EA047546B1 - STABILIZED FORMULA CONTAINING ANTI-PCSK9 ANTIBODIES - Google Patents

Description

Field of invention

The present invention relates to antibody-based medicinal products. More specifically, the present invention relates to the field of medicinal products comprising a human antibody that specifically binds human proprotein convertase subtilisin/kexin type 9 protease (PCSK9).

List of sequences

A text file containing a sequence listing that complies with the requirements of ST.25 is attached to this specification. The contents of the text file are incorporated into this document by reference. The attached paper copy of the sequence listing, identical in content to the text file that complies with the requirements of ST.25, is part of this specification.

State of the art

Macromolecular drug products (e.g., antibodies) must be formulated not only so that the molecules can be administered to patients, but also so that they are stable during storage and subsequent use. For example, antibody drug products in liquid solutions are prone to degradation, aggregation, or undesirable chemical modification if the solution is not formulated properly. The stability of a given antibody in a liquid formulation depends not only on the nature of the excipients used in the formulation, but also on the amounts and relative proportions of the various excipients.

Moreover, considerations other than stability must be taken into account when preparing liquid antibody formulations. Examples of such additional considerations include the viscosity of the solution and the concentration of antibody that can be achieved in a particular formulation, as well as the visual quality or attractiveness of the formulation. Therefore, special care must be taken when developing an antibody drug formulation to produce a formulation that is stable, contains sufficient concentration of antibody, and has acceptable viscosity and other properties that allow the formulation to be conveniently administered to patients.

Antibodies to the human proprotein convertase subtilisin/kexin type 9 protease protein (PCSK9) are one example of a therapeutically important macromolecule that needs to be properly formulated into a pharmaceutical formulation. Anti-PCSK9 antibodies are clinically used to treat or prevent diseases such as hypercholesterolemia and other dyslipidemias, as well as other abnormalities. Examples of anti-PCSK9 antibodies are described in, among other things, WO 2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, US Patent No. 7,572,618, WO 2010/077854, US 2010/0166768, and US 2011/0065902.

Although anti-PCSK9 antibodies are known to those skilled in the art, there remains a need for new pharmaceutical formulations containing αanti-PCSK9 antibodies that are sufficiently stable and suitable for administration to patients.

Brief description of the invention

The present invention addresses the above-mentioned need by providing medicaments comprising a human antibody that specifically binds human proprotein convertase subtilisin/kexin type 9 protease (PCSK9).

In one aspect, a liquid drug formulation is provided comprising: (i) a human antibody that specifically binds human proprotein convertase subtilisin/kexin type 9 protease (PCSK9); (ii) a buffer; (iii) an organic auxiliary solvent; (iv) a stabilizer; and in some cases (v) a viscosity reducer.

In one embodiment, the antibody is used at a concentration of about 50 ± 7.5 mg/ml to about 200 ± 30 mg/ml. In another embodiment, the antibody is used at a concentration of about 50 mg/ml ± 7.5 mg/ml. In another embodiment, the antibody is used at a concentration of about 100 mg/ml ± 15 mg/ml. In another embodiment, the antibody is used at a concentration of about 150 mg/ml ± 22.5 mg/ml. In another embodiment, the antibody is used at a concentration of about 175 mg/ml ± 26.25 mg/ml. In another embodiment, the antibody is used at a concentration of about 200 mg/ml ± 30 mg/ml.

In one embodiment, the antibody comprises one or more of the amino acid sequences of SEQ ID NOs:1-8. In one embodiment, the antibody comprises (a) a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions 1, 2, and 3 (HCDR1HCDR2-HCDR3), each comprising the sequence of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (b) a light chain variable region (LCVR) comprising light chain complementarity determining regions 1, 2, and 3 (LCDR1-LCDR2-LCDR3), each comprising the sequence of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. In a specific embodiment, the antibody comprises HCVRs and LCVRs each comprising the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:5, respectively.

In one embodiment, the pH of the liquid preparation is about 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 particular embodiment, the pH

- 1 047546 liquid preparation is about pH 6.0±0.3. In one embodiment, the liquid pharmaceutical buffer comprises one or more buffers with an effective buffering capacity range from about pH 5.5 to about pH 7.4 or a pKa of about 6.0.

In one embodiment, the buffer is histidine. In one embodiment, the histidine has a concentration of 5 mM ± 0.75 mM to 50 mM ± 7.5 mM. In one embodiment, the histidine has a concentration of 10 mM ± 1.5 mM, or about 10 mM. In one embodiment, the histidine has a concentration of 20 mM ± 3 mM, or about 20 mM. In one embodiment, the histidine has a concentration of 40 nM ± 6 mM, or about 40 nM.

In one embodiment, the organic auxiliary solvent is a nonionic polymer containing polyoxyethylene. In some embodiments, the organic auxiliary solvent is any or more of polysorbate 20, poloxamer 188, and polyethyleneglycol 3350. In a particular embodiment, the organic auxiliary solvent is polysorbate 20.

In one embodiment, the organic co-solvent has a concentration of from about 0.005%±0.00075% to about 1%±0.15% weight to volume or w/v, where, for example, 0.1 g/mL = 10% and 0.01 g/mL = 1%. In one embodiment, the organic co-solvent is polysorbate 20, which is present at a concentration of about 0.2%±0.03% w/v. In another embodiment, the organic co-solvent is polysorbate 20, which is present at a concentration of 0.01%±0.0015% w/v or about 0.01% w/v.

In one embodiment, the stabilizer is a sugar. In one embodiment, the sugar is selected from the group consisting of sucrose, mannitol, and trehalose. In a particular embodiment, the stabilizer is sucrose.

In one embodiment, the stabilizer has a concentration of from 1%±0.15% w/v to 20%±3% w/v. In a particular embodiment, the stabilizer is sucrose at a concentration of 5%±0.75% w/v or about 5% w/v. In another particular embodiment, the stabilizer is sucrose at a concentration of 10%±1.5% w/v or about 10% w/v. In another particular embodiment, the stabilizer is sucrose at a concentration of 12%±1.8% w/v or about 12% w/v.

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 sodium acetate. In one embodiment, the viscosity reducer is L-arginine hydrochloride.

In one embodiment, the concentration of the viscosity reducer is from 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.

In one embodiment, the viscosity of the solution or reconstituted lyophilized pharmaceutical preparation at 25°C does not exceed about 15 cP±10%. In one embodiment, the viscosity at 25°C is from 1.0 cP±10% to 18 cP±10%. In one embodiment, the viscosity at 25°C is 1.6 cP±10%, 1.7 cP±10%, 3.3 cP±10%, 3.5 cP±10%, 4.8 cP±10%, 6.0 cP±10%, 7.0 cP±10%, 7.1 cP±10%, 7.2 cP±10%, 7.9 cP±10%, 8.9 cP±10%, 10.0 cP±10%, 10.6 cP±10%, 11.4 cP±10%, 11.6 cP±10%, 11.8 cP±10%, 12.4 cP±10%, 13.9 cP±10%, 14.0 cP±10%, 15.5 cP±10% or 17.9 cP±10%.

In one embodiment, the osmolality of the liquid drug preparation is from 100 ± 15 mOsm/kg to 460 ± 69 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 103 ± 15 mOsm/kg or about 103 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 195 ± 29 mOsm/kg mlm about 195 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 220 ± 33 mOsm/kg or about 220 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 330 ± 50 mOsm/kg mlm about 330 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 435±65 mOsm/kg, or about 435 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 440±66 mOsm/kg, or about 440 mOsm/kg. In one embodiment, the osmolality of the liquid drug preparation is 458±69 mOsm/kg, or about 458 mOsm/kg.

In one embodiment, after three months of storage of the liquid drug product at -80°C, at least 96% or at least 97% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by size exclusion chromatography. In one embodiment, after three months of storage of the liquid drug product at -80°C, at least 56% of the non-basic and non-acidic form (i.e., the main peak or the main charged form) of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by ion exchange chromatography.

In one implementation, after three months of storage of a liquid medicinal product at -30°C,

- 2 047546 liquid medicinal product releases at least 96% or at least 97% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody, as confirmed by size exclusion chromatography. In one embodiment, after three months of storage of the liquid medicinal product at -30°C, at least 56% of the major charged form of the anti-PCSK9 antibody is released from the liquid medicinal product, as confirmed by ion exchange chromatography.

In one embodiment, after three months of storage of the liquid drug product at -20°C, at least 96% or at least 97% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by size exclusion chromatography. In one embodiment, after three months of storage of the liquid drug product at -20°C, at least 56% of the major charged form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by ion exchange chromatography.

In one embodiment, after six months of storage of the liquid drug product at 5°C, at least 96% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by size exclusion chromatography. In one embodiment, after three months of storage of the liquid drug product at 5°C, at least 58 or 59% of the major charged form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by ion exchange chromatography.

In one embodiment, after six months of storage of the liquid drug product at 25°C, at least 94% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by size exclusion chromatography. In one embodiment, after six months of storage of the liquid drug product at 25°C, at least 45% or 47% of the non-basic and non-acidic form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by ion exchange chromatography.

In one embodiment, after 28 days of storage of the liquid drug product at 45°C, at least 91% or 92% of the non-aggregated and non-degraded form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by size exclusion chromatography. In one embodiment, after 28 days of storage of the liquid drug product at 45°C, at least 35% or 37% of the non-basic and non-acidic form of the anti-PCSK9 antibody is recovered from the liquid drug product, as determined by ion exchange chromatography.

In one aspect, a liquid formulation is provided comprising: (i) from 50±7.5 mg/mL to 175±26 mg/mL of a human antibody that specifically binds 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 at a pH of about 5.3 to about 6.7. The anti-PCSK9 antibody of the present aspect comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), such that the combination of HCVR/LCVR comprises heavy and light chain complementarity determining regions (HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3) that comprise the amino acid sequences of SEQ ID NOs:2-3-4/SEQ ID NOs:6-7-8, respectively. In a specific embodiment, the anti-PCSK9 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), each of which comprises the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:5, respectively (hereinafter mAb-316P).

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) polysorbate 20; and (iv) 6%±0.9% (w/v) sucrose at a pH of 6.0±0.3. In one embodiment of this particular formulation, the viscosity is about 1.7 cP. In one embodiment of this particular formulation, the osmolality is 220±44 mosmol/kg.

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) polysorbate 20; and (iv) 12% ± 2.4% (w/v) sucrose at a pH of 6.0 ± 0.3. In one embodiment of this particular formulation, the viscosity is about 3.5 cP. In one embodiment of this particular formulation, the osmolality is 440 ± 88 mosmol/kg.

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) polysorbate 20; and (iv) 10% ± 1.5% (w/v) sucrose at pH 6.0 ± 0.3. In one embodiment of this particular formulation, the viscosity is about 6 cP. In one embodiment of this particular formulation, the osmolality is 435 ± 65.25 mosmol/kg. In one embodiment of this particular formulation, after storage of the formulation at 45°C for 28 days, > 92% of the antibody remains native and > 35% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at 25°C for six months, >94% of the antibody remains native and >45% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at 5°C for six months, >96% of the antibody remains native and >58% of the antibody retains the major charged form. In one embodiment of the present specific formulation

- 3 047546 after storing the preparation at -20°C for twelve months, >97% of the antibody remains native and >56% of the antibody retains the major charged form. In one embodiment of the present specific preparation, after storing the preparation at -30°C for twelve months, >97% of the antibody remains native and >56% of the antibody retains the major charged form. In one embodiment of the present specific preparation, after storing the preparation at -80°C for twelve months, >97% of the antibody remains native and >56% of the antibody retains the major charged form.

In some embodiments of the present particular formulation, >85% of the antibody retains its biological activity after 28 days at 45°C, >82% after 28 days at 37°C, and/or >98% after 28 days at 25°C.

In some embodiments of the present particular formulation, >85% of the antibody retains its biological activity after six months at -20°C, >70% after six months at -30°C, and/or >79% after six months at -80°C. In some embodiments of the present particular formulation, >81% of the antibody retains its biological activity after eight freeze-thaw cycles and/or >84% of the antibody retains its biological activity after 120 minutes of shaking.

In another embodiment of the present aspect, the liquid formulation comprises (i) 175±26.25 mg/mL mAb316P; (ii) 10±1.5 mM histidine; (iii) 0.01%±0.0015% (w/v) polysorbate 20; (iv) 5%±0.75% (w/v) sucrose; and (v) 50±7.5 mM arginine at 6.0±0.3. In one embodiment of this particular formulation, the viscosity is about 10.6 cP. In one embodiment of this particular formulation, the osmolality is 330±50 mosmol/kg. In one embodiment of this particular formulation, after storage of the formulation at 45°C for 28 days, >91% of the antibody remains native and >38% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at 25°C for six months, >94% of the antibody remains native and >47% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at 5°C for six months, >96% of the antibody remains native and >59% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at -20°C for three months, >96% of the antibody remains native and >56% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at -30°C for three months, >96% of the antibody remains native and >56% of the antibody retains the major charged form. In one embodiment of the present specific formulation, after storing the formulation at -80°C for three months, >96% of the antibody remains native and >56% of the antibody retains the major charged form.

In one aspect, the liquid drug of any of the above embodiments is placed in a container. In one embodiment, the container is a polycarbonate vial. In another embodiment, the container is a glass vial. In one embodiment, the glass is borosilicate glass type 1 with a butyl rubber stopper coated with fluorocarbons. In another embodiment, the container is a microinjector. In another embodiment, the container is a syringe. In a specific embodiment, the syringe plunger is coated with fluorocarbons. In one specific embodiment, the syringe is a long 1 ml glass syringe containing less than 500 ppb tungsten, with a 27-G needle, a fluorocarbon-coated butyl rubber stopper, and a latex-free, non-cytotoxic rubber cap. In a more specific embodiment, the syringe is a 1 ml long glass NUOVA OMPI syringe with a 27-G thin-walled needle, a FLUROTEC 4023/50 coated butyl rubber stopper and an FM 27 protective rubber cap. In another specific embodiment, the syringe is a 1 ml or 3 ml plastic syringe with a 27-G needle. In a more specific embodiment, the plastic syringe is supplied by BECTON DICKINSON.

In one aspect, there is provided a pharmaceutical preparation 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 the HCVD of SEQ ID NO:1 and the LCVD of SEQ ID NO:5, (b) more than 90% of the antibodies in the preparation have a molecular weight of 155 kDa±1 kDa, (c) more than 50% of the antibodies in the preparation have an isoelectric point of about 8.5, and (d) from 75 to 90% of the antibodies in the preparation fucosylated.

In one embodiment, the pharmaceutical preparation comprises (a) 175 mg/mL+26.25 mg/mL of the anti-PCSK9 antibody of the previous 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.

In one aspect, there is provided a pharmaceutical preparation 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, (d) 10% w/v ± 1.5% sucrose, wherein (i) the antibody comprises the HCVD of SEQ ID NO:1 and the LCVD of SEQ

- 4 047546

ID NO:5, (ii) more than 90% of the antibodies in the preparation have a molecular weight of 155 kDa+1 kDa, (iii) more than 50% of the antibodies in the preparation have an isoelectric point of approximately 8.5, and (iv) from 75 to 90% of the antibodies in the preparation are fucosylated.

In one embodiment, the pharmaceutical preparation comprises (a) 150 mg/ml+22.5 mg/ml of the anti-PCSK9 antibody of the previous paragraph, (b) 10 mM+1.5 mM histidine, pH 6+0.3, (c) 0.2% w/v+0.03% polysorbate 20, (d) 10% w/v±1.5% sucrose in water.

In one aspect, there is provided a pharmaceutical preparation 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 HCVD of SEQ ID NO:1 and LCVD of SEQ ID NO:5, (ii) more than 90% of the antibodies in the preparation have a molecular weight of 155 kDa+1 kDa, (iii) more than 50% of the antibodies in the preparation have an isoelectric point of about 8.5, and (iv) from 75 to 90% of the antibodies in the preparation are fucosylated.

In one embodiment, the pharmaceutical preparation comprises (a) 150 mg/mL+22.5 mg/mL of the anti-PCSK9 antibody of the previous 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.

In one aspect, there is provided a pharmaceutical preparation 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, (d) 12% w/v+1.8% sucrose, wherein (i) the antibody comprises HCVD of SEQ ID NO:1 and LCVD of SEQ ID NO:5, (ii) more than 90% of the antibodies in the preparation have a molecular weight of 155 kDa+1 kDa, (iii) more than 50% of the antibodies in the preparation have an isoelectric point of about 8.5, and (iv) from 75 to 90% of the antibodies in the preparation are fucosylated.

In one embodiment, the pharmaceutical preparation comprises (a) 100 mg/ml+15 mg/ml of the anti-PCSK9 antibody of the previous 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.

In one aspect, there is provided a pharmaceutical preparation 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, (d) 6% w/v+0.9% sucrose, wherein (i) the antibody comprises HCVD of SEQ ID NO:1 and LCVD of SEQ ID NO:5, (ii) more than 90% of the antibodies in the preparation have a molecular weight of 155 kDa+1 kDa, (iii) more than 50% of the antibodies in the preparation have an isoelectric point of about 8.5, and (iv) from 75 to 90% of the antibodies in the preparation are fucosylated.

In one embodiment, the pharmaceutical preparation comprises (a) 50 mg/ml+7.5 mg/ml of the anti-PCSK9 antibody of the previous 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.

In one aspect, a method is provided for preparing a lyophilized preparation that comprises an anti-PCSK9 antibody and less than 0.3% water. The method comprises the following steps: (a) mixing water, an anti-PCSK9 antibody, histidine, sucrose and polysorbate 20 in a glass vial, (b) then maintaining the mixture at about 5°C for about 60 min, (c) then decreasing the temperature at a rate of about 0.5°C per min, (d) then maintaining the mixture at about 45°C for about 120 min, (e) then decreasing the atmospheric pressure to about 100 mTorr, (f) then increasing the temperature at a rate of about 0.5°C per min, (g) then maintaining the mixture at about -25°C for about 78 h, (h) then increasing the temperature at a rate of 0.2°C per min, (i) then maintaining the mixture at about 35°C for about 6 h, (j) then decreasing the temperature at a rate of about 0.5°C, (k) and then keeping the mixture at approximately 25° for approximately 60 minutes before transferring it for storage.

In one embodiment, the method also comprises the following steps: (l) filling a glass vial containing the mixture after step (k) with nitrogen gas, and (m) sealing the vial under pressure at a level of about 80% of atmospheric pressure. In one embodiment, the mixture is brought to a temperature of 2-8°C after step (i), (j) or (k) and before the step of sealing the vial.

In some embodiments, in step (a), the concentration of the anti-PCSK9 antibody is 50 mg/mL+7.5 mg/mL, the concentration of histidine is 10 mM+1.5 mM (pH 6.0), the concentration of polysorbate 20 is 0.1%+0.015%, and the concentration of sugar is 6%+0.9%. In one embodiment, the anti-PCSK9 antibody comprises HCDR1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7, and LCDR3 of SEQ ID NO:8.mAb-316P. In one embodiment, the anti-PCSK9 antibody comprises HCVD of SEQ ID NO:1 and LCVD of SEQ ID NO:5. In one embodiment, (i) the antibody comprises the HCVD of SEQ ID NO:1 and the LCVD of SEQ ID NO:5, (ii) more than 90% of the antibodies in the mixture have a molecular weight of 155 kDa+1 kDa, (iii) more than 50% of the antibodies in the mixture have an isoelectric point of about 8.5, and (iv) from 75% to 90% of the antibodies in the mixture are fucosylated.

In one aspect, a method is provided for preparing a lyophilized preparation that contains an anti-PCSK9 antibody and less than 0.3% water, which is prepared in accordance with the method in the previous aspect.

- 5 047546

In one aspect, there is provided a pharmaceutical preparation that comprises a lyophilized pharmaceutical preparation of the previous aspect diluted in water. In one embodiment, the pharmaceutical preparation comprises 50 mg/mL±7.5 mg/mL 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 preparation comprises 100 mg/mL±15 mg/mL 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 preparation comprises 150 mg/mL ± 22.5 mg/mL 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 preparation comprises 175 mg/mL ± 26.25 mg/mL 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.

In some embodiments, the anti-PCSK9 antibody comprises the HCVD of SEQ ID NO:1 and the LCVD of SEQ ID NO:5, and (b) more than 90% of the antibodies in the mixture have a molecular weight of 155 kDa±1 kDa, (c) more than 50% of the antibodies in the mixture have an isoelectric point of about 8.5, and (d) from 75 to 90% of the antibodies in the mixture are fucosylated.

In one aspect, a medicinal preparation of any of the above embodiments is provided, wherein said preparation is placed in a container. In one embodiment, a vial is used as a container, which in some embodiments is a glass vial. In another embodiment, a syringe is used as a container. In some applications, a syringe made of low-tungsten glass is used as a syringe. In one embodiment, a NUOVA OMPI long glass syringe with a volume of 1 ml with a thin-walled needle of size 27-G, with a butyl rubber stopper with a FLUROTEC 4023/50 coating and a protective rubber cap FM 27 is used as a syringe.

In one aspect, a kit is provided that includes a medicinal product of any of the above aspects, a container, and instructions for use. In one embodiment, a pre-filled syringe is used as the container. In a specific embodiment, a NUOVA OMPI long glass syringe with a volume of 1 ml with a thin-walled needle of size 27-G, with a butyl rubber stopper with a FLUROTEC 4023/50 coating and a protective rubber cap a FM 27 is used as the syringe.

Other embodiments of the present invention will become apparent from the detailed description given below.

Detailed description of the invention

Before describing the present invention, it should be noted that the present invention is not limited to the specific methods and experimental conditions set forth, since such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not limiting, since the scope of the present invention is limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the term "about" with respect to a particular numerical value or range of values referred to means that the value may deviate from the value by no more than 1%. For example, as used herein, the expression "about" 100 encompasses 99 and 101, and all values in between (i.e., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or identical to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are hereby incorporated by reference in their entirety.

Pharmaceutical formulations

As used herein, the term pharmaceutical formulation/preparation means a mixture of at least one active ingredient (e.g., a small molecule, macromolecule, compound, etc., which is capable of exerting a biological effect on humans or animals) and at least one inactive ingredient, which in combination with the active ingredient or one or more inactive ingredients is suitable for therapeutic use in humans or animals. The term formulation/preparation as used herein, unless specifically stated otherwise, means a pharmaceutical formulation/preparation. The present invention relates to pharmaceutical formulations comprising at least one therapeutic polypeptide. According to certain embodiments of the present invention, the therapeutic polypeptide is an antibody or antigen-binding fragment thereof that specifically binds to human proprotein convertase subtilisin/kexin type 9 (PCSK9). More specifically, the present invention includes pharmaceutical formulations that comprise: (i) a human antibody that

- 6 047546 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 in some cases (v) a viscosity reducer, which is a salt. Specific examples of components and compositions included in the present invention are described in detail below.

Antibodies that specifically bind to PCSK9.

The pharmaceutical compositions of the present invention may include a human antibody or antigen-binding fragment thereof that specifically binds to human PCSK9. As used herein, the term PCSK9 means human proprotein convertase, which belongs to the proteinase K subfamily of the secretory subtilase family. Available data indicate that PCSK9 increases plasma LDL levels by binding to the receptor for low-density lipoprotein particles and stimulating its degradation. An exemplary amino acid sequence of human PCSK9 is provided in SEQ ID NO:9. Antibodies to human PCSK9 are described in patent application publications US 2010/0166768, US 2011/0065902 and WO 2010/077854.

As used herein, the term "antibody" is generally intended to refer to immunoglobulin molecules composed of four polypeptide chains, with two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, and to refer to multimers thereof (e.g., IgM); however, immunoglobulin molecules containing only heavy chains (i.e., without light chains) are also included within the scope of the term "antibody". Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and V _L regions can be subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions of higher conservation, called framework regions (FRs). Each VH and V _L region is formed by three CDRs and four FRs, arranged from amino-terminal to carboxyl-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Unless otherwise specifically stated, the term antibody as used herein shall be understood to include complete antibody molecules as well as antigen-binding fragments thereof. As used herein, the term antigen-binding portion or antigen-binding fragment of an antibody (or simply an antibody portion or antibody fragment) refers to one or more fragments of an antibody that retain the ability to specifically bind to human PCSK9 or an epitope thereof.

As used herein, the term "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies that have different antigen specificity (e.g., an isolated antibody that specifically binds human PCSK9 is substantially free of antibodies that specifically bind antigens other than human PCSK9).

The term "specifically bind" or the like means that the antibody or antigen-binding fragment thereof forms a complex with the antigen that is relatively stable under physiological conditions. Specific binding can be described by a dissociation constant of at least about 1 x 10 ^-6 M or higher. Methods for determining specific binding of molecules are known to those skilled in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. An isolated antibody that specifically binds human PCSK9 may, however, exhibit cross-reactivity to 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 and also to one or more other antigens are considered to specifically bind to human PCSK9. Moreover, an isolated antibody may be substantially free of other cellular materials or chemical reagents.

Examples of anti-human PCSK9 antibodies that can be included in the pharmaceutical compositions of the present invention are provided in US Patent Application Publications Nos. US 2010/0166768, US 2011/0065902 and WO 2010/077854, the disclosures of which are incorporated herein by reference in their entirety.

According to certain embodiments of the present invention, the anti-human PCSK9 mAb-316P antibody is a human IgG1 comprising a heavy chain variable region that has the IGHV3-23 subtype and a light chain variable region that has the IGKV4-1 subtype (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).

In some embodiments, the anti-human PCSK9 antibody mAb-316P comprises at least

- 7 047 546 at least one amino acid substitution that results in a charge change on the outer surface of the antibody relative to the IGKV4-1 germline sequence. The IGKV4-1 germline sequence and the amino acid position numbers provided herein are in accordance with the International Immunogenetics Information System (IMGT) 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 outer surface comprises a complementarity determining region (CDR). In some embodiments, the amino acid substitution or substitutions are selected from the group consisting of substitution of an uncharged polar amino acid for a basic amino acid within CDR1 (e.g., at position 32) of IGKV4-1. Unique permutations in the antibody charge distribution, particularly at the interface with the medium (e.g. in the CDR), are expected to create unpredictable conditions for maintaining or improving the stability of the antibody in solution.

In some embodiments, the anti-human PCSK9 mAb-316P antibody comprises at least one amino acid substitution that results in a charge alteration within the framework region of the antibody variable region relative to the IGHV3-23 germline sequence or the IGKV4-1 germline sequence. In some embodiments, the amino acid substitution or substitutions are selected from the group consisting of (a) a substitution of a polar amino acid within framework region 3 (FR3) (e.g., at position 77) of IGHV3-23 with a hydrophobic amino acid, and (b) a substitution of a basic amino acid within framework region 2 (FR2) (e.g., at position 51) of IGKV4-1 with a polar amino acid. Changes in the folding ability of the peptide chain, particularly within the framework region that affects the interface between the CDRs and the solvent, would be expected to create unpredictable conditions for maintaining or improving the stability of the antibody in solution.

According to a number of embodiments of the present invention, the anti-human PCSK9 antibody or antigen-binding fragment thereof comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 4. In some embodiments, the anti-human PCSK9 antibody or antigen-binding fragment thereof comprises HCVD of SEQ ID NO: 1.

According to a number of embodiments of the present invention, the anti-human PCSK9 antibody or antigen-binding fragment thereof comprises the light (kappa) chain complementarity determining region (LCDR) 1 of SEQ ID NO: 6, LCDR2 of SEQ ID NO: 7, and LCDR3 of SEQ ID NO: 8. In some embodiments, the anti-human PCSK9 antibody or antigen-binding fragment thereof comprises the LCVD of SEQ ID NO: 5.

A non-limiting example of an antibody used in the examples is called mAb-316P. Such an antibody, which was also called H4H098P in U.S. Patent 7,608,693. mAb-316P (H4H098P) comprises the amino acid sequence pair HCVR/LCVR with SEQ ID NOs: 1/5, as well as the domains HCDR1-HCDR2HCDR3/LCDR1-LCDR2-LCDR3, represented by the sequences SEQ ID NOs: 2-3-4/SEQ ID NOs: 6-7-8.

The amount of antibody or antigen-binding fragment thereof contained in the pharmaceutical compositions of the present invention can be varied depending on the desired specific properties of the compositions, as well as the particular circumstances and purposes for which the compositions are intended to be used. In certain embodiments, the pharmaceutical compositions are liquid preparations that can contain from 50 ± 7.5 mg/ml to 250 ± 37.5 mg/ml antibody; from 60 ± 9 mg/ml to 240 ± 36 mg/ml antibody; from 70 ± 10.5 mg/ml to 230 ± 34.5 mg/ml antibody; from 80 ± 12 mg/ml to 220 ± 33 mg/ml antibody; from 90 ± 13.5 mg/ml to 210 ± 31.5 mg/ml antibody; from 100±15 mg/ml to 200±30 mg/ml antibody; from 110±16.5 mg/ml to 190±28.5 mg/ml antibody; from 120±18 mg/ml to 180±27 mg/ml antibody; from 130±19.5 mg/ml to 170±25.5 mg/ml antibody; from 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 may 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 120 mg/ml; About 125 mg/ml; About 130 mg/ml; About 135 mg/ml; About 140 mg/ml; About 145 mg/ml; About 150 mg/ml; About 155 mg/ml; About 160 mg/ml; About 165 mg/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 thereof that specifically binds to human PCSK9.

Excipients and pH

The medicinal preparations of the present invention may also contain one or more excipients. As used herein, the term "excipient" means any non-drug agent added to the preparation to achieve the required consistency, viscosity or stabilizing effect.

In some embodiments, the pharmaceutical preparation of the present invention comprises

- 8 047546 at least one organic auxiliary solvent of a kind and in such amounts that stabilize the human PCSK9 antibody upon rough handling or agitation, such as shaking. In some embodiments, stabilizing means preventing the formation of more than 3% of aggregated antibody from the total amount of antibody (on a molar basis) upon rough handling. In some embodiments, rough handling means shaking a solution containing the antibody and the organic auxiliary solvent for about 60 minutes or about 120 minutes.

In some embodiments, the organic co-solvent is a non-ionic surfactant, such as an alkyl polyethylene oxide. Representative non-ionic surfactants that may be included in the formulation 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 PLURONIC F68.

The amount of nonionic surfactant contained in the pharmaceutical compositions of the present invention can be varied depending on the specific properties desired of the compositions and the specific circumstances and purposes for which the compositions are intended to be used. In some embodiments, the compositions can contain from 0.01%±0.0015% to 0.2%±0.03% surfactant. For example, the compositions of the present invention can contain 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 0.20%; about 0.21%; about 0.22%; or about 0.23% polysorbate 20 or poloxamer 188.

The pharmaceutical compositions of the present invention may also contain one or more stabilizers of a type and in amounts that stabilize the human PCSK9 antibody under conditions of heat stress. In some embodiments, stabilize means maintaining more than about 91% of the antibody in its native conformation under conditions where a solution containing the antibody and the thermal stabilizer is maintained at a temperature of about 45°C for up to about 28 days. In some embodiments, stabilize means aggregating less than about 6% of the antibody under conditions where a solution containing the antibody and the thermal stabilizer is maintained at a temperature of about 45°C for about 28 days. As used herein, the term native means the basic form of the antibody, which is determined by size exclusion chromatography and is typically an intact antibody monomer.

In some embodiments, the thermal stabilizer is a sugar or sugar alcohol selected from sucrose, trehalose and mannitol, or any combination thereof, the amount of which in the preparation can be varied depending on the particular circumstances and purposes for which the compositions are intended to be used. In some embodiments, the preparations can contain from about 3% to about 14% sugar or sugar alcohol; from about 4% to about 13% sugar or sugar alcohol; from about 5% to about 12% sugar or sugar alcohol; from about 6% to about 11% sugar or sugar alcohol; from about 7% to about 10% sugar or sugar alcohol; from about 8% to about 9% sugar or sugar alcohol; from about 4% to about 6% sugar or sugar alcohol; from about 5% to about 7% sugar or sugar alcohol; from about 9% to about 11% sugar or sugar alcohol; or from about 11% to about 13% sugar or sugar alcohol. For example, the pharmaceutical preparations of the present invention may contain 4%±0.6%; 5%±0.75%; 6%±0.9%; 7%±1.05%; 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 (e.g., sucrose, trehalose, or mannitol).

The pharmaceutical compositions of the present invention may also comprise a buffer or buffer system that serves to maintain a stable pH and promotes stabilization of the human PCSK9 antibody. In some embodiments, by stabilizing means aggregating less than about 4.5%±0.5% or less than 6.0%±0.5% of the antibody under conditions where the solution containing the antibody and the buffer is maintained at a temperature of about 45°C for about 28 days. In some embodiments, stabilizing means aggregating less than about 3%±0.5% or less than 2.6%±0.5% of the antibody under conditions where the solution containing the antibody and the buffer is maintained at a temperature of about 37°C for about 28 days. In some embodiments, by stabilizing is meant that at least 91%±0.5% or at least 92%±0.5% of the antibody, as determined by size exclusion chromatography, remains in its native conformation under conditions where a solution containing the antibody and buffer is maintained at a temperature of about 45°C for about 28 days. In some embodiments, by stabilizing is meant that at least 94%±0.5% or at least 95%±0.5% of the antibody, as determined by size exclusion chromatography, remains in its native conformation.

- 9 047546 under conditions where the solution containing the antibody and the buffer is maintained at a temperature of about 37°C for about 28 days. By native or native conformation is meant the proportion of the antibody that has not aggregated or degraded. This is typically determined by an assay that determines the relative particle size of the antibody, such as by size exclusion chromatography. Unaggregated and undegraded antibody elutes in a fraction that is identical to the native antibody and is typically the major fraction of the eluate. Aggregated antibody elutes in a fraction that indicates sizes larger than the native antibody. Degraded antibody elutes in a fraction that indicates sizes smaller than the native antibody.

In some embodiments, by stabilizing is meant that at least 38%±0.5% or at least 29%±0.5% of the antibody, as determined by cation exchange chromatography, remains in its major charged form under conditions where a solution containing the antibody and a buffer is maintained at a temperature of about 45°C for about 28 days. In some embodiments, by stabilizing is meant that at least 46%±0.5% or at least 39%±0.5% of the antibody, as determined by cation exchange chromatography, remains in its major charged form under conditions where a solution containing the antibody and a buffer is maintained at a temperature of about 37°C for about 28 days. By major charge or major charged form is meant the fraction of the antibody that elutes from the ion exchange resin in a major peak flanked on one side by more basic peaks and on the other side by more acidic peaks.

The pharmaceutical formulations of the present invention may have a pH from about 5.2 to about 6.4. For example, the formulations of the present invention may 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.

In some embodiments, the buffer or buffer system comprises at least one buffer with a buffering capacity range that fully or partially overlaps the pH range of 5.5-7.4. In one embodiment, the buffer has a pKa of about 6.0±0.5. In some embodiments, the buffer is a histidine buffer. In some embodiments, the 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 mM 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 some embodiments, the buffer system comprises histidine at a concentration of 10 mM±1.5 mM at a pH of 6.0±0.3.

The pharmaceutical formulations of the present invention may also contain one or more excipients that serve to maintain a low viscosity or to reduce the viscosity of formulations containing high concentrations of the anti-PCSK9 antibody drug substance (e.g., typically >150 mg/mL antibody). In some embodiments, the formulation includes arginine in amounts sufficient to maintain the viscosity of the liquid formulation at a level of less than 20±3 cP, less than 15±2.25 cP, or less than 11±1.65 cP. In some embodiments, the formulations contain arginine in an amount sufficient to maintain a viscosity at a level of no greater than 10.6±1.59 cP. In some embodiments, the pharmaceutical composition of the present invention comprises arginine, preferably in the form of L-arginine hydrochloride at a concentration of 10 mM±1.5 mM to 90 mM±13.5 mM, 20 mM±3 mM to 80 mM±12 mM, 30 mM±4.5 mM to 70 mM±10.5, 40 mM±6 mM to 60 mM±9 mM, or 50 mM±7.5 mM.

Examples of compositions

According to one aspect of the present invention, the pharmaceutical formulation is a generally physiologically isotonic, low-viscosity liquid formulation that 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±26.25 mg/mL; (ii) a buffer system that maintains the pH at about 6.0±0.3; (iii) a sugar that also serves as a thermal stabilizer; (iv) an organic auxiliary solvent that protects the structural integrity of the antibody; and (v) an amino acid salt that serves to maintain a sufficiently low viscosity for injection of a suitable volume for subcutaneous administration.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises a substituted heavy chain of the variable region of the IGHV3-23 type and a substituted light chain of the variable region of the IGLV4-1 type (e.g., mAb-316P) at a concentration of from about 50±7.5 mg/mL to about 175±26.25 mg/mL; (ii) a buffer system containing histidine that maintains a pH of about 6.0±0.3; (iii) sucrose; (iv) a nonionic detergent, such as polysorbate; and in some cases (v) an arginine salt.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises HCDR1 of SEQ

- 10 047546

ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7, and LCDR3 of SEQ ID NO:8, at a concentration of 175±26.25 mg/mL; (ii) histidine at a concentration of 10 mM±1.5 mM, which provides a pH of 6.0±0.3; (iii) sucrose at a concentration of 5% w/v±0.75% w/v; (iv) polysorbate 20 at a concentration of 0.01% w/v±0.0015% w/v; and (v) L-arginine hydrochloride at a concentration of 50 mM±7.5 mM.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises HCDR1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7, and LCDR3 of SEQ ID NO:8, at a concentration of 150+22.5 mg/mL; (ii) histidine at a concentration of 10 mM+1.5 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 10% w/v±1.5% w/v; and (iv) polysorbate 20 at a concentration of 0.2% w/v+0.03% w/v. or 0.01% w/v + 0.0015% w/v.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises HCDR1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7, and LCDR3 of SEQ ID NO:8, at a concentration of about 100+15 mg/mL; (ii) histidine at a concentration of 20 mM+3 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 12% w/v±1.8% w/v; and (iv) polysorbate 20 at a concentration of 0.2% w/v+0.03% w/v. or 0.01% w/v + 0.0015% w/v.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises HCDR1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7, and LCDR3 of SEQ ID NO:8, at a concentration of 50+7.5 mg/mL; (ii) histidine at a concentration of 10 mM+1.5 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 6% w/v+0.9% w/v; and (iv) polysorbate 20 at a concentration of 0.1% w/v+0.015% w/v. or 0.01% w/v + 0.0015% w/v.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:5 at a concentration of 175+26.25 mg/mL; (ii) histidine at a concentration of 10 mM+1.5 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 5% w/v+0.75% w/v; (iv) polysorbate 20 at a concentration of 0.01% w/v+0.0015% w/v; and (v) L-arginine hydrochloride at a concentration of 50 mM+7.5 mM.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:5 at a concentration of about 150+22.5 mg/mL; (ii) histidine at a concentration of 10 mM+1.5 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 10% w/v+1.5% w/v; and (iv) polysorbate 20 at a concentration of 0.2% w/v+0.03% w/v or 0.01% w/v+0.0015% w/v.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:5 at a concentration of about 100+15 mg/mL; (ii) histidine at a concentration of 20 mM+3 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 12% w/v+1.8% w/v; and (iv) polysorbate 20 at a concentration of 0.2% w/v+0.03% w/v or 0.01% w/v+0.0015% w/v.

According to one embodiment, the pharmaceutical composition comprises: (i) a human IgG1 antibody that specifically binds human PCSK9 and that comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:5 at a concentration of about 50+7.5 mg/mL; (ii) histidine at a concentration of 10 mM+1.5 mM, which provides a pH of 6.0+0.3; (iii) sucrose at a concentration of 6% w/v+0.9% w/v; and (iv) polysorbate 20 at a concentration of 0.1% w/v+0.015% w/v or 0.01% w/v+0.0015% w/v.

Additional non-limiting examples of pharmaceutical compositions within the scope of the present invention are disclosed elsewhere herein, including in the illustrative examples provided below.

Stability and viscosity of pharmaceutical formulations

The pharmaceutical formulations of the present invention typically exhibit high levels of stability. The term stable, as used herein with respect to the pharmaceutical formulations, means that the antibodies in the pharmaceutical formulations retain an acceptable level of chemical structure or biological function after storage under specified conditions. A formulation may be stable even though the antibody contained therein does not retain 100% of its chemical structure or biological function after storage for a specified period.

- 11 047546 period of time. Under certain circumstances, retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of an antibody after storage for a certain period of time may be considered stable.

Stability can be measured, among other things, by the detectable percentage of native antibody that remains in the formulation after storage for a certain period of time at a certain temperature. The percentage of native antibody can be determined, among other things, by size exclusion chromatography (e.g., high performance liquid size exclusion chromatography [size exclusion HPLC]), wherein native is defined as non-aggregated and non-degraded antibody. As used herein, the expression "acceptable level of stability" means that after storage for a certain period of time at a given temperature, at least 90% of the native form of the antibody can be detected in the formulation. In some embodiments, after storage for a certain time at a certain temperature, 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. The specified period of time after which stability is measured may 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 target temperature at which the pharmaceutical formulation may be stored when determining stability may be any temperature from about -80°C to about 45°C, such as 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, the pharmaceutical formulation may be considered stable if, after 6 months of storage at 5°C, more than about 95, 96, 97, or 98% of the native antibody is detected by size-exclusion HPLC. The pharmaceutical formulation may be considered stable if, after 6 months of storage at 25°C, more than about 94, 95, 96, 97, or 98% of the native antibody is detected by size-exclusion HPLC. The pharmaceutical formulation may be considered stable if after 28 days of storage at 45°C, more than about 91, 92, 93, 94, 95, 96, 97, or 98% of the native antibody is detected by size-exclusion HPLC. The pharmaceutical formulation may be considered stable if after three months of storage at -20°C, more than about 96, 97, or 98% of the native antibody is detected by size-exclusion HPLC. The pharmaceutical formulation may be considered stable if after three months of storage at -30°C, more than about 96, 97, or 98% of the native antibody is detected by size-exclusion HPLC. The pharmaceutical formulation may be considered stable if after three months of storage at -80°C, more than about 96, 97, or 98% of the native antibody is detected by size-exclusion HPLC.

Stability can be measured, among other things, by the detectable percentage of antibody that forms aggregates in the formulation after storage for a certain period of time at a certain temperature, wherein stability is inversely proportional to the percentage of aggregate formed. The percentage of aggregated antibody can be determined, among other things, by size exclusion chromatography (e.g., high performance liquid size exclusion chromatography [size exclusion HPLC]). As used herein, the expression "acceptable level of stability" means that after storage for a certain period of time at a given temperature, no more than 6% of the antibody can be detected in the formulation in aggregated form. In some embodiments, after storage for a certain time at a certain temperature, no more than about 6, 5, 4, 3, 2, 1, 0.5, or 0.1% of aggregated antibody can be detected in the formulation. The specified period of time after which stability is measured may 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 target temperature at which the pharmaceutical formulation can be stored when determining stability can be any temperature from about -80°C to about 45°C, such as 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, the pharmaceutical formulation can be considered stable if less than about 3, 2, 1, 0.5, or 0.1% of the antibody in aggregated form is detected after six months of storage at 5°C. The pharmaceutical formulation can also be considered stable if less than about 4, 3, 2, 1, 0.5, or 0.1% of the antibody in aggregated form is detected after six months of storage at 25°C. The 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% of the antibody in aggregated form is detected. The pharmaceutical formulation may also be considered stable if after three months of storage at -20, -30, or -80°C, less than about 3, 2, 1, 0.5, or 0.1% of the antibody in aggregated form is detected.

- 12 047546

Stability can be measured, inter alia, by the detectable percentage of antibody that is converted to the more acidic fraction by ion exchange (the acidic form) compared to the major fraction of the antibody (the major charged form), with stability being inversely proportional to the proportion of antibody in the acidic form. Without intending to be bound by theory, deamidation of an antibody can result in an increased negative charge on the antibody and hence a higher acidity compared to non-deamidated antibody (see, e.g., Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99(8):5283-5288). The percentage of acidified antibody can be determined, inter alia, by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [cation exchange HPLC]). As used herein, the expression "acceptable level of stability" means that after storage for a certain period of time at a given temperature, no more than 49% of the antibody in a more acidic form can be detected in the preparation. In some embodiments, an acceptable level of stability means that after storage for a certain period of time at a certain temperature, no more than about 49, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5 or 0.1% of the antibody in an acidic form can be detected in the preparation. The predetermined period of time after which the stability is measured may 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 predetermined temperature at which the pharmaceutical formulation can be stored when determining stability may be any temperature from about -80 °C to about 45 °C, such as storage at about -80 °C, about -30 °C, about -20 °C, about 0 °C, about 4-8 °C, about 5 °C, about 25 °C or about 45 °C. For example, a pharmaceutical formulation may be considered stable if, after three months of storage at -80, -30, 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 the 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 the more acidic form. A pharmaceutical formulation may 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 the 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 is in the more acidic form.

Other methods can be used to evaluate the stability of the compositions of the present invention, such as differential scanning calorimetry (DSC) to determine thermal stability, controlled shaking to determine mechanical stability, and absorbance at about 350 nm or about 405 nm to determine solution turbidity. For example, a composition of the present invention can be considered stable if, after 6 months or more of storage at a temperature of from about 5°C to about 25°C, the change in OD ₄₀₅ of the composition is less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less) compared to the OD ₄₀₅ of the composition at the initial time.

Measuring biological activity or binding affinity of an antibody to its target can also be used to assess stability. For example, a formulation of the present invention can be considered stable if, after storage at, for example, 5, 25, 45°C, etc. for a certain period of time (for example, from 1 to 12 months), the anti-PCSK9 antibody contained in the formulation binds to PCSK9 with an affinity that is at least 90, 95% or more of the binding affinity of the antibody before said storage. Binding affinity can be determined, for example, by ELISA or plasmon resonance. Biological activity can be determined by assaying PCSK9 activity, for example, by interacting a cell that expresses PCSK9 with a formulation containing an anti-PCSK9 antibody. Binding of the antibody to such a cell can be measured directly, for example, by FACS analysis. Alternatively, subsequent PCSK9 system activity may be measured in the presence of the antibody and compared to PCSK9 system activity in the absence of the antibody. In some embodiments, PCSK9 may be endogenous to the cell. In other embodiments, PCSK9 may be ectopically expressed in the cell.

Additional methods for assessing the stability of an antibody in a formulation are provided in the examples below.

Liquid pharmaceutical preparations of the present invention may, in some embodiments, have low or moderate levels of viscosity. As used herein, the term viscosity refers to kinematic viscosity or absolute viscosity. Kinematic viscosity is a measure of the resistive flow of a liquid under the influence of gravity. If two liquids of equal volume are placed in identical capillary viscometers and allowed to flow

- 13 047546 under the action of gravity, a viscous liquid will take longer to flow through a capillary than a liquid with a lower viscosity. For example, if it takes 200 s for one liquid to flow completely out while another liquid takes 400 s, the second liquid is considered twice as viscous as the first on the kinematic viscosity scale. Absolute viscosity, sometimes called dynamic or simple viscosity, is the product of the kinematic viscosity and the density of the liquid (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is lVt, where L is length and T is time. Kinematic viscosity is usually expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm ² /s, which is equal to 1 cSt. Absolute viscosity is expressed in centipoise (cP). In the SI system, the unit of absolute viscosity is the millipascal second (mPa-s), where 1 cP = 1 mPa-s.

As used herein, a low viscosity characteristic for a liquid formulation of the present invention will correspond to an absolute viscosity of less than about 15 cP. For example, a liquid formulation of the present invention will have a low viscosity if, when recorded using standard viscosity measurement techniques, the formulation is found to have an absolute viscosity of about 15 cP, about 14 cP, about 13 cP, about 12 cP, about 11 cP, about 10 cP, about 9 cP, about 8 cP or less. As used herein, a medium viscosity characteristic for a liquid formulation of the present invention will correspond to an absolute viscosity in the range between about 35 cP and about 15 cP. For example, a liquid formulation of the present invention will have a medium viscosity if, when recorded using standard viscosity measurement techniques, the formulation is found to have an absolute viscosity of about 34 cP, about 33 cP, about 32 cP, about 31 cP, about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP, about 21 cP, about 20 cP, about 19 cP, 18 cP, about 17 cP, about 16 cP, or about 15.1 cP.

As is evident from the examples below, the inventors of the present invention have unexpectedly found that by adding arginine to the antibody formulation at a concentration of from about 25 mM to about 100 mM, liquid formulations with low to medium viscosity can be obtained containing high concentrations of the anti-human PCSK9 antibody (e.g., from about 100 mg/mL to at least 200 mg/mL). In addition, it was also found that the viscosity of the formulation can be further reduced by reducing the sucrose content to less than about 10%.

Containers and routes of administration

The pharmaceutical compositions of the present invention can be placed in any container suitable for storing drugs and other medicinal products. For example, the pharmaceutical compositions can be placed in a sealed and sterilized plastic or glass container with a certain volume, such as a vial, ampoule, syringe, cartridge or bottle. Various types of vials can be used to store the compositions of the present invention, including, for example, transparent and opaque (e.g., amber) glass or plastic vials. Likewise, syringes of any type can be used to store or administer the pharmaceutical composition of the present invention.

The pharmaceutical compositions of the present invention can be filled into syringes with a regular tungsten content or syringes with a low tungsten content. It will be appreciated by those skilled in the art that the process of manufacturing glass syringes typically involves using a hot tungsten rod to form an opening in the glass through which liquids will be drawn and dispensed. This process results in the deposition of trace amounts of tungsten on the interior surface of the syringe. Subsequent washing and other manufacturing steps can be used to reduce the tungsten content of the syringe. As used herein, the term regular tungsten content means that the syringe contains at least 500 ppb of tungsten. The term low tungsten content means that the syringe contains less than 500 ppb of tungsten. For example, in accordance with the present invention, a low tungsten content syringe contains less than about 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 parts per billion of tungsten.

Rubber plungers used in syringes, as well as rubber stoppers used to close vials, may be coated to prevent contamination of the drug content of the syringe or vial, or to ensure its stability. Thus, the pharmaceutical compositions of the present invention, according to certain embodiments, can be placed in a syringe with a coated plunger or in a vial that is sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a film of fluorocarbons. Examples of coated stoppers or plungers suitable for use with vials and syringes containing the pharmaceutical compositions of the present invention are given, for example, in U.S. Pat. Nos. 4,997,423; 5,908,686; 6,286,699; and 7,226,554, the contents of which are hereby incorporated by reference in their entirety. Specific examples of coated rubber stoppers and pistons that can be used in the context of the present invention are commercially available under the trade name

- 14 047546 FluroTec® brand, supplied by West Pharmaceutical Services, Inc. (Lionville, Pa.). FluroTec® is an example of a fluorocarbon coating that is used to minimize or prevent drug adhesion to rubber surfaces.

According to certain embodiments of the present invention, pharmaceutical compositions can be placed in a low tungsten syringe with a fluorocarbon coated plunger.

The pharmaceutical compositions can be administered to the patient parenterally, for example by injection (e.g. subcutaneously, intravenously, intramuscularly, intraperitoneally, etc.), or by transdermal, transmucosal, nasal, inhalation or oral administration. For subcutaneous administration of the pharmaceutical composition of the present invention, a wide variety of designs of reusable syringe pens and automatic injection devices can be used. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable injection pens or automatic injection devices that can be used for subcutaneous administration of the pharmaceutical composition of the present invention include, among others, the SOLOSTAR™ injection pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), the SURECLICK™ automatic device (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and the HUMIRA™ injection pen (Abbott Labs, Abbott Park, Ill.).

It is also possible to use a microinjector to deliver the pharmaceutical compositions of the present invention. As used herein, the term microinjector means a subcutaneous administration device designed to slowly infuse large volumes (e.g., up to 2.5 ml or more) of a drug composition over an extended period of time (e.g., about 10, 15, 20, 25, 30 or more minutes). See, e.g., U.S. Patent No. 6,629,949; U.S. Patent No. 6,659,982; and Meehan et al., J. Controlled Release 46:107-116 (1996). Microinjectors are particularly useful for delivering large doses of drug proteins present in high concentrations (e.g., about 100, 125, 150, 175, 200 or more mg/ml) or viscous solutions.

In one embodiment, a liquid pharmaceutical preparation containing about 175 mg/mL±26.25 mg/mL of an anti-PCSK9 antibody is administered subcutaneously in a volume of about 1.14 mL±0.17 mL in a pre-filled syringe. In one embodiment, the syringe is a 1 mL long glass syringe with a 27-G thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle cap. In one embodiment, the syringe is a 1 mL OMPI long glass syringe with a 27-G needle, an FM27 rubber needle cap, and a FLUROTEC® 4023/50 coated rubber plunger.

In one embodiment, a liquid pharmaceutical preparation containing about 150 mg/mL±22.5 mg/mL of an anti-PCSK9 antibody is administered subcutaneously in a volume of about 1 mL±0.15 mL in a pre-filled syringe. In one embodiment, the syringe is a 1 mL long glass syringe with a 27-G thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle cap. In one embodiment, the syringe is a 1 mL OMPI long glass syringe with a 27-G needle, an FM27 rubber needle cap, and a FLUROTEC® 4023/50 coated rubber plunger.

Therapeutic use of pharmaceutical formulations

The pharmaceutical compositions of the present invention are, inter alia, used to treat, prevent or alleviate the symptoms of any disease or disorder associated with PCSK9 activity, including diseases or disorders mediated by PCSK9. Examples of non-limiting diseases and disorders that can be treated or prevented by administering the pharmaceutical compositions of the present invention include various dyslipidemias, such as hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, familial hyperlipidemia, dysbetalipoproteinemia, familial dysbetalipoproteinemia, hypertriglyceridemia and familial hypertriglyceridemia.

Examples

The following examples are intended to provide those skilled in the art with a full disclosure and description of how to formulate and use the methods and preparations of the present invention and are not intended to limit the scope of what the inventors consider to be the subject of their invention. Every effort has been made to ensure accuracy with respect to the variables used (e.g., amounts, temperatures, etc.), but some experimental errors and variations must be taken into account. Unless otherwise indicated, parts are defined as parts in moles, molecular weights, and

- 15 047546 som is called the average molecular weight, the temperature is given in degrees Celsius, and the pressure is at or close to atmospheric pressure.

Initial formulation development steps included screening organic co-solvents, thermal stabilizers, and buffers in liquid and lyophilized formulations of mAb-316P (the invention's anti-PCSK9 antibody) to identify excipients that would be compatible with the protein and enhance its stability while maintaining near-physiological osmolality and low viscosity for intravenous and subcutaneous administration. Buffer conditions were also analyzed to determine the optimal pH for maximizing protein stability.

Example 1: development of anti-PCSK1 mAb-316P.

A variety of buffers, organic co-solvents, and thermal stabilizers were screened to identify excipients that enhance the stability of the PCSK9 antibody. Buffer conditions were also analyzed to determine the optimal pH for maximizing antibody stability. The results of these studies were used to develop a stable liquid formulation as well as a stable lyophilized formulation suitable for clinical use for both intravenous (IV) and subcutaneous (SC) administration. For the lyophilized formulation, a single dual-use formulation was developed that can be reconstituted with sterile water for injection (WFI) to a concentration of either 50 mg/mL for IV or 100 mg/mL for SC administration. Once reconstituted to 50 mg/mL, the drug can be further diluted in an IV infusion bag containing 0.9% sodium chloride for IV injection. For the liquid formulation, the concentration of mAb-316P was prepared at 175 ± 27 mg/mL and 150 ± 23 mg/mL. In one embodiment, 175 ± 27 mg/mL mAb-316P was prepared in 10 ± 1.5 mM histidine (pH 6.0 ± 0.3), 0.01% ± 0.0015% polysorbate 20, 5% ± 0.75% sucrose. In one embodiment, 150 ± 23 mg/mL mAb-316P was prepared 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: buffer and pH of anti-PCSK1 mAb-316P.

For liquid solutions, the effect of pH and buffer type on the stability of PCSK9 antibodies was studied. 2 mg/mL anti-PCSK9 mAb-316P was incubated at 45°C in one of the following 10 mM buffers: acetate (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, each of the 0.35 mL liquid formulations was placed in a 2 mL borosilicate glass type 1 vial with a FLUROTEC® 4432/50 coated butyl rubber stopper. Total recovered mAb-316P was determined using reversed phase chromatography. Percentage of native versus aggregated mAb-316P was determined using size exclusion chromatography. Percentage of acidic and basic mAb-316P was determined using cation exchange chromatography. Maximum protein stability was monitored using size exclusion chromatography (SEC) and cation exchange chromatography (CEC) data for the anti-PCSK9 mAb316P formulation in 10 mM histidine buffer at pH 6.0.

The optimal pH for mAb-316P was then determined by incubating 10 mg/mL mAb-316P at 45°C in histidine buffer over a range from pH 5.5 to pH 6.5. Maximum protein stability was monitored by SEC and COX for mAb-316P formulations in histidine buffer at pH 6.0 (Table 2). The results of these analyses showed that the main pathways of protein degradation are associated with the formation of aggregates, degradation products, and various charged forms. Based on these results, 10 mM histidine buffer at pH 6.0 was selected for the preparation of liquid and lyophilized mAb-316P formulations.

The results of the formulation variant studies indicate that under basic conditions (pH > 6.5), anti-PCSK9 mAb-316P can undergo deamidation in solution. In contrast, at pH < 5.5, an increased rate of formation of molecular weight variants of mAb-316P was observed. Based on these data, the pH of the buffer used for the mAb-316P formulation was maintained between pH 5.7 and pH 6.3. Similar results were obtained for the accelerated stability studies of mAb-316P in this pH range.

Example 3: Selecting stress protection products for shaking.

Various co-solvents were separately studied for their ability to minimize particle formation in mAb-316P-containing mixtures due to shaking stress. Shaking turbidity analysis of the drug product showed an increase in optical density (OD) at 405 nm after 120 min of shaking of a solution containing mAb-316P (0.35 mL of a 25 mg/mL mAb316P solution, 10 mM histidine, pH 6.0 ± 0.2 in a 2 mL type 1 borosilicate glass vial with a FLUROTEC® 4432/50 coated butyl rubber stopper) (Table 3). The formulation with all co-solvents studied prevented an increase in turbidity due to shaking. However, 20% PEG 300, 10% PEG 300, and 20% propylene glycol significantly reduced the thermal stability of mAb316P as determined by SEC (Table 4; same mAb-316P concentration, buffer, and capacity as in the above shake study). Formulations with polysorbate 20, polysorbate 80, Pluronic F68, and PEG 3350 had no noticeable effect on the thermal stability of mAb-316P as determined by SEC and CEC, making these cosolvents suitable for use in αntu-PCSK9 formulations.

- 16 047546 mAb-316P. Polysorbate 20 was chosen as the organic auxiliary solvent for the preparation of lyophilized and liquid formulations of mAb-316P, since it provided high stability characteristics during shaking and thermal stability studies of mAb316P.

Example 4: Selecting protection against thermal stress.

Various excipients were analyzed separately and were selected from a list including sugars, amino acids, and inorganic salts to optimally enhance the thermal stability of mAb-316P. A summary of some of the thermal stabilizers included in the study is provided in Table 5. For these experiments, the thermal stabilizers were added to a solution of 20 mg/mL mAb-316P in 10 mM histidine (0.35 mL in a 2 mL type 1 borosilicate glass vial with a FLUROTEC® 4432/50 coated butyl rubber stopper). Formulations containing sucrose, sorbitol, mannitol, and trehalose showed the lowest degradation of mAb-316P based on SEC analysis. However, an unexpected increase in turbidity was observed in the sorbitol formulations compared to the sucrose, trehalose, and mannitol formulations (Table 5). Although no effect on the formation of the different charge forms of anti-PCSK9 mAb-316P was observed for sucrose, trehalose, and mannitol, mannitol appeared to destabilize the protein over multiple freeze-thaw cycles. Thus, the stability of mAb-316P in sucrose and trehalose formulations was approximately equal.

Example 5: lyophilized composition.

A lyophilized formulation was developed to improve the stability of anti-PCSK9 mAb-316P, particularly with respect to different charge forms, and to increase the maximum delivered concentrations of mAb-316P. Various lyoprotectants were added to 0.7 mL of a 50 mg/mL solution of mAb316P, 10 mM histidine in a 2 mL type 1 borosilicate glass vial with a FLUROTEC® 4432/50 coated butyl rubber stopper, lyophilized, and analyzed for their ability to stabilize the lyophilized mAb-316P upon incubation at 50°C. The lyophilized residue was diluted to 100 mg/mL mAb-316P prior to analysis. The two lyophilized formulations with the highest stability according to the EC and KOC data contained: 1) 6% sucrose or 2) 2% sucrose and 2% arginine. For the preparation of the drug product mAb-316P, 6% sucrose was chosen. Thus, the lyophilized drug product based on anti-PCSK9 mAb-316P was obtained by lyophilization of an optimized buffered aqueous solution of the drug containing 10 mM histidine, pH 6.0±0.1, 0.1% (w/v) polysorbate 20, 6% (w/v) sucrose and 50 mg/ml anti-PCSK9 mAb-316P. The results of the analysis of the stability of such a lyophilized product during storage and stress are presented in Table 7.

The lyophilization cycle of anti-pPCSK9 mAb-316P was designed based on the measured Tg' of the formulation (low-temperature glass transition temperature), which was determined using modulated differential scanning calorimetry at reduced ambient temperature (mDSC). During initial drying, the product temperature should not rise above the Tg', which was measured to be -27.9°C.

To obtain the lyophilized mAb-316P preparation, 5.3 ml of 50 mg/ml mAb-316P, 10 mM histidine (pH 6.0), 0.1% polysorbate 20, 6% sucrose were placed in type 1 glass vials and lyophilized in the following sequence.

1. Temperature of the preparation during loading: 5-25°C.

2. Initial holding: 60 min at 5°C.

3. Freezing speed (time): 0.5°C/min (100 min).

4. Holding: 120 min at -45°C.

5. Vacuum setting: 100 mTorr.

6. Heating rate (time) for primary drying: 0.5°C/min (40 min).

7. Temperature of the preparation during primary drying: -25°C.

8. Primary drying time: 78 hours.

9. Heating rate (time) for secondary drying: 0.2°C/min (300 min).

10. Temperature of the preparation during secondary drying: 35°C.

11. Secondary drying time: 6 hours.

12. Cooling speed (time): 0.5°C/min (20 min).

13. Holding: 60 min at 25°C.

14. Filling with nitrogen gas.

15. Vacuum sealing: 80% of atmospheric pressure (608000 mTorr).

If long-term storage is required after secondary drying and before capping, the temperature in the lyophilizer is set at 2-8°C. The lyophilized drug product obtained during the above-described final cycles had a good appearance, low moisture content (0.3%), dissolution time less than 4 minutes, and no signs of turbidity of the diluted solution.

The appearance of the lyophilized residue did not change if the drug mAb-316P

- 17 047546 (mAb-316P DP) was incubated for 2 months at 50°C or stored for 3 months at 5°C. There was no effect on the pH, appearance, or turbidity of the reconstituted mAb-316P drug and no significant differences in the amounts of mAb-316P recovered. After 2 months of incubation at 50°C, lyophilized mAb-316P drug was 1.1% more degraded by SEC-HPLC and 8.3% more degraded by KOC-HPLC. There was no detectable degradation of lyophilized mAb-316P drug upon storage for 3 months at 5°C. No detectable decrease in bioactivity was observed in the anti-PCSK9 bioassays for any of the stressed samples.

Example 6: liquid and reconstituted preparations of mAb-316P.

There are two methods for reconstituting lyophilized mAb-316P drug product depending on the route of administration. For intravenous administration, mAb-316P drug product is diluted with 5.0 mL sterile WFI to form 5.3 mL of a solution containing 50 mg/mL mAb-316P, 10 mM histidine, pH 6.0, 0.1% (w/v) polysorbate 20, and 6% (w/v) sucrose. For subcutaneous administration, mAb-316P drug product is diluted with 2.3 mL sterile WFI to form 2.7 mL of a solution containing 100 mg/mL REGN727, 20 mM histidine, pH 6.0, 0.2% (w/v) polysorbate 20, and 12% (w/v) sucrose. The volume available for withdrawal is 4.8 ml for IV and 2.0 ml for SC administration; the remainder of the diluted solution 0.7 ml is left in the vial for SC.

Alternatively, liquid mAb-316P is prepared as a liquid formulation without the intermediate lyophilization step. Liquid mAb-316P formulations contain 150 mg/mL (±15%) or 175 mg/mL (±15%) anti-PCSK9 mAb-316P in 10 ±1.5 mM histidine (pH 6.0 ± 0.3), polysorbate 20 at 0.01% ± 0.0015% or 0.2% ± 0.03%, sucrose at 5% ± 0.75% or 10% ± 1.5%, and, for the 175 mg/mL formulation, arginine at 50 ± 7.5 mM.

Ahtu-PCSK9 mAb-316P was stable under sterile filtration. A Millipore MILLIPAK filtration system was used for the preparation of clinical preparations, and a filter of similar composition (Millipore MILLEX DURAPORE) was used for research. Compared with storage in a glass vial, the stability of the composition of the mAb-316P drug (mAb-316P FDS) changed insignificantly when stored in a polypropylene tube, a polystyrene tube, a polycarbonate tube, or a glass vial with a steel ball valve (Table 8).

Example 7: Liquid preparations in pre-filled syringes.

Formulation development studies were conducted to prepare highly concentrated liquid formulations of mAb-316P that could be used in prefilled syringes (PFS) for subcutaneous administration. Results obtained during the development stage of the lyophilized REGN727 formulation demonstrated that the optimal buffer, pH, excipient, and thermal stabilizer were histidine, pH 6.0, polysorbate 20, and sucrose, respectively (see above). These same excipients were used to formulate the 150 mg/mL and 175 mg/mL mAb-316P formulations. To reduce the viscosity of the formulation, arginine was added to the 175 mg/mL mAb-316P formulation. The stress stability of 150 and 175 mg/mL mAb-316P drug formulations was studied in Nuova OMPI 1 mL long glass prefill syringes (PFS) and compared to the stability of 150 and 175 mg/mL drug formulations in glass control vials. Following incubation of 150 or 175 mg/mL mAb-316P drug formulations at 45°C, no differences in physical or chemical degradation were observed between the OMPI PFS and the glass control vial. These data indicate that the 150 and 175 mg/mL anti-PCSK9 mAb-316P drug formulations are stable enough for use in PFS.

Example 8: Stability of mAb-316P drug formulations.

Stability studies were conducted to determine the storage and stress stability of the 150 and 175 mg/mL mAb-316P formulations. Turbidity analysis and RP-HPLC were used to assess the physical stability of mAb316P. Physical stability is defined as the recovery of soluble forms of anti-PCSK9 mAb-316P in solution. Protein loss can be due to protein precipitation or surface adsorption. The presence of particles in solution can be monitored visually or by optical density (OD) measurements at 405 nm (turbidity measurements). In the latter assay, an increase in OD indicates an increase in turbidity due to particulate matter formation. The presence of particulate matter, as measured by OD measurements, indicates that the sample is unable to remain stable. Recovery of mAb-316P is monitored by RP-HPLC. In RP-HPLC analysis, the anti-PCSK9 mAb-316P antibody eluted from a reversed-phase column as a single peak. The concentration of each sample tested was determined from the area of the eluted peak of the mAb316P antibody compared to a calibration curve constructed using mAb316P standards with a fixed protein load.

Chemical stability is determined by the retention of the chemical structure of the anti-PCSK9 antibody (mAb-316P) in the sample. Chemical instability can be largely attributed to the formation of covalently modified forms of the protein (e.g. covalent aggregates, products

- 18 047546 degradation products or different charged forms), as well as non-covalently modified forms of the protein (e.g. non-covalent aggregates). To date, the only reported degradation products of mAb-316P have been compounds that differ in either molecular weight or charge. Higher and lower molecular weight degradation products can be separated from native mAb-316P by SEC-HPLC. The percentage of native mAb-316P in a size-exclusion chromatography method is determined by the ratio of the native peak area to the total area of all peaks of the mAb-316P antibody.

The different charged forms of mAb-316P are separated from native mAb-316P using cation exchange chromatography. Peaks that elute from the KOC HPLC column with retention times before the main peak are labeled as acidic peaks, and those that elute from the KOC HPLC column with retention times after the main peak are labeled as basic peaks. The percentage of mAb-316P that is degraded in the cation exchange chromatography method is determined by the change in the relative percentage of the areas of the main, acidic, and basic peaks compared to the sum of the areas of all mAb-316P peaks.

Accelerated stability testing of mAb-316P was performed by exposing the antibody to various stressors. These tests utilize extreme handling conditions that the drug formulation may be exposed to during drug manufacturing. The mAb-316P formulation was packaged in 5 mL polycarbonate vials for shaking, freeze/thaw cycling, and simulated frozen storage conditions. For high temperature stress stability testing, mAb-316P formulations were packaged in glass vials.

Example 9: Formulation stability studies (FDS) of pharmaceutical products during storage.

The 150 mg/mL mAb-316P formulation (FDS; 0.5 mL in 5 mL polycarbonate vial; 150 mg/mL mAb-316P antibody, 10 mM histidine (pH 6.0), 0.2% polysorbate 20, and 10% sucrose) was physically and chemically stable when stored at <-20°C for 12 months. No detectable loss of mAb-316P or detectable chemical degradation of the antibody was detected by size exclusion chromatography and ion exchange chromatography. More than 97% of the recovered mAb-316P was in the native structure by size exclusion chromatography, and more than 56% of the recovered mAb-316P was in the major charged form by cation exchange chromatography. The results are shown in Table 9.

The formulation of 175 mg/mL mAb-316P (FDS; 0.75 mL in 5 mL polycarbonate vial; 175 mg/mL mAb-316P antibody, 10 mM histidine (pH 6.0), 0.01% polysorbate 20, and 5% sucrose) was physically and chemically stable when stored at <-20°C for 3 months. No detectable loss of mAb-316P or detectable chemical degradation of the antibody was detected by size exclusion chromatography and ion exchange chromatography. More than 96% of the recovered mAb-316P was in the native structure, as determined by size exclusion chromatography, and more than 56% of the recovered mAb-316P was in the major charged form, as determined by cation exchange chromatography. The results are shown in Table 10.

Example 10: studies of the stability of drug formulations under stress.

Stress stability studies were performed for the 150 mg/ml mAb-316P (FDS) formulation (0.35 ml–0.5 ml 150 mg/ml mAb-316P, 10 mM histidine (pH 6.0), 0.2% polysorbate 20, 10% sucrose) and the 175 mg/ml mAb-316P formulation (0.5 ml–1.7 ml 175 mg/ml mAb-316P, 10 mM histidine (pH 6.0), 0.01% polysorbate 20, 5% sucrose, 50 mM arginine). High-temperature studies were conducted in 2 mL type 1 borosilicate glass vials with FLUROTEC® 4432/50 coated butyl rubber stoppers; other studies were conducted in 5 mL polycarbonate vials. The 150 mg/mL and 175 mg/mL anti-PCSK9 mAb-316P formulations were physically and chemically stable after shaking for 2 hours. The solution remained visibly clear, and no protein loss or formation of different molecular weights or different charged species was observed (Tables 11 and 12). MAb-316P was also physically and chemically stable after eight freeze-thaw cycles at -80°C and thawing to room temperature. After eight freeze/thaw cycles, the protein solution remained visibly clear and no protein loss was observed. Analysis by SEC or KOC did not reveal the formation of compounds with different molecular weights (soluble aggregates or degradation products) or different charged forms, respectively.

Although the 150 and 175 mg/mL formulations of anti-PCSK9 mAb-316P were physically stable when incubated at 37°C or 45°C for 28 days, some chemical degradation was observed (Tables 11 and 12). The results of stress testing showed that the major degradation pathways involved the formation of aggregates, degradation products, and various charge species. As expected, the degradation rate of anti-PCSK9 mAb-316P was slower at 37°C than at 45°C. No significant changes in the physical

- 19 047546 the therapeutic or chemical stability of drug formulations of 150 or 175 mg/ml mAb-316P when incubated for 28 days at 25°C.

Example 11: Storage stability of a medicinal product (DP).

The 150 mg/mL mAb-316P formulation contains 10 mM histidine, pH 6.0, 0.01% polysorbate 20, 10% sucrose, and 150 mg/mL α-anti-PCSK9 mAb-316P antibody. The 175 mg/mL mAb-316P formulation contains 10 mM histidine, pH 6.0, 0.01% polysorbate 20, 5% sucrose, 50 mM arginine, and 175 mg/mL anti-PCSK9 mAb-316P antibody. No changes in the physical and chemical stability of 150 mg/mL or 175 mg/mL mAb-316P (DP) were observed when stored at 5°C for 6 months in a pre-filled syringe (PFS; 1 mL OMPI long glass syringe with a 27-gauge thin-wall needle and FM27 rubber needle guard with a FLUROTEC® 4023/50 coated rubber plunger) (Tables 13 and 14). The solutions remained visually clear, no protein loss was observed, and no changes in pH were observed after exposure to the listed stress factors. In addition, no significant changes were observed in compounds with different molecular weights or different charge forms as determined by SEC and COX, respectively.

Example 12: Drug stability (DP) under stress.

The stability of 150 mg/mL mAb-316P and 175 mg/mL mAb-316P formulations was analyzed by incubating pre-filled syringes at 25 and 45°C. Each of the respective formulations was physically stable when incubated for 28 days at 45°C or for 6 months at 25°C (Tables 13 and 14). The solutions remained visually clear, no protein loss was observed, and no change in pH was observed after exposure to the listed stress factors. However, aggregates and various charged forms were detected when the protein was incubated at 45 and 25°C. These stress tests indicate that these are the major degradation pathways for the drug. For the 150 mg/mL drug product, the proportion of mAb-316P aggregates increased by 1.9% after 28 days of incubation at 45°C, and the proportion of acidic forms increased by 19.1%. A decrease in the level of chemical degradation was observed when the protein was incubated at 25°C. An increase in the relative amount of aggregates was observed by 0.8%, and the proportion of acidic forms by 10.3% after 6 months of incubation at 25°C. For the 175 mg/mL drug product, the proportion of mAb-316P aggregates increased by 1.8% after 28 days of incubation at 45°C, and the proportion of acidic forms increased by 17.0%. A decrease in the level of chemical degradation was observed when the protein was incubated at 25°C. An increase in the relative amount of aggregates by 0.7% and acidic forms by 9.4% was observed after 6 months of incubation at 25°C. For both the 150 mg/mL and 175 mg/mL formulations, the proportion of aggregates did not show significant changes in the stability of mAb-316P after 1 month of incubation at 25°C.

Example 13: Filling volumes.

The injectable volume of the prefilled syringe (PFS) containing 150 mg/mL REGN727 is 1.0 mL. The injectable volume of the PFS containing 175 mg/mL REGN727 is 1.14 mL. No overages were included in either PFS because the dead volume of the syringe is negligible (0.005 to 0.01 mL).

Example 14: Stability of mAb-316P during storage in different materials.

Ahtu-PCSK9 mAb-316P was stable under sterile filtration. A MILLIPORE MILLIPAK filtration unit was used for the study and for preparation of clinical preparations. Compared with storage in glass vials, the stability of the 150 and 175 mg/mL mAb-316P drug formulations did not change significantly when stored in a polypropylene tube, a polystyrene tube, a polycarbonate tube, or a glass vial with a stainless steel ring (Tables 15 and 16). Although degradation was observed upon incubation of the drug formulation at 40°C for 14 days, no significant differences in the extent of mAb-316P degradation were observed between the control, glass vial, and exposure to plastic containers and stainless steel.

Example 15 Anti-PCSK9 mAb-316P antibody assay.

At least two lots of mAb-316P (lot 1 and lot 2) were analyzed by size exclusion chromatography coupled with multiple angle laser light scattering (SEC LALS), an analytical technique that allows the molar mass of a protein or glycoprotein to be estimated. Lots 1 and 2 had respective molar masses of 154.5 and 154.6 kDa. In the other lots, molar masses ranged from 154.4 kDa to 154.8 kDa (average approximately 155 kDa) for the major peak that eluted from the SEC support. This major peak accounted for approximately 96.7-99.2% of the total peak area of the protein and corresponds to intact mAb-316P monomer (i.e., native according to the terminology of this document).

mAb-316P was analyzed by capillary isoelectric focusing (CIEF) to determine the isoelectric points of the major constituent isoforms. The pI and average peak area (% of total peak area) for mAb-316P samples by CIEF are shown in Table 17. Each batch contained a major peak (peak 5) with a calculated pI of approximately 8.5, which was present at a concentration of

- 20,047,546 peaks 66.4% and 68.0% in batches 1 and 2, respectively. The predominant form (peak 5) most likely represents the intact, fully glycosylated antibody lacking the C-terminal lysine (i.e., the major charged form according to the terminology of this document).

Mass spectrometric (MS) analysis of the recovered tryptic peptide maps of mAb-316P for batch 1 and batch 2 confirmed the presence of a single glycosylation site, Asn298, in the Fc domain of both batches. The major covalently linked glycan forms of this glycosylation site are listed in Table 18. Overall, both batches were found to contain complex 2antennary glycans, the majority of which are fucosylated at Asn298. However, the relative amount of fucosylated agalactosyl (G0) containing sugar chains in batch 2 was slightly higher than that of such glycoform in batch 1. In contrast, the relative amounts of fucosylated digalactosyl (G2) and fucosylated monogalactosyl (G1) containing sugar chains in batch 2 were lower than that of such sugar chain structures in batch 1. LC/MS analysis of the two drug samples (peak 16) also revealed 2.9 and 8.4% of the heavy chain peptide lacking the glycan site at Asn298 in batch 1 and batch 2, respectively.

Glycan profiles obtained by HPLC after the release of oligosaccharides from each of two batches of mAb-316P were analyzed. In each chromatogram, oligosaccharide derivatives were divided into two main groups: non-fucosylated 2-antennary forms and fucosylated 2-antennary forms. Within each group (fucosylated and non-fucosylated), oligosaccharides were further subdivided into digalactosyl (G2), monogalactosyl (G1), and agalactosyl (G0) forms. Structural assignments of oligosaccharides were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Integration of each peak in the two chromatograms showed that the level of fucosylation in the two batches of mAb-316P was generally quite high, with batches 1 and 2 showing 80.0 and 86.9% fucosylation.

Despite the similar overall fucosylation percentage of the two batches, there were quantitative differences in the relative abundance of each glycan form present in the two batches (Table 19). In batch 1, the peak area percentages of 34.4, 39.6, and 11.7%, respectively, were recorded for the fucosylated glycan structures G0, G1, and G2. In contrast, the peak area percentages in batch 2 were 45.8, 33.3, and 7.1% for the fucosylated glycoform structures G0, G1, and G2, respectively, indicating differences in the degree of galactosylation between the two batches. The high-mannose glycan peak (man5 glycan) (peak 2) was recorded with a relative abundance of 1.8–2.9% compared to the total amount of sugar chain recorded in both batches (Table 19). A total of nine unidentified peaks with peak area percentages between 0.5 and 1.5% were also detected in both lots analyzed by this method, and they represent <3% of the total glycan peak area detected in each lot. Analysis of an equimolar mixture of the two lots revealed no new peaks, and the area percentages for all peaks in the mixture correlated well with the expected values from separate analysis of each lot (Table 19).

Table 1

Effect of buffer and pH on the stability of mAb-316P at 45°C for 28 days

pH/buffer Turbidity ¹ Total (mg/ml) % native 0 % a gr. % home % sour % main without incubation ² 0,00 1,8 98.1 0,3 57,0 33,0 10,0 pH 8.0, Tris 0,00 1,7 88.9 0.8 15.2 82.4 2,4 pH 8.0, phosphate 0,01 2.0 89.4 1,3 n.d. n.D. n.D. pH 7.5, phosphate 0,01 1.9 91.7 0.9 n.d. n.D. n.D. pH 7.0, phosphate 0,01 2,1 92.4 0,7 16.7 78.8 4,5 pH 6.5, phosphate 0,00 2.0 93.2 0,6 25,0 68.3 6,6 pH 6.0, phosphate 0,00 1,8 93.9 0,3 29.8 62.3 8.0 pH 6.0, histidine 0,00 1,9 94.5 0,0 36.9 54.0 9.1 pH 6.0, succinate 0,00 1,9 93.1 0.4 31.9 59.8 8.3 pH 6.0, citrate 0,00 1.9 95.1 0,4 32.1 58.1 9.9 pH 5.5, citrate 0,00 2,1 94.6 0.4 28,0 62,0 9.9 pH 5.5, acetate 0,00 2.0 92.4 0,2 34.5 56.9 8.5 pH 5.0, acetate 0,00 1,8 93,0 0,2 31,1 57.5 11.4

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

² Mean values of unincubated samples for all 12 formulations.

- 21 047546

Table 2

Effect of pH on 10 mg/mL mAb-316P, 10 mM histidine at 45°C for 28 days

pH/buffer Turbidity ¹ Total (mg/ml) % native % agr. % home % sour % main without incubation 2 0,00 9.5 98.1 0,2 57.8 31,1 11,1 pH 5.5 0,01 9.5 94.3 0,5 34.7 52.2 13.1 pH 6.0 0,01 9.7 94.7 0.9 37,5 51.8 10.6 pH 6.5 0,01 10.1 93.4 1,8 35.7 55.2 9.1

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

² Mean values of unincubated samples for all 3 formulations.

Table 3

Effect of auxiliary solvents on 25 mg/mL mAb-316P under shaking for 120 min

Organic auxiliary solvent Turbidity ¹ Total (mg/ml) % native 0 % agr. % home % sour % main No Shaking ² 0,00 25.6 97.3 0,5 50.6 39.2 10.3 Without auxiliary solvent 0,10 25.6 97.3 0,5 51.1 39,0 9.9 0.2% polysorbate 20 0,01 24.7 97.2 0,5 51.1 38.9 10.1 0.2% polysorbate 80 0,01 24.7 97.3 0,5 50.9 38.9 10.2 0.2% Pluronic F68 0,01 25.2 96.9 0,5 50.5 39.2 10.3 3% PEG 3350 0,01 25.3 97.1 0,5 50.7 39,0 10.2 1.5% PEG 3350 0,01 24.9 97.1 0,5 50.8 39.1 10.1 20% PEG 300 0,00 26.7 97,0 0,6 48.8 40,5 10.7 10% PEG 300 0,01 25.7 97.2 0,5 49.9 39.8 10.3 20% propylene glycol 0,01 25.8 96.9 0,5 51.1 38.6 10.3

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

² Average values of samples without shaking for all 9 formulations.

Table 4

Effect of auxiliary solvents on 25 mg/mL mAb-316P incubated at 45°C for 28 days

Organic auxiliary solvent Muddy ¹ rn Total (mg/ml) % native % agr. % home % sour % main without incubation ² 0,00 6.1 25.6 97.3 0,5 50.6 39.2 10.3 Without auxiliary solvent 0,02 6.2 24.9 94.5 0,7 34.8 54.7 10,5 0.2% polysorbate 20 0,03 6.2 24.3 94.6 0,5 35.2 54.3 10,5

- 22 047546

0.2% polysorbate 80 0,02 6.2 24.3 94.8 0,6 35.1 54.5 10.4 0.2% Plutonic F68 0,03 6.1 24.6 94.7 0,6 33.8 55.6 10.6 3% PEG 3350 0,03 6.2 24.8 95,0 0,7 35.9 53.5 10.6 1.5% PEG 3350 0,02 6.2 24.4 94.8 0,6 36,0 53.5 10,5 20% PEG 300 0,12 4.8 25.4 88.8 4.2 n.d. n.D. n.D. 10% PEG 300 0.09 5.4 25,0 93.7 0.9 24.1 67,0 8.9 20% propylene glycol OLYA 0,03 6.1 24.6 89.9 5,6 34.6 54.2 11,2

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

² Average values of samples without shaking for all 9 formulations.

Table 5

Effect of stabilizer on 20 mg/ml mAb-316P, 10 mM histidine at 45°C for 28 days

Excipient Visually Muddy ¹ rn Total (mg/ml) % native % a gr. % main I % sour I am % main without incubation ^{2 3} Good 0,00 6.0 20.2 97.7 0,5 51.1 39.3 9.6 Without stabilizer Good 0,02 6.1 19.8 93.6 2.0 33.5 56.5 10.1 150 mM NaCI Not good 0,03 6.0 20,0 91,0 4.6 35.3 51,0 13.6 20% sucrose Good 0,04 6.0 22.6 94.4 1,0 32.1 57,0 10.9 20% sorbitol Good 0.16 5.8 21.8 94.3 1,0 23.5 67.4 9.1 10% mannitol Good 0,02 6.0 21.2 94.8 0.9 34.4 54.5 11,1 20% trehalose Good 0,05 6.0 22.7 94.7 0,5 33.2 56.6 10.2 5% glycerin Good 0,10 5.9 20.8 90.2 5.4 n.d. n.D. n.D. 3% arginine Good 0,02 6.1 21,1 92.9 3.0 37,5 49.2 13.3 3% glycine Good 0,03 6.1 19.8 93.7 1,7 31,0 58,0 10.9

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

² Average values of unincubated samples for all 9 formulations.

Table 6

Effect of lyoprotectants on lyophilized mAb-316P incubated at 50°C for 28 days

Excipient Visually Muddy ¹ rn Total (mg/ml) % native % agr. % main % sour I am % main without incubation Good 0,00 6.0 100 97.1 1,2 51,0 38.3 10.7 Without Negod 0.09 6.1 100 70,0 28,5 24.2 35.1 40.7 lyoprotector en 2% sucrose Good 0,02 6.1 104 90.7 7.4 37.4 39.1 23.6 6% sucrose Good 0,00 6.1 105 96.1 1,8 46.8 39.2 14.1 2% sugar, 2% Gly Good 0,02 6.0 114 94.5 3,4 40.9 42.4 16.8 2% sugar, 2% Arg Good 0,00 5.9 109 95.9 2.0 47.2 38.4 14.4

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

² Lyophilized drug product diluted to 100 mg/mL REGN727 prior to analysis.

³ Average values for the starting material of 4 compositions.

- 23 047546

Table 7

Stability of lyophilized medicinal product diluted to 100 mg/ml

Storage temperature No storage 5°С 4 months. 25°С 3 months. 40 °C 3 months. 50°С 2 months. Turbidity ¹ 0,00 0,01 0,01 0,02 0,02 pH 6.2 6.3 6.2 6.2 6.2 % of total allocation 100 104 100 98 105 % native 96,0 96.5 96.2 95.7 94.9 % of the aggregate 1,7 1,4 1,7 2,4 2.9 % home 50.6 51.5 49.2 46.2 43.5 % sour 38,0 37.9 38.2 39.5 40.1 % main 11.4 10,5 12.7 14.3 16.2 Bioassay (% stage std.) ² 146 NP NP NP 152

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

TL _ _ _ _ GP ^G\G\P/ _ ___ ____ _ __

Acceptance criteria: 50-200% of the reference standard.

Table 8

Compatibility 50 mg/ml mAb-316P after 14 days at 40°C and 75% humidity

Storage No storage Glass Polypropylene Polystyrene Polycarbonate Stainless steel Turbidity ¹ 0,00 0,00 0,01 0,00 0,01 0,01 % of the amount 100 98 99 104 103 98 % native 97,0 96.3 96.3 96.2 96.2 96.1 % of the aggregate 2.0 1.9 1.9 2.0 2.0 2.0 % home 50.8 45.7 44.9 45.2 45.6 45.5 % sour 38.1 42.8 43.6 43,0 42.5 43.2 % main 11,2 11,5 11.6 11.9 11.8 11,2

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

Table 9

Stability of 150 mg/mL anti-PCSK9 mAb-316P for 12 months at -80°C

Storage temperature Turbidity (OD 405 nm) Control 0.00 -80°С 0,00 -30°С 0.01 -20°С 0.01 % total mAb-316P release 100 104 108 111 % allocation of native 97.5 97.3 97.2 97.2 % allocation of aggregates 1,7 1,8 1,8 1.9 % highlighting of the main 56.2 56.5 56.4 56.3 % acid secretion 26.5 25.7 25.7 25.4 % allocation of the main 17.4 17.9 17.9 18.2

Table 10

Stability of 175 mg/ml anti-PCSK9 mAb-316P for 3 months at -80°C

Storage temperature Control -80°С -30°С -20°С Turbidity (OD 405 nm) ¹ 0,00 0,01 0,01 0,01 % Total Recovery of REGN727 (RP HPLC) 100 100 104 101 % recovery of native REGN727 (SEC HPLC) 96.2 96.5 96.4 96.3 % recovery of aggregated REGN727 (SEC HPLC) 2.7 2,5 2.6 2.7 % recovery of the main REGN727 (KOH HPLC) 58.5 56.2 56.6 56.7 % recovery of acidic REGN727 (KOH HPLC) 29.4 29.4 29.4 29.4 % recovery of the main REGN727 (KOH HPLC) 12.2 14.5 14,0 13.9

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

- 24 047546

Table 11

Stability of 150 mg/ml mAb-316P FDS ¹ under stress conditions

Stress test No stress a ² Shake it 45°C incubation 37°C incubation 25°C incubation Zamor,/otta willow. Time of exposure to IA 0 min 60 min 120 min 14 days 28 days 14 days 28 days 14 days 28 days 8 cycles Appearance Good Good Goden Good Good Good Good Good Good Good Turbidity ³ 0,00 0,00 0,00 0,02 0,03 0,00 0,01 0,00 0,00 0,00 pH % total 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 O 100 101 102 98 98 99 98 100 99 101 selection % selection 97.4 97.5 97.2 94.2 92.4 95.7 95.1 96.7 96.2 97.1 native % allocation 1,6 1,7 2,1 3,4 4.1 2,4 2.6 2.0 2,2 1,7 aggregates % highlighting the main % 53.6 53.7 54.7 38.7 29,0 46.3 39.5 51.5 49.1 53.9 acid secretion 27.2 26.1 25.9 39.5 48.6 31.9 36.9 27.8 28.9 26.5 % allocation of the main 19.3 20.2 19,5 21.8 22.5 21.8 23.7 20.7 22,0 19.6 Bioassay (% relative act.) ⁴ 84 NP 84 NP 85 NP 82 NP 98 81

¹ 10 mM histidine, pH 6.0, 0.2% polysorbate 20, 10% sucrose, 150 mg/ml αanti-PCSK9 mAb-316P.

² Unstressed values represent the average of both stability studies.

³ Turbidity is defined as the relative change in OD at 405 nm compared to the parent substance.

⁴ Percentage of relative activity; acceptance criteria: 50-200% of the reference standard.

Table 12

Stability of 175 mg/mL mAb-316P FDS ¹ under stress conditions

Stress test No Stress ² Shaking 45°C incubation 37°C incubation 25°C incubation Frozen/thawed. Time 0 60 120 14 days 28 days 14 days 28 days 14 31 days 8 impacts min min min day cycles Appearance Good Good Good Good Good Good Good nd Good Good

- 25 047546

Turbidity ³ pH 0,00 6,0 0,00 6,0 0,01 6,0 0,01 6,1 0.03 6.1 0,00 6,0 0,01 6,0 n.D. n.D. 0,00 6,0 0,01 6,0 % of total 100 96 98 101 98 100 96 n.D. 99 98 allocation % allocation 96.5 96.3 96.4 94.5 91.7 95.7 94.7 n.D. 96.2 96.1 native % selection 2,5 2,5 2,5 3.6 5.2 2.8 3.0 n.D. 2.6 2,3 aggregates % allocation 59.5 58.3 59,0 47.1 38.4 56.3 46.9 n.D. 58.2 59.3 main % selection 30.1 29.3 29.4 39.5 47.6 32.3 39.8 N.D. 30.9 29,5 acid % discharge 10.3 12.4 11.6 13.4 14.1 11.4 13.4 N.D. 11,0 11,1 Main Bioassay (% rel. 0,00 0,00 0,01 0,01 0,03 0,00 0,01 N.D. 0,00 0,01

act.) ⁴ _____________________________________________________________________________________________________________________ ¹ 10 mM histidine, pH 6.0, 0.01% polysorbate 20, 5% sucrose, 50 mM arginine, 175 mg/ml anti-PCSK9 mAb-316P.

² Unstressed values represent the average of both stability studies.

³ Turbidity = change in OD at 405 nm compared to the parent substance.

⁴ Percentage of relative activity; acceptance criteria: 50-200% of the reference standard.

Table 13

Stability of 150 mg/mL mAb-316P DP in PFS

Storage 0 5°С 6 months. 25°С 6 months. 45°С 28 days Appearance Good Good Good Good Turbidity ¹ 0,00 0,00 0,00 0,02 rn 6.1 6.1 6.1 6.1 % of total allocation 100 102 102 97 % allocation of native 96.6 96.1 94.2 92.4 % allocation of aggregates 2,4 2.6 3.2 4.3 % highlighting of the main 58.4 58.5 45.7 35.8 % acid secretion 31.8 31.3 42.1 50.9 % allocation of the main 9.8 10.2 12.2 13.4 ¹ Turbidity = change OD on 405 nm by comparison with the original

substance.

Table 14

Stability of 175 mg/mL mAb-316P DP in PFS

Storage 0 5°С 6 months. 25°С 6 months. 45°С 28 days Appearance Good Good Good Good Turbidity ¹ 0,00 0,00 0,02 0,04 rn 6.1 6.1 6.1 6.1 % of total allocation 100 103 101 100 % allocation of native 96.7 96.3 94.6 91.6 % allocation of aggregates 2,3 2,4 3.0 5.4 % highlighting of the main 59.1 59.7 47.1 37.7 % acid secretion 31.2 30.6 40.6 48.2 % allocation of the main 9.7 9.7 12.3 14.2

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

- 26 047546

Table 15

Compatibility 150 mg/ml mAb-316P1 after 14 days at 40°C

Storage No storage Glass Glass Polycarbonate Polypropylene Polystyrene Stainless steel Turbidity ¹² 0,00 0,00 0,00 0,01 0,01 0,01 pH 6.0 6.1 6.0 6.0 6.0 6.0 % of total allocation 100 97 104 99 105 104 % allocation of native 97.4 95.9 95.8 95.7 95.7 95.5 % of total allocation 1,7 2,5 2.6 2.7 2.6 2.7 % Main Recovered 53.3 45.8 45.8 44.7 45.6 45.1 % Acid Recovered 26.9 33,0 32.9 33.8 33,0 33.5 % Basic Recovered 19.8 21.3 21.6 21.6 21,5 21.4

¹ 10 mM histidine, pH 6.0, 0.2% polysorbate 20, 10% sucrose, 150 mg/ml anti-mAb-.

¹ Turbidity = change in OD at 405 nm compared to the parent substance.

Table 16

Compatibility 175 mg/ml mAb-316P1 after 14 days at 40°C

Storage No storage Glass Glass Polycarbonate Polypropylene Polystyrene Stainless steel Turbidity ³ 0,00 0,00 0,00 0,00 0,00 0,01 RN 6.1 6.1 6.1 6.0 6.1 6.1 % of total allocation 100 98 104 100 105 98 % allocation of native 96.6 95.3 95.2 95.1 95.2 94.9 % allocation of aggregates 2,4 3.0 3.1 3.2 3.1 3,3 % highlighting of the main 57.7 51.3 51,0 50.6 51,0 50.4 % acid secretion 30,0 34.5 34.5 35,0 34.5 35.2 % allocation of the main 12.3 14.2 14.5 14.4 14.4 14.4

¹ 10 mM histidine, pH 6.0, 0.01% polysorbate 20, 5% sucrose, 50 mM arginine and 175 mg/mL mAb ² Turbidity = change in OD at 405 nm compared to parent compound. Table 17

Heterogeneity of mAb-316P charge according to KIEF data

Party 1 Part 2 Batch 1:Batch 2 (1:1 mix) Peak No. r| Peak area, % r| Peak area, % r| Peak area, % 1 7.99 (0.01) 0.8(0.1) 7.99 (0.01) 0.8 (0.0) 7.98 (0.01) 0.7 (0.1) 2 8.15 (0.00) 2.6 (0.1) 8.15(0.01) 2.6 (0.1) 8.14(0.01) 2.6 (0.1) 3 8.29 (0.00) 6.9(0.1) 8.29 (0.00) 7.0 (0.0) 8.28 (0.01) 6.9 (0.1) 4 8.42 (0.00) 18.4 (0.2) 8.42 (0.00) 18.6 (0.2) 8.42 (0.01) 18.5 (0.1) 5 8.54 (0.01) 66.4 (0.3) 8.55 (0.01) 68.0 (0.3) 8.54 (0.00) 67.3 (0.1) 6 8.65 (0.00) 4.0(0.1) 8.65 (0.00) 2.8 (0.1) 8.64 (0.01) 3.4 (0.0) 7 8.82 (0.00) 0.9(0.1) 8.81 (0.00) 0.3 (0.1) 8.81 (0.01) 0.6 (0.1)

- 27 047546

Table 18

Glycosylated peptides mAb-316P

Heavy Received Mass (Yes)

No. chain peak Fragment Lot 1 t Comments Party 2 15a 294-302 2957,15 2957,19 (Fuc)i(GlcNAc)2(Man) ₃ (GlcNAc)2(Gal)2 294-302 2795,11 2795,11 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc) ₂ (Gal)i 294-302 2404.94 2404.94 (GlcNAc) ₂ (Man)5 294-302 2592,02 2591,96 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc)i(Gal)i 294-302 2267,94 2267,92 (Fuc)i(GlcNAc) ₂ (Man) ₂ (GlcNAc)i 294-302 2121,82 2121,80 (GlcNAc) ₂ (Man) ₂ (GlcNAc)i 15b 294-302 2283,91 2283,91 (GlcNAc) ₂ (Man) ₃ (GlcNAc)i 294-302 2429,98 2429,98 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc)i 294-302 2486,99 2486,97 (GlcNAc) ₂ (Man) ₃ (GlcNAc) ₂ 294-302 2633,05 2633,06 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc) ₂ 16 294-302 1188,52 1188,53 non-glycosylated NST site 17a 290-302 3439,44 3439,44 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc) ₂ (Gal)2 290-302 3278,40 3277,35 (Fuc)i(GlcNAc)2(Man) ₃ (GlcNAc) ₂ (Gal)i 290-302 2887,20 2887,26 (GlcNAc) ₂ (Man)5 17b 290-302 2766,24 2766,21 (GlcNAc) ₂ (Man) ₃ (GlcNAc)i 290-302 2969,34 2969,25 (GlcNAc) ₂ (Man) ₃ (GlcNAc) ₂ 290-302 2912,28 2912,25 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc)i 290-302 3115,32 3115,35 (Fuc)i(GlcNAc) ₂ (Man) ₃ (GlcNAc) ₂

Table 19

Integrated peak areas of glycans, according to capillary electrophoresis data

Peak area of batch ^a % Peak area of batch 2, % Peak area of batch mixture 1:1, % Identification of glycan ¹ Glycan ² structure 1 8.6 (0.2) 6.8(0.1) 7.6 (0.2) G0-Fuc U 2 1.8 (0.0) 2.9(0.1) 2.3 (0.1) Map5 3 0.7 (0.0) 0.5 (0.0) 0.6 (0.0) A small peak was detected 4 Below PO Below PO Below PO A small peak was detected 5 Below PO Below PO Below PO A small peak was detected 6 34.4 (0.1) 45.8 (0.2) 40.4 (0.2) G0 7 1.5 (0.0) 0.7 (0.0) 1.1 (0.0) A small peak was detected 8 Below PO 0.9 (0.0) 0.6 (0.0) A small peak was detected 9 Below PO 0.7 (0.0) 0.6 (0.0) A small peak was detected 10 29.5(0.1) 24.7 (0.0) 27.0(0.1) G1 (1-6) 11 10.1 (0.1) 8.6 (0.0) 9.3 (0.1) G 1(1-3) and/or G2-Fuc 12 Below PO Below PO Below PO A small peak was detected 13 Below PO Below PO Below PO A small peak was detected 14 11.7(0.0) 7.1 (0.1) 9.4 (0.2) G2

¹ G#-fuc and G# denote non-fucosylated and fucosylated glycans, respectively. The symbol # represents 0, 1, or 2.

² Glycan designation: ▼ fucose, N-acetylglucosamine, · mannose, ♦ galactose.

LO = limit of determination.

Claims (22)

1. A kit for treating, preventing or mitigating a disease or disorder associated with human proprotein convertase subtilisin/kexin type 9 (PCSK9), comprising a liquid pharmaceutical formulation, a container and instructions, wherein the liquid pharmaceutical formulation comprises: (a) from 50±7.5 mg/ml to 250±37.5 mg/ml of an antibody or antigen-binding fragment thereof that specifically binds human proprotein convertase subtilisin/kexin type 9 (PCSK9), wherein the antibody comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7, and LCDR3 of SEQ ID NO:8; (b) from 5±0.75 mM to 50±7.5 mM histidine (pH 6.0±0.3); (c) 0.01±0.0015% w/v polysorbate 20; and (d) 10±1.5% sucrose; wherein the container comprises a syringe, wherein the syringe is pre-filled with a liquid pharmaceutical composition. 2. The kit according to item 1, wherein the syringe is a syringe with a normal tungsten content. 3. The kit of claim 1, wherein the syringe is a low tungsten content syringe. 4. The kit according to claim 1, wherein the syringe comprises a coated plunger. 5. The kit according to item 4, wherein the coated piston is coated with a fluorocarbon film. 6. The kit of claim 1, wherein the syringe is a low tungsten syringe, and wherein the syringe comprises a coated plunger. 7. The kit according to claim 1, wherein the syringe is a long glass syringe with a volume of 1 ml, containing a thin-walled needle of size 27-G, a rubber plunger with a fluorocarbon coating and a rubber needle cap. 8. The kit according to claim 1, wherein the antibody comprises a heavy chain variable domain (HCVD) comprising SEQ ID NO:1 and a light chain variable domain (LCVD) comprising SEQ ID NO:5. 9. The kit according to claim 1, wherein more than 90% of the antibodies have a molecular weight of 155 kDa±1 kDa; more than 50% of the antibodies have an isoelectric point of about 8.5; and from 75 to 90% of the antibodies are fucosylated. 10. The kit of claim 1, wherein at least 91% of the antibody has a native conformation after 28 days at 45°C. 11. The kit of claim 1, wherein at least 35% of the antibody remains in the major charged form after 28 days at 45°C. 12. The kit of claim 1, wherein at least 94% of the antibody has a native conformation after 6 months at 25°C. 13. The kit of claim 1, wherein at least 45% of the antibody remains in the major charged form after 6 months at 25°C. 14. The kit of claim 1, wherein at least 96% of the antibody has a native conformation after 6 months at 5°C. 15. The kit of claim 1, wherein at least 58% of the antibody remains in the major charged form after 6 months at 5°C. 16. The kit of claim 1, wherein at least 96% of the antibody remains in the major charged form after three months at -20, -30 or -80°C. 17. The kit of claim 1, wherein at least 56% of the antibody remains in the major charged form after three months at -20, -30 or -80°C. 18. The kit of claim 1, wherein the composition contains approximately 175 mg/ml of antibody. 19. The kit of claim 1, wherein the composition contains approximately 150 mg/ml of antibody. 20. The kit of claim 1, wherein the composition contains approximately 100 mg/ml of antibody. 21. The kit of claim 1, wherein the composition contains approximately 75 mg/ml of antibody. 22. The kit of claim 1, wherein the composition contains approximately 50 mg/ml of antibody.