally the major elution fraction. Aggregated antibody elutes under a fraction that indicates a larger size than native antibody. Degraded antibody elutes under a fraction that indicates a smaller size than the native antibody.
[00076] In some embodiments, what is meant by "stabilizes" is where at least 38% ± 0.5% or at least 29% ± 0.5% of the antibody is in its main charge form as determined by cation exchange chromatography, when the solution containing the antibody and the buffer is kept at around 45oC for up to 28 days. In some embodiments, what is meant by "stabilizes" is where at least 46% ± 0.5% or at least 39% ± 0.5% of the antibody is in its main charge form as determined by exchange chromatography. cationic, when the solution containing the antibody and the buffer is kept at about 37oC for up to about 28 days. By "main charge" or "main charge form" what is meant is the fraction of antibody that elutes from an ion exchange resin in the main peak, which is usually flanked by more "basic" peaks on one side and more "acidic" peaks on the other side.
[00077] The pharmaceutical formulations of the present invention can have a pH of from about 5.2 to about 6.4. For example, the formulations of the present invention can have a pH of about 5.5; about 5.6; about 5.7; about 5.8; about 5.9; about 6.0; about 6.1; about 6.2; about 6.3; about 6.4; or about 6.5. In some embodiments, the pH is 6.0 ± 0.4; 6.0 ± 0.3; 6.0 ± 0.2; 6.0 ± 0.1, about 6.0; or 6.0.
[00078] In some embodiments, the buffer or buffering system comprises at least one buffer that has a buffering range that totally or partially overlaps the pH range 5.5 - 7.4. In one embodiment, the buffer has a pKa of about 6.0 ± 0.5. In certain embodiments, the buffer comprises a histidine buffer. In certain embodiments, histidine is present at a concentration of 5 mM ± 0.75 mM to 15 mM ± 2.25 mM, 6 mM ± 0.9 mM to 14 mM ± 2.1 mM, 7 mM ± 1.05 to 13 mM ± 1.95 mM; 8 mM ± 1.2 mM to 12 mM ± 1.8 mM; 9 mM ± 1.35 mM to 11 mM ± 1.65 mM, 10 mM ± 1.5 mM, or about 10 mM. In certain embodiments, the buffering system comprises 10 mM ± 1.5 mM histidine at a pH of 6.0 ± 0.3.
[00079] The pharmaceutical formulations of the present invention may also comprise one or more excipients that serve to maintain a reduced viscosity or to decrease the viscosity of formulations that contain a high concentration of anti-PCSK9 antibody drug substance (e.g., generally, > 150 mg/ml antibody). In some embodiments, the formulation comprises arginine in an amount sufficient to maintain the viscosity of the liquid formulation to less than 20 ± 3 cPoise, less than 15 ± 2.25 cPoise, or less than 11 ± 1.65 cPoise. In some embodiments, the formulation comprises arginine in an amount sufficient to maintain viscosity at or below 10.6 ± 1.59 cPoise. In certain embodiments, the pharmaceutical formulation of the present invention contains arginine, preferably as L-arginine hydrochloride, at a concentration of 10 mM ± 1.5 mM to 90 mM ± 13.5 mM, 20 mM ± 3 to 80 mM ± 12 mM, 30 mM ± 4.5 mM to 70 mM ± 10.5 mM, 40 mM ± 6 mM to 60 mM ± 9 mM, or 50 mM ± 7.5 mM. EXEMPLARY FORMULATIONS
[00080] According to one aspect of the present invention, the pharmaceutical formulation is a liquid formulation of low viscosity, generally physiologically isotonic, which comprises: (i) a human antibody that specifically binds to human PCSK9 (e.g., mAb -316P), at a concentration of 50 mg/ml ± 7.5 mg/ml, 100 mg/ml ± 15 mg/ml, 150 mg/ml ± 22.5 mg/ml, or 175 mg/ml ± 26, 25mg/ml; (ii) a buffering system that provides sufficient buffering at approximately pH 6.0 ± 0.3; (iii) a sugar that functions, inter alia, as a thermal stabilizer; (iv) an organic co-solvent that protects structural integrity, if the antibody; and (v) an amino acid salt, which serves to maintain a manageable viscosity for injection in a convenient volume for subcutaneous administration.
[00081] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9 and which comprises a substituted IGHV3-23-like heavy chain variable region and a substituted light chain-like variable region substituted IGLV4-1 (e.g. mAb-316P) at a concentration of 50 ± 7.5 mg/ml to about 175 ± 26.25 mg/ml; (ii) a buffer system comprising histidine, which effectively buffers at about pH 6.0 ± 0.3; (iii) sucrose; (iv) a non-ionic detergent, such as a polysorbate, and optionally, (v) an arginine salt.
[00082] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises an HCDR1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO: 4, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8, at a concentration of 175 mg/ml ± 26.25 mg /ml; (ii) histidine in 10 mM ± 1.5 mM, which buffers under pH 6.0 ± 0.3; (iii) sucrose 5% w/v ± 0.75% w/v; (iv) 0.01% w/v polysorbate 20 ± 0.0015% w/v; and (v) 50 mM ± 7.5 mM L-arginine hydrochloride.
[00083] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises an HCDR1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7, and an LCDR3 of SEQ ID NO:8, at a concentration of about 150 mg/ml ± 22, 5 mg/ml; (ii) 10 mM ± 1.5 mM histidine, which buffers under pH 6.0 ± 0.3; (iii) 10% w/v sucrose; 1.5% w/v; and (iv) polysorbate 20 at 0.2% w/v ± 0.03% w/v or 0.01% w/v ± 0.0015% w/v.
[00084] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises an HCDR1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7, and an LCDR3 of SEQ ID NO:8, at a concentration of about 100 mg/ml ± 15 mg µg/ml, (ii) histidine in about 20 mM ± 3 mM, which buffers a pH 6.0 ± 0.3, (iii) sucrose 12% w/v ± 1.8% w/v; and (iv) polysorbate 20 at 0.2% w/v ± 0.03% w/v or 0.01% w/v ± 0.0015% w/v.
[00085] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises an HCDR1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO: 4, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8, at a concentration of about 50 mg/ml ± 7, 5 mg/ml; (ii) 10 mM ± 1.5 mM histidine, which buffers at pH 6.0 ± 0.3; (iii) 6% w/v sucrose; 0.9% w/v; and (iv) polysorbate 20 at 0.1% w/v ± 0.015% w/v or 0.01% w/v ± 0.0015% w/v.
[00086] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody that specifically binds to human PCSK9, and which comprises a heavy chain variable domain of SEQ ID NO: 1, and a variable domain of light chain of SEQ ID NO: 5, at a concentration of 175 mg/ml ± 26.25 mg/ml; (ii) 10 mM ± 1.5 mM histidine, which buffers at pH 6.0 ± 0.3; (iii) sucrose 5% w/v ± 0.75% w/v; (iv) 0.01% w/v polysorbate 20 ± 0.0015% w/v; and (v) 50 mM ± 7.5 mM L-arginine hydrochloride.
[00087] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises a heavy chain variable domain of SEQ ID NO: 1, and a domain light chain variable of SEQ ID NO: 5, at a concentration of about 150 mg/ml ± 22.5 mg/ml; (ii) 10 mM ± 1.5 mM histidine, which buffers at pH 6.0 ± 0.3; (iii) sucrose 10% w/v ± 1.5% w/v; and (iv) polysorbate 20 at 0.2% w/v ± 0.03% w/v or 0.01% w/v ± 0.0015% w/v.
[00088] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises a heavy chain variable domain of SEQ ID NO: 1, and a variable domain of light chain SEQ ID NO: 5, at a concentration of about 100 mg/ml ± 15 mg/ml; (ii) about 20 mM ± 3 mM histidine, which buffers at pH 6.0 ± 0.3; (iii) sucrose 12% w/v ± 1.8% w/v; and (iv) polysorbate 20 at 0.2% w/v ± 0.03% w/v or 0.01% w/v ± 0.0015% w/v.
[00089] According to an embodiment, the pharmaceutical formulation comprises: (i) a human IgG1 antibody which specifically binds to human PCSK9, and which comprises a heavy chain variable domain of SEQ ID NO: 1, and a variable domain of light chain of SEQ ID NO: 5, at a concentration of about 50 mg/ml ± 7.5 mg/ml; (ii) 10 mM ± 1.5 mM histidine, which buffers at pH 6.0 ± 0.3; (iii) 6% w/v sucrose ± 0.9% w/v; and (iv) polysorbate 20 at 0.1% w/v ± 0.015% w/v or 0.01% w/v ± 0.0015% w/v.
[00090] Additional, non-limiting pharmaceutical formulation examples encompassed by the present invention are presented elsewhere in this report, including, the working examples presented below. STABILITY AND VISCOSITY OF PHARMACEUTICAL FORMULATIONS
[00091] The pharmaceutical formulations of the present invention typically exhibit high levels of stability. The term "stable" as used in this report in reference to pharmaceutical formulations means that the antibodies in pharmaceutical formulations retain an acceptable degree of chemical structure or biological function after storage under defined conditions. A formulation may be stable even though the antibody contained in them does not retain 100% of its chemical structure or biological function after storage for a defined period of time. Under certain circumstances, maintaining about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of an antibody structure or function after storage for a period of time defined can be considered as "stable".
[00092] Stability can be measured, inter alia, by determining the percentage of native antibody that remains in the formulation after storage for a defined period of time at a defined temperature. The percentage of native antibody can be determined by, among others, size exclusion chromatography (e.g., high performance size exclusion liquid chromatography [SE-HPLC]), such that native means non-aggregated and non-degraded. An "acceptable degree of stability" as the term is used herein means that at least 90% of the native form of the antibody can be detected in the formulation after storage for a defined period of time at a given temperature. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native form of the antibody can be detected in the formulation. after storage for a set amount of time at a set temperature. The defined amount of time after which stability is measured can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more. The set temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80°C to about 45°C, e.g., storage, at about -80°C, about -30 °C, about -20°C, about 0°C, about 4°-8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if, after 6 months of storage at 5°C, greater than about 95%, 96%, 97% or 98% of native antibody is detected by SE-HPLC. A pharmaceutical formulation can also be considered stable, if after 6 months of storage at 25°C, greater than about 94%, 95%, 96%, 97% or 98% of native antibody is detected by SE-HPLC. A pharmaceutical formulation may also be considered stable if after 28 days of storage at 45°C, greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% of native antibody is detected by SE-HPLC. A pharmaceutical formulation may also be considered stable if after three months of storage at -20°C, greater than about 96%, 97%, or 98% of the native antibody is detected by SE-HPLC. A pharmaceutical formulation can also be considered stable if after three months of storage at -30°C, greater than about 96%, 97% or 98% of native antibody is detected by SE-HPLC. A pharmaceutical formulation can also be considered stable if after three months of storage at -80°C, greater than about 96%, 97% or 98% of native antibody is detected by SE-HPLC.
[00093] Stability can be measured, inter alia, by determining the percentage of antibody that forms in an aggregate in the formulation after storage for a defined period of time, under a defined temperature, where stability is inversely proportional to the percentage of aggregate that is formed. The percentage of aggregated antibody can be determined by, among others, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). An "acceptable degree of stability", as that of the expression is used in this report, means that at most 6% of the antibody is in an aggregated form detected in the formulation after storage for a defined period of time at a given temperature. In certain embodiments, an acceptable degree of stability means that at most about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody can be detected in an aggregate in the formulation after storage for a defined period of time at a given temperature. The defined period of time after which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months , at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80°C to about 45°C, for example, storage at about -80°C, about -30° C, about -20°C, about 0°C, about 4°C - 8°C, about 5°C, about 25°C, about 35°C, about 37°C or about 45 °C. For example, a pharmaceutical formulation can be considered stable if, after six months of storage at 5°C, less than about 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detected in an aggregate form. A pharmaceutical formulation may also be considered stable if, after six months of storage at 25°C, less than about 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detected in an aggregated form. A pharmaceutical formulation may also be considered stable if after 28 days of storage at 45°C, less than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1 % antibody is detected in an aggregated form. A pharmaceutical formulation may also be considered stable if, after three months of storage at -20°C, -30°C, or -80°C less than about 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detected in an aggregated form.
[00094] Stability can be measured, inter alia, by determining the percentage of antibody that migrates in a more acidic fraction during ion exchange ("acid form") than in the main antibody fraction ("main charge form" ), where stability is inversely proportional to the antibody fraction in the acidic form. While not wishing to be bound by theory, antibody deamidation can cause the antibody to become more negatively charged and thus more acidic relative to the non-deamidated antibody (see, for example, Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99(8):5283-5288). The percentage of "acidified" antibody can be determined by, among others, ion exchange chromatography (eg cation exchange high performance liquid chromatography [CEX-HPLC]). An "acceptable degree of stability" as that of the term is used in this report means that at most 49% of the antibody is in a more acidic form detected in the formulation after storage for a defined period of time at a defined temperature. In certain embodiments, an acceptable degree of stability means at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody can be detected in an acidic form in the formulation after storage for a defined period of time at a given temperature. The defined period of time after which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80°C to about 45°C, for example, storage at about -80°C, about -30°C about -20°C, about 0°C, about 4°C -8°C, about 5°C, about 25°C, or about 45°C. For example, a pharmaceutical formulation that can be considered stable if, after three months of storage at -80°C, -30°C, or 20°C less than about 30%, 29%, 28%, 27%, 26 %, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form. A pharmaceutical formulation may also be considered stable if, after six months of storage at 5°C, less than about 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24% , 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form. A pharmaceutical formulation can also be considered stable if, after six months of storage at 25°C, less than about 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19% , 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5% or 0.1% of the antibody is in a more acidic form. A pharmaceutical formulation may also be considered stable if, after 28 days of storage at 45°C, less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41% , 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24 %, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody can be detected in a more acidic form.
[00095] Other methods can be used to assess the stability of the formulations of the present invention, such as, for example, Differential Scanning Calorimetry (DVC) to determine thermal stability, controlled agitation to determine mechanical stability, and absorbance at about 350 nm or about 405 nm to determine solution turbidities. For example, a formulation of the present invention can be considered stable if, after 6 or more months of storage at about 5°C to about 25°C, the change in OD405 of the formulation is less than about 0.05 (e.g., 0, 04, 0.03, 0.02, 0.01, or less) from the OD405 of the formulation at zero time.
[00096] Measurement of the biological activity or binding affinity of the antibody to its target can also be used to assess stability. For example, a formulation of the present invention may be considered to be stable if, after storage at, for example, 5oC, 25oC, 45oC, etc. for a defined period of time (eg, 1 to 12 months), the anti-PCSK9 antibody contained in the formulation binds with PCSK9 with an affinity that is at least 90%, 95%, or more of the antibody's binding affinity. before storage. Binding affinity can be determined by, for example, ELISA or plasmon resonance. Biological activity can be determined by a PCSK9 activity assay, such as, for example, contacting a cell that expresses PCSK9 with the formulation comprising the PCSK9 antibody. Antibody binding as such to a cell can be measured directly, such as, for example, via FACS analysis. Alternatively, the downstream activity of the PCSK9 system can be measured in the presence of antibody, and compared to the activity of the PCSK9 system in the absence of antibody. In some embodiments, PCSK9 can be endogenous to the cell. In other embodiments, PCSK9 can be ectopically expressed in the cell.
[00097] Additional methods for evaluating the stability of an antibody in the formulation are demonstrated in the Examples presented below.
[00098] The liquid pharmaceutical formulations of the present invention may, in certain embodiments, exhibit low to moderate levels of viscosity, "Viscosity" as used in this report may be "kinematic viscosity" or "absolute viscosity". "Kinematic viscosity" is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume are placed in identical capillary viscometers and allowed to flow by gravity, a viscous fluid takes longer than a less viscous fluid to flow through the capillary. For example, if one fluid takes 200 seconds to complete its flow and another fluid takes 400 seconds, the second fluid is twice as viscous as the first on a kinematic viscosity scale. "Absolute viscosity", sometimes called dynamic or simple viscosity, is the product of kinematic viscosity and fluid density (Absolute Viscosity = Kinematic Viscosity x Density). The kinematic viscosity dimension is L2/T where L is a length and T is time. Commonly, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm2/s, which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is the milliPascal-second (mPa-s), where 1 cP = 1 mPa-s.
[00099] As used in this report, a low level of viscosity, in reference to a fluid formulation of the present invention, will exhibit an absolute viscosity of minus about 15 cPoise (cP). For example, a fluid formulation of the invention will be considered to exhibit "low viscosity" if, when measured using standard viscosity measurement techniques, the formulation exhibits an absolute viscosity of approximately 15 cP, approximately 14 cP, approximately 13 cP, approximately 12 cP, approximately 11 cP, approximately 10 cP, approximately 9 cP, approximately 8 cP, or less. As used in this report, a moderate level of viscosity, in reference to a fluid formulation of the present invention, will exhibit an absolute viscosity of between approximately 35 cP and approximately 15 cP. For example, a fluid formulation of the invention will be considered to exhibit "moderate viscosity" if, when measured using standard viscosity measurement techniques, the formulation exhibits an absolute viscosity of approximately 34 cP, approximately 33 cP, approximately 32 cP, approximately 31 cP, approximately 30 cP, approximately 29 cP, approximately 28 cP, approximately 27 cP, approximately 26 cP, approximately 25 cP, approximately 24 cP, approximately 23 cP, approximately 22 cP, approximately 21 cP, approximately 20 cP, approximately 19 cP, approximately 18 cP, approximately 17 cP, approximately 16 cP, or approximately 15.1 cP.
[000100] As illustrated in the examples below, the present inventors have made the surprising discovery that low to moderate viscosity liquid formulations comprising high concentrations of anti-human PCSK9 antibody (e.g., from approximately 100 mg/ml to at least 200 mg/ ml) can be obtained by formulating the antibody with arginine from approximately 25 mM to approximately 100 mM. Furthermore, it has further been found that the viscosity of the formulation can be lowered to an even greater degree by adjusting the sucrose content by at least about 10%. RECIPIENTS AND METHODS OF ADMINISTRATION
[000101] The pharmaceutical formulations of the present invention can be contained in any container suitable for storing drugs and other therapeutic compositions. For example, pharmaceutical formulations can be contained in a sealed and sterilized plastic and glass container having a defined volume such as a vial, ampoule, syringe, cartridge or glass. Different types of vials can be used to contain the formulations of the present invention, including, for example, clear and opaque (e.g., amber) glass or plastic vials. Also, any type of syringe can be used to contain or administer the pharmaceutical formulations of the present invention.
[000102] The pharmaceutical formulations of the present invention can be contained in syringes of "normal tungsten" or syringes of "low tungsten". As will be appreciated by those skilled in the art, the process of producing glass syringes generally involves the use of a hot tungsten rod which functions to pierce the glass thereby creating an orifice from which liquid can be drawn and expelled. from the syringe. This process results in the deposition of trace amounts of tungsten on the inner surface of the syringe. Subsequent washing and other processing steps can be used to reduce the amount of tungsten in the syringe. As used in this report, the term "normal tungsten" means that the syringe contains more than or equal to 500 parts per billion (ppb) of tungsten. The term "low tungsten" means the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe in accordance with the present invention may contain less than approximately 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or less ppb of tungsten.
[000103] The rubber plungers used in syringes, and the rubber stoppers used to close the openings of vials, can be coated to prevent contamination of the medicinal contents of the syringe or vial, or to preserve its stability. Thus, pharmaceutical formulations of the present invention, according to certain embodiments, can be contained in a syringe that comprises a coated plunger, or in a vial that is sealed with a coated rubber stopper. For example, the plunger or cap can be coated with a fluorocarbon film. Examples of coated stoppers or plungers suitable for use with vials and syringes containing the pharmaceutical formulations of the present invention are mentioned in, for example, United States Patent Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, the contents of which are incorporated by reference in this report in its entirety. Particular exemplary coated rubber closures and plungers that can be used in the context of the present invention are commercially available under the tradename "FluroTec®", available from West Pharmaceutical Services, Inc. (Lionville, PA). FluroTec® is an example of a fluorocarbon coating used to minimize or prevent drug product from adhering to rubber surfaces.
[000104] According to certain embodiments of the present invention, pharmaceutical formulations may be contained in a low tungsten syringe comprising a fluorocarbon coated plunger.
[000105] Pharmaceutical formulations can be administered to a patient via parenteral routes such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal, pulmonary or oral administration. Numerous reusable pen transfer devices or auto-injectors can be used to subcutaneously transfer the pharmaceutical formulations of the present invention. Examples include, but are not limited to, AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALO-GTM pen, HUMALIN 70 pen /30TM (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes , NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM and OPTICLIKTM (Sanofi-aventis, Frankfurt, Germany). Examples of available pen or auto-injector transfer devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTARTM pen (Sanofi-aventis), the FLEXPENTM (Novo Nordisk) and the KWIKPENTM ( Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, LP), and the HUMIRATM pen (Abbott Labs. Abbott Park, IL), ).
[000106] The use of a microinfuser to transfer the pharmaceutical formulations of the present invention is also considered in this report. As used in this report, the term "microinfuser" means a subcutaneous transfer device designed to slowly deliver large volumes (eg, up to about 2.5 mL or more) of a therapeutic formulation over an extended period of time (for example, example, approximately 10, 15, 20, 25, 30 or more minutes). See, for example, U.S. 6,629,949; U.S. 6.659,982; and Meehan et al., J. Controlled Release 46: 107-116 (1996). Microinfusors are particularly useful for transferring large doses of therapeutic proteins contained in high concentrations (eg, about 100, 125, 150, 175, 200 or more mg/ml) or viscous solutions.
[000107] In one embodiment, liquid pharmaceutical formulation containing approximately 175 mg/mL ± 26.25 mg/mL of anti-PCSK9 antibody is administered subcutaneously in a volume of approximately 1.14 mL ± 0.17 mL in a filled syringe . In one embodiment, the syringe is a 1 mL long glass syringe filled with a 27-gauge thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle shield. In one embodiment, the syringe is a 1 mL OMPI long glass syringe fitted with a 27 gauge needle, an FM27 rubber needle shield, and a FLUROTEC® coated 4023/50 rubber plunger.
[000108] In one embodiment, a liquid pharmaceutical formulation containing approximately 150 mg/mL ± 22.5 mg/mL of anti-PCSK9 antibody is administered subcutaneously in a volume of approximately 1 mL ± 0.15 mL in a pre-filled syringe . In one embodiment, the syringe is a 1 mL long glass syringe filled with a 27-gauge thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle shield. In one embodiment, the syringe is a 1 mL OMPI long glass syringe fitted with a 27 gauge needle, an FM27 rubber needle shield, and a FLUROTEC® coated 4023/50 rubber plunger. THERAPEUTIC USES OF PHARMACEUTICAL FORMULATIONS
[000109] The pharmaceutical formulations of the present invention are useful, inter alia, for the treatment, prevention or amelioration of any disease or disorder associated with PCSK9 activity, including PCSK9-mediated diseases or disorders. Non-limiting examples of diseases and disorders that can be treated or prevented by administering the pharmaceutical formulations of the present invention include various dyslipidemias, such as, for example, hypercholesterolemia, hypercholesterolemia, hyperlipidemia, familial hyperlipidemia, dysbetalipoproteinemia, familial dysbetalipoproteinemia, hypertriglyceridemia, and familial hypertriglyceridemia. EXAMPLES
[000110] The following examples are presented in order to provide those skilled in the art with a complete description and description of how to make and use the methods and compositions of the invention, and it is not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (eg quantities, temperature, etc.), but some experimental errors and deviations must be considered. Unless otherwise noted, parts are parts by moles, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric pressure.
[000111] Initial formulation development activities involved selection of organic co-solvents, thermal stabilizers, and buffers in liquid and lyophilized formulations of mAb-316P (anti-PCSK9 antibodies of the invention) to identify excipients that are compatible with the protein and enhance its stability , while maintaining physiological osmolality and low viscosity for intravenous and subcutaneous injection. Buffer conditions were also examined to determine the optimum pH for maximum protein stability. EXAMPLE 1: MAB-316p ANTI-PCSK1 FORMULATION Development
[000112] Various buffers, organic co-solvents, and thermal stabilizers have been selected to identify excipients that enhance the stability of the PCSK9 antibody. Buffer conditions were also examined to determine the optimum pH for maximum antibody stability. Results generated from these studies were used to develop a stable liquid formulation as well as a stable lyophilized formulation suitable for clinical use, for intravenous (IV) or subcutaneous (SC) administration. Regarding lyophilized drug product, a single or dual-use formulation was developed that can be reconstituted with sterile water for injection (WFI) at a concentration of 50 mg/mL for IV or 100 mg/mL for SC administration. Once reconstituted at 50 mg/mL, the drug product can be further diluted in an IV bag containing 0.9% sodium chloride for IV release. Regarding the liquid formulation, mAb-316P was formulated at 175 ± 27 mg/mL and 150 ± 23 mg/mL. In one embodiment, mAb-316P at 175 ± 27 mg/mL is formulated in 10 ± 1.5 mM histidine (pH 6.0 ± 0.3), 0.01% ± 0.0015% polysorbate 20.5% ± 0.75% sucrose. In one embodiment, mAb-316P at 150 ± 23 mg/mL is formulated in 10 ± 1.5 mM histidine (pH 6.0 ± 0.3), 0.2% ± 0.03% or 0.01 % ± 0.0015% polysorbate 20, 10% ± 1.5% sucrose. EXAMPLE 2: MAB-316P ANTI-PCSK1 BUFFER AND pH
[000113] The effect of pH and buffer type on the stability of PCSK9 antibodies was examined in liquid formulations. 2 mg/ml of anti-PCSK9 mAb-316P were incubated at 45°C in 10 mM each of acetate buffer (pH 5.0-5.5), citrate (pH 5.5-6.0), succinate (pH 6.0), histidine (pH 6.0), phosphate (pH 6.0-7.5), or Tris (pH 8.0) to assess the effect of buffer and pH on the thermal stability of the protein (Table 1 ). For this experiment, the liquid formulations were maintained as 0.35 mL in a type 1 borosilicate glass vial with a capacity of 2 mL with a 4432/50 butyl rubber stopper coated with FLUROTEC®. The total amount of mAb-316P recovered was determined using reverse phase chromatography. The percentage of native versus aggregated form of mAb-316P was determined using size exclusion chromatography. The percentage of acidic and basic species of mAb-316P was determined using cation exchange chromatography. Maximum protein stability was observed, as determined by both size exclusion chromatography (SE) and cation exchange chromatography (CEX), when anti-PCSK9 mAb316P was formulated in 10 mM histidine buffer at pH 6.0.
[000114] The optimal pH of mAb-316P was then determined by incubating 10 mg/ml of mAb-316P at 45oC in histidine buffer between pH 5.5 and pH 6.5. Maximum protein stability was observed, as determined by SE and CEX, when mAb-316P was formulated in buffered histidine at pH 6.0 (Table 2). These analyzes also revealed that the major protein degradation pathways were the formation of aggregates, cleavage products and charge variants. Based on these results, 10 mM histidine buffer at pH 6.0 was chosen to develop a liquid and lyophilized formulation of mAb-316P.
[000115] Results from formulation development studies indicate that under basic conditions (pH > 6.5), mAb-316P anti-PCSK9 in solution can undergo deamidation reactions. Conversely, at pH < 5.5, a high rate of formation of molecular weight variants of mAb-316P was observed. Based on these data, the pH buffer used for the mAb-316P formulation is maintained between pH 5.7 and pH 6.3. The accelerated stability of mAb-316P is similar over this pH range. EXAMPLE 3: SELECTION OF PROTECTORS AGAINST AGGTION VOLTAGE
[000116] Several co-solvents were individually tested for their ability to minimize the formation of particulates in mixtures containing mAb-316P due to agitation stress. Turbidity analysis of stirred drug substance demonstrated an increase in optical density (OD) under 405 nm when a solution containing mAb-316P (0.35 ml of 25 mg/ml mAb-316P, 10 mM histidine, pH 6.0 ± 0.2 in a 2 mL capacity type 1 borosilicate glass vial with a 4432/50 butyl rubber stopper coated with FLUROTEC®) was vortexed for 120 minutes (Table 3). Formulation with any of the evaluated co-solvents appeared to have prevented the agitation-induced increase in turbidity. However, 20% PEG 300, 10% PEG 300, and 20% propylene glycol significantly decreased the thermal stability of mAb-316P, as determined by SE (Table 4; same mAb-316P concentration, buffer, and conditions container as in the vortex study, above). Formulations with polysorbate 20, polysorbate 80, Pluronic F68, and PEG 3350 had no significant effect on the thermal stability of mAb-316P, as determined by SE and CEX, making these cosolvents suitable for the anti-PCSK9 mAb-316P formulation. Polysorbate 20 was chosen as the organic co-solvent for development of both a lyophilized and liquid formulation of mAb-316P because it demonstrated good stability attributes, both in agitation and in thermal studies of mAb-316P. EXAMPLE 4: SELECTION OF PROTECTORS AGAINST THERMAL STRESS
[000117] Several excipients that are selected from a varied list containing sugars, amino acids and inorganic salts, have been individually tested to optimally increase the thermal stability of mAb-316P. A summary of some of the thermal stabilizers that were examined is presented in Table 5. For these experiments, the “thermal stabilizer” excipients were included in a solution of 20 mg/mL mAb-316P in 10 mM histidine ( 0.35 ml in a type 1 borosilicate glass bottle with a capacity of 2 ml with a 4432/50 butyl rubber stopper coated with FLUROTEC®). Formulations containing sucrose, sorbitol, mannitol and trehalose showed at least amount of mAb-316P degradation as determined by SE analysis. However, those formulations containing sorbitol showed a surprising increase in turbidity compared to those formulations containing sucrose, trehalose, and mannitol (Table 5). Although sucrose, trehalose and mannitol were observed to have no effect on the formation of charge variants of the anti-PCSK9 mAb-316P, mannitol was observed to destabilize the protein during several freeze-thaw cycles. Thus, mAb-316P exhibits similar stability when formulated with sucrose or trehalose. EXAMPLE 5: LYOPHILLED FORMULATION
[000118] A lyophilized formulation was developed to increase the stability of anti-PCSK9 mAb-316P, particularly with respect to loading variants, and to increase the maximum transferable concentration of mAb-316P. Various lyoprotectants were combined with 0.7 mL of 50 mg/mL mAb-316P, 10 mM histidine, in a type 1 borosilicate glass vial with a capacity of 2 mL with a 4432/50 butyl rubber stopper coated with FLUROTEC®, lyophilized and examined for its ability to stabilize lyophilized mAb-316P when incubated at 50°C. Prior to analysis, the lyophilized product cake was reconstituted to 100 mg/ml mAb-316P. The two lyophilized formulations with the greatest stability as determined by SE and CEX contained: 1) 6% sucrose or 2) 2% sucrose plus 2% arginine. 6% sucrose was chosen for the mAb-316P drug product formulation. Thus, the lyophilized anti-PCSK9 mAb-316P drug product was produced by lyophilization in an optimized buffered aqueous formulation containing 10 mM histidine, pH 6.0 ± 0.1, 0.1% (w/v) of polysorbate 20, 6% (w/v) sucrose, and 50 mg/ml anti-PCSK9 mAb-316P. The storage and tension stability of these lyophilized formulations is shown in Table 7.
[000119] The lyophilization cycle of anti-PCSK9 mAb-316P was developed based on the measured Tg’ (frozen glass transition temperature) of the formulation, which was measured using subenvironment-modulated Differential Scanning Calorimetry (mDSC). The product temperature should not go above the Tg' during primary drying, which was determined to be -27.9°C.
[000120] Lyophilized mAb-316P was produced by filling 5.3 ml of the formulation with 50 mg/ml mAb-316P, 10 mM histidine (pH 6.0), 0.1% polysorbate 20, 6% sucrose in 20 mL of 20 mL type 1 glass vials and lyophilization according to the following steps:1. Required shelf temperature during charging: 5-25°C2. Initial Hold at: 5°C for 60 minutes3. Ascending rate (time) for freezing 0.5°C/minute (100 minutes) 4. Hold at: -45°C for 120 minutes5. Vacuum Set Point 13.33 Pa6. Ascending rate (time) for heating to primary drying: 0.5°C/minute (40 minutes)7. Primary Drying Shelf Temperature: -25°C8. Extension of Primary Drying: 78 hours9. Ascending rate (time) from heating to secondary drying: 0.2°C/minute (300 minutes)10. Secondary Drying shelf temperature: 35°C11. Extension of Secondary Drying: 6 hours12. Ascending rate (time) for cooling: 0.5°C/minute (20 minutes) 13. Hold at: 25°C for 60 minutes*14. Filling with nitrogen gas15. Vacuum plugging: 80% atmospheric pressure (81.06 Pa)
[000121] When extensive storage is required after secondary drying and before the buffering step, the shelf temperature of the lyophilizer is brought to 2-8oC. Lyophilized drug product that was produced using the final cycles described above had good cake appearance, low moisture content (0.3%), reconstitution time less than 4 minutes, and no turbidity of the reconstituted solution.
[000122] The appearance of the freeze-dried cake was not affected when the drug product mAb-316P (mAb-316P) was incubated for 2 months at 50°C, or stored for 3 months at 5°C. There was no effect on pH, appearance, or turbidity of the reconstituted mAb-316P drug product, and there was no significant difference in the amount of recovered mAb-316P. After 2 months of incubation at 50°C, the lyophilized drug product mAb-316P was 1.1% more degraded as determined by SE-HPLC and 8.3% more degraded as determined by CEX-HPLC. No significant degradation was observed when the lyophilized drug product mAb-316P was stored for 3 months at 5°C. No significant potency loss, as determined using an anti-PCSK9 bioassay, was observed for any of the voltage samples. EXAMPLE 6: MAB-316P LIQUID AND RECONSTITUTED
[000123] There are two methods to reconstitute lyophilized mAb-316P drug product depending on the route of administration. For IV administration, mAb-316P drug product is reconstituted with 5.0 mL of sterile WFI resulting in 5.3 mL of solution containing 50 mg/mL of mAb-316P, 10 mM histidine, pH 6.0, 0.1% (w/v) of polysorbate 20, and 6% (w/v) of sucrose. For SC administration, mAb-316P drug product is reconstituted with 2.3 mL of sterile WFI resulting in a 2.7 mL solution containing 100 mg/mL of REGN727, 20 mM histidine, pH 6.0, 0.2% (p /v) of polysorbate 20, and 12% (w/v) of sucrose. The volume available for withdrawal is 4.8 mL for IV and 2.0 mL for SC injection; an excess of 0.7 mL of reconstituted solution is contained in the SC bottle.
[000124] In the alternative, liquid mAb-316P is formulated as a liquid formulation, without the intervention step of lyophilization. Liquid formulations of mAb-316P are each 150 mg/ml (± 15%) or 175 mg/ml (± 15%) of anti-PCSK9 mAb-316P, in 10 ± 1.5 mM histidine (pH 6 ,0 ± 0.3), polysorbate 20 in 0.01% ± 0.0015% or 0.2% ± 0.03%, sucrose at 5% ± 0.75% or 10% ± 1.5%, and , in the case of the 175 mg/ml formulation, 50 ± 7.5 mM arginine.
[000125] Anti-PCSK9 mAb-316P was found to be stable when sterile filtered. A Millipore MILLIPAK filtration unit was used in the production of the clinical supplies, while a filter of identical composition was used in research studies (Millipore MLLEX DURAPORE). Compared to storage in glass vials, the stability of formulated mAb-316P drug substance (mAb-316P FDS) was not significantly affected when stored in a polypropylene tube, a polystyrene tube, a polycarbonate tube, or a vial glass housing containing a stainless steel ball bearing (Table 8). EXAMPLE 7: LIQUID FORMULATION IN FILLED SYRINGES
[000126] Formulation development studies were conducted with the goal of developing a high concentration liquid mAb-316P formulation that can be used in filled syringes (PFS) for SC delivery. Results from the development phase of the lyophilized formulation REGN727 demonstrated that the optimal buffer, pH, organic co-solvent, and thermal stabilizer were histidine, pH 6.0, polysorbate 20, and sucrose, respectively (supra). These same excipients were used to develop both the 150 mg/mL and 175 mg/mL drug product formulations of mAb-316P. Arginine was added to the 175 mg/mL version of the mAb-316P drug product to reduce the viscosity of the formulation. Tension stability of 150 and 175 mg/mL of mAb-316P drug product was examined in Nuova OMPI filled 1 mL long glass syringes (PFS) and compared with stability of 150 and 175 mg/mL of control drug product. , glass jars. No significant difference in the amount of physical or chemical degradation was observed after incubation of 150 or 175 mg/mL of the mAb-316P drug product at 45°C between the WIPO PFS and the control glass vial. These data indicate that the anti-PCSK9 mAb-316P drug product formulations of 150 and 175 mg/ml anti-PCSK9 mAb-316P are stable enough for use in PFS. EXAMPLE 8: STABILITY OF DRUG SUBSTANCE FORMULATED MAB-316P
[000127] Stability studies were performed to determine both storage and tension stability of mAb-316P formulations of 150 and 175 mg/mL. Turbidity and RP-HPLC assays were used to assess the physical stability of mAb-316P. Physical stability is defined as the recovery of soluble forms of the anti-PCSK9 mAb-316P in solution. Protein loss can be due to protein precipitation or surface adsorption. The presence of particulates in the solution can be detected by visual inspection measurements or by optical density (OD) under 405 nm (turbidity measurements). In this last trial, an increase in OD indicates an increase in turbidity, due to the formation of particulates. The presence of particulates as determined by optical density (OD) measurements indicates that the sample was unable to maintain stability. Recovery of mAb-316P is measured by RP-HPLC. In the RP-HPLC assay, the anti-PCSK9 mAb-316P antibody is eluted from the reversed phase column as a single peak. The concentration of each test sample is determined from the peak area of mAb-316P antibody eluted compared to a calibration curve generated using mAb-316P standards of defined protein loads.
[000128] Chemical stability refers to the integrity of the chemical structure of the anti-PCSK9 antibody (mAb-316P) in a sample. Most chemical instability can be attributed to the formation of covalently modified forms of the protein, (eg, covalent aggregates, cleavage products, or charge variants) and noncovalently modified forms of the protein (eg, non-covalent aggregates). To date, the only mAb-316P degradation products that are detected are species that differ in molecular weight or charge. The higher and lower molecular weight degradation products can be separated from native mAb-316P by SE-HPLC. The percentage of native mAb-316P in the size exclusion chromatography method is determined by the ratio of the native peak area to the total area of all mAb-316P antibody peaks.
[000129] Charge variant forms of mAb-316P are resolved from native mAb-316P using cation exchange chromatography. Peaks that elute from the CEX-HPLC column with retention times earlier than the main peak are labeled as "acid peaks", while those that elute from the CEX-HPLC column with retention times later than the main peak are labeled "basic peaks". The percentage of mAb-316P degraded in the cation exchange chromatographic method is determined by the change in the relative percentage of areas of main, acidic, basic peaks compared to the total area of all peaks of mAb-316P.
[000130] Evaluation of mAb-316P under accelerated conditions was performed by subjecting the antibody to a variety of strain tests. These tests represent the extreme handling conditions that the formulated drug substance may be subjected to during drug product production. Formulated drug substance from mAb-316P was filled into 5 mL polycarbonate vials for shaking, freeze/thaw cycles, and frozen storage conditions. Formulated drug substance of mAb-316P was filled into glass vials to examine high temperature stress stability. EXAMPLE 9: DRUG SUBSTANCE STORAGE STABILITY STUDIES FORMULATED
[000131] Formulated drug substance mAb-316P 150 mg/mL (FDS; 0.5 mL in 5 mL polycarbonate bottle; mAb-316P antibody 150 mg/mL, 10 mM histidine (pH 6.0), polysorbate 20 to 0.2% and 10% sucrose) was found to be physically and chemically stable when stored at < -20oC for 12 months. No significant loss of mAb-316P was observed and no significant chemical degradation was detected by size exclusion or ion exchange chromatography. More than 97% of the recovered mAb-316P was of the "native" structure as determined by size exclusion, and more than 56% of the recovered mAb-316P was of the "main charge variant" as determined by cation exchange. The results are summarized in Table 9.
[000132] Formulated drug substance mAb-316P 175 mg/ml (FDS; 0.75 ml in 5 ml polycarbonate bottle; mAb-316P antibody 175 mg/ml, 10 mM histidine (pH 6.0), polysorbate 20 to 0.01%, 5% sucrose, and 50 mM arginine) was found to be physically and chemically stable when stored at < -20oC for 3 months. No significant loss of mAb-316P was observed and no significant chemical degradation was detected by size exclusion or ion exchange chromatography. More than 96% of the recovered mAb-316P was of the "native" structure as determined by size exclusion, and more than 56% of the recovered mAb-316P was of the "main charge variant" as determined by cation exchange. The results are summarized in Table 10. EXAMPLE 10: DRUG SUBSTANCE TENSION STABILITY STUDIES FORMULATED
[000133] Voltage stability studies were performed on the formulated drug substance 150 mg/ml mAb-316P (FDS) (0.35 ml - 0.5 ml of 150 mg/ml mAb-316P, 10 mM histidine (pH) 6.0), 0.2% polysorbate 20, 10% sucrose) and the formulated drug substance mAb-316P of 175 mg/ml (0.5 ml - 1.7 ml of 175 mg/ml of mAb-316P , 10 mM histidine (pH 6.0), 0.01% polysorbate 20, 5% sucrose, 50 mM arginine). High temperature studies were conducted in a type 1 borosilicate glass with a capacity of 2 mL, 4432/50 butyl rubber stopper coated with FLUROTEC®; the remaining studies were performed in a 5 mL polycarbonate bottle. The formulated 150 mg/ml and 175 mg/ml anti-PCSK9 mAb-316P drug substance were found to be physically and chemically stable when vortexed for two hours. The solution remained visibly clear, no protein loss occurred and no molecular weight species or charge variants were formed (Tables 11 and 12). MAb-316P was also observed to be both physically and chemically stable when subjected to eight cycles of freezing at -80oC and thawing at room temperature. After the eight freeze/thaw cycles, the protein solution remained visibly clear and no loss of protein was observed. No molecular weight (soluble aggregates or cleavage products) or charge variant forms were detected by either SE or CEX assay, respectively.
[000134] Although the formulated drug substance anti-PCSK9 mAb-316P 150 and 175 mg/mL were physically stable when incubated at 37°C or 45°C for 28 days, some chemical degradation, however, was observed (Tables 11 and 12). These stress tests indicated that the main degradation pathways were the formation of aggregates, cleavage products, and charge variants. As expected, the degradation rate of anti-PCSK9 antibody mAb-316P antibody was slower at 37°C than at 45°C. There was no significant change in physical or chemical stability of the formulated drug substance mAb-316P of 150 or 175 mg/mL when incubated at 25°C for 28 days. EXAMPLE 11: DRUG PRODUCT STORAGE STABILITY (PF)
The 150 mg/ml mAb-316P drug product consists of 10 mM histidine, pH 6.0, 0.01% polysorbate 20, 10% sucrose, and 150 mg/ml of PCSK9 mAb-316P antibody. The 175 mg/ml mAb-316P drug product consists of 10 mM histidine, pH 6.0, 0.01% polysorbate 20, 5% sucrose, 50 mM arginine, and 175 mg anti-PCSK9 mAb-316P antibody /mL. There is no change in physical and chemical stability of either the 150 mg/mL or 175 mg/mL mAb-316P drug product (PF) when stored at 5°C for 6 months in the filled syringe (PFS; long glass syringe 1 mL WIPO with a 27-gauge thin-walled needle and FM27 rubber needle guard closed with a FLUROTEC® coated 4023/50 rubber plunger) (Table 13 and Table 14). The solutions remained visibly clear, no loss of protein was observed, and there was no change in pH after these stresses. Furthermore, no significant changes in species molecular weight or charge variants were detected by SE and CEX, respectively. EXAMPLE 12: PHARMACEUTICAL PRODUCT TENSION STABILITY (PF)
[000136] The voltage stabilities of mAb-316P drug product 150 mg/ml and mAb-316P drug product 175 mg/ml were examined by incubating the filled syringes at 25°C and 45°C. Each respective drug product was physically stable when incubated at 45oC for 28 days or incubated at 25oC for 6 months (Tables 13 and 14). The solution remained visibly clear, no loss of protein was observed, and no change in pH occurred after these stresses. However, aggregates and charge variants were detected when the protein was incubated at 45oC and 25oC. This stress test indicates that these are the major drug product degradation pathways. Of the 150 mg/ml drug product, mAb-316P aggregate increased by 1.9% and acidic species increased by 19.1% after incubation at 45oC for 28 days. A reduced level of chemical degradation was detected when the protein was incubated at 25oC. There is a 0.8% increase in the relative amount of aggregate and a 10.3% increase in acidic species after 6 months of incubation at 25oC. From the drug product of 175 mg/ml; mAb-316P aggregate increased by 1.8% and acidic species increased by 17.0% after incubation at 45oC for 28 days. A reduced level of chemical degradation was detected when the protein was incubated at 25oC. There is a 0.7% increase in aggregate and a 9.4% increase in acidic species after 6 months of incubation at 25oC. For both the 150 mg/mL drug product and the 175 mg/mL drug product, there is no significant change in mAb-316P stability after 1 month of incubation at 25oC. EXAMPLE 13: FILL VOLUMES
[000137] The injectable volume of a filled syringe (PFS) containing the drug product of 150 mg/mL REGN727 is 1.0 mL. The injectable volume of a PFS containing 175 mg/mL REGN727 drug product is 1.14 mL. No excess is included in any PFS because the dead volume in the syringe is negligible (0.005 to 0.01 mL). EXAMPLE 14: STABILITY OF MAB-316P STORAGE MATERIALS
[000138] Anti-PCSK9 mAb-316P was found to be stable when sterile filtered. A MILLIPORE MILLIPAK filtration unit was used for research studies and in the production of clinical supplies. Compared to storage in glass vials, the stability of formulated drug product mAb-316P of 150 and 175 mg/mL was not significantly affected when stored in a polypropylene tube, a polystyrene tube, a polycarbonate tube, or in a glass bottle containing a stainless steel gasket (Table 15 and Table 16). Although degradation was observed when the formulated drug substance was incubated at 40°C for 14 days, no significant difference was observed in the amount of degradation of mAb-316P between the control, glass vial and exposure to plastic and steel containers stainless. EXAMPLE 15: MAB-316P ANTI-PCSK9 ANTIBODY CHARACTERIZATION
[000139] At least two lots of mAb-316P (Lot 1 and Lot 2) were analyzed using size exclusion chromatography and multi-angle laser light scattering (SEC-MALLS), an analytical method that provides an estimate of molar mass of a protein or glycoprotein. Lots 1 and 2 had respective molar masses of 154.5 and 154.6 kDa. Other lots had molar masses ranging from 154.4 to 154.8 kDa (mean approximately 155 kDa) for the main peak of species eluted from the SE matrix. This major peak represented approximately 96.7-99.2% of the total protein peak area and corresponds to the intact monomer of mAb-316P (ie, "native" as used in this report).
[000140] mAb-316P was analyzed by means of capillary isoelectric focusing (CIEF) to determine the isoelectric points for the main constituent isoforms. The pI and mean peak area (% of total peak area) of mAb-316P samples determined by cIEF are summarized in Table 17. Each lot exhibited one major species (peak 5) with a calculated pI of approximately 8.5. which was present in 66.4% and 68.0% for lots 1 and 2, respectively. The dominant species (peak 5) most likely represents fully glycosylated intact antibody that lacks the C-terminus lysine (ie, "major charge form" as used in this report).
[000141] Mass spectrometric (MS) analysis of the reduced mAb-316P tryptic maps of Lot 1 and Lot 2 resulted in the confirmation of a single glycosylation site, Asn298, in the Fc domain in both lots. The major glycan forms covalently attached to this glycosylation site are summarized in Table 18. Overall, both lots were determined to have biantennary complex glycans, with most of them fucosylated at Asn298. However, the relative amount of fucosylated agalactosyl (G0) containing sugar chain species in Lot 2 was slightly higher than the amount of this glycoform in Lot 1. Conversely, the relative amounts of fucosylated digalactosyl (G2) and fucosylated monogalactosyl (G1) containing the sugar chain forms in Lot 2 were reduced compared to the relative amounts of these sugar chain structures in Lot 1. Analysis of the LC/MS results of the two drug substance samples (peak 16) also identified 2 .9% and 8.4% heavy chain peptide lacking glycan occupancy in Asn298 in Lots 1 and 2, respectively.
[000142] Glycan profiles generated by HPLC after release of oligosaccharides from each of the two lots of mAb-316P were analyzed. In each chromatogram, the derived oligosaccharides were separated into two main groups: non-fucosylated bi-branched species and bi-branched fucosylated species. Within each group (fucosylated versus non-fucosylated), oligosaccharides were further separated into digalactosyl (G2), monogalactosyl (G1), and agalactosyl (G0) forms. Structural assignments of oligosaccharides were obtained via MALDI-TOF mass spectrometry. Integration of each peak into the two chromatograms revealed that the level of fucosylation of the two lots of mAb-316P was generally high, with 80.0% and 86.9% of fucosylation observed in lots 1 and 2, respectively.
[000143] Although the total percentage of fucosylation of the two lots was similar, there were quantitative differences in the relative abundance of each of the glycan forms present in two lots (Table 19). For lot 1, peak area percentages of 34.4%, 39.6% and 11.7% were determined for the fucosylated glycan structures G0, G1 and G2, respectively. In contrast, peak area percentages for Lot 2 were 45.8%, 33.3%, and 7.1% for the fucosylated glycoform structures G0, G1 and G2, respectively, indicating differences in the extent of galactosylation between the two lots. A high peak of mannose glycan (man5 glycan) (peak 2) was detected at a relative abundance of 1.8 - 2.9% relative to the total amount of sugar chain observed in both batches (Table 19). A total of nine unidentified peaks with peak area percentages ranging from 0.5% to 1.5% were also detected in both batches analyzed by this method and represent <3% of the total detected glycan peak areas in each batch. Analysis of an equimolar co-mix of the two lots produced no new peaks and the peak area percentages of all peaks in the co-mix well correlated with the expected values based on the individual analysis of each lot (Table 19).

权利要求:
Claims (11)
[0001]
1. Liquid pharmaceutical formulation, characterized in that it comprises: (a) from 50 ± 7.5 mg/ml to 250 ± 37.5 mg/ml of an antibody that specifically binds to the proprotein convertase subtilisin/ human kexin type 9 (PCSK9), wherein the antibody comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO: 4, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8; (b) 10 ± 1.5 mM histidine ( pH 6.0 ± 0.3); (c) 0.01 ± 0.0015% w/v polysorbate 20; and (d) 10 ± 1.5 % w/v sucrose.
[0002]
2. Liquid pharmaceutical formulation according to claim 1, characterized in that the antibody comprises a heavy chain variable region (HCVD) comprising SEQ ID NO: 1, and a light chain variable region (LCVD) comprising SEQ ID NO: 5.
[0003]
3. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the formulation comprises 175 mg/ml of the antibody.
[0004]
4. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the formulation comprises 150 mg/ml of the antibody.
[0005]
5. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the formulation comprises 100 mg/ml of the antibody.
[0006]
6. Liquid pharmaceutical formulation, according to claim 1 or 2, characterized by the fact that the formulation comprises 75 mg/ml of the antibody.
[0007]
7. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the formulation comprises 50 mg/ml of the antibody.
[0008]
8. Liquid pharmaceutical formulation, characterized in that it comprises: (a) 75 mg/ml of an antibody that specifically binds to proprotein convertase human subtilisin/kexin type 9 (PCSK9), wherein the antibody comprises a determinant region of heavy chain complementarity (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO: 4, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8;(b) histidine (pH 6.0 ± 0.3), at a concentration of 8 ± 12 mM;( c) 0.01 ± 0.0015% w/v polysorbate 20; and (d) 10 ± 1.5 % w/v sucrose.
[0009]
9. Liquid pharmaceutical formulation according to claim 8, characterized in that the antibody comprises a heavy chain variable region (HCVD) comprising SEQ ID NO: 1, and a light chain variable region (LCVD) comprising SEQ ID NO: 5.
[0010]
10. Liquid pharmaceutical formulation, characterized in that it comprises: (a) 150 mg/ml of an antibody that specifically binds to proprotein convertase human subtilisin/kexin type 9 (PCSK9), wherein the antibody comprises a determinant region of heavy chain complementarity (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, an HCDR3 of SEQ ID NO: 4, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8; (b) histidine (pH 6.0 ± 0.3), at a concentration of 6 ± 0.9 mM ;(c) 0.01 ± 0.0015% w/v polysorbate 20; and (d) 10 ± 1.5 % w/v sucrose.
[0011]
11. Liquid pharmaceutical formulation according to claim 10, characterized in that the antibody comprises a heavy chain variable region (HCVD) comprising SEQ ID NO: 1, and a light chain variable region (LCVD) comprising SEQ ID NO: 5.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-02| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-09-08| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-06-29| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/07/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161512666P| true| 2011-07-28|2011-07-28|

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US61/512,666|2011-07-28|

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PCT/US2012/048574|WO2013016648A2|2011-07-28|2012-07-27|Stabilized formulations containing anti-pcsk9 antibodies|

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