Granted Patent B2

US 12,312,394 · App. 17/254,508 · Granted May 27, 2025

Inventors: Hunter F. Malanson (Wallingford, CT); Kyle A. Zingaro (Southington, CT); Anjil Giri (Hamden, CT); Justin Weaver (Manchester, CT); Abraham Friedman (West Hartford, CT); Jeffrey William Hunter (Wallingford, CT); Saranya Sivanandam (Norwalk, CT); Jeffrey Zugates (Hamden, CT); Rahul Godawat (Woodbridge, CT)

Assignee: Alexion Pharmaceuticals, Inc.

C07K16/065B01D15/362B01D15/363B01D15/3809C07K16/18C07K2317/565C07K2317/92C07K2317/94

Abstract

Claims (174)

1. A method of producing an anti-C5 antibody, wherein the method comprises

a. culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium;

b. a recovery step comprising filtering the cell culture production medium through a depth filter;

c. purification by Protein A affinity chromatography;

d. a low pH viral inactivation step;

e. purification by cation exchange chromatography;

f. purification by anion exchange chromatography;

g. a virus reduction filtration step; and

h. a concentration and diafiltration step, wherein the concentration and diafiltration step involves using a polysorbate 80 buffer,

wherein the anti-C5 antibody comprises CDR1, CDR2 and CDR3 heavy chain sequences as set forth in SEQ ID NOs: 19, 18 and 3, respectively, and CDR1, CDR2 and CDR3 light chain sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively, and

wherein steps a. to h. are performed sequentially in order.

2. The method of claim 1 , wherein the anti-C5 antibody:

(a) comprises the heavy chain variable region set forth in SEQ ID NO:12 and the light chain variable region set forth in SEQ ID NO:8;

(b) comprises a heavy chain constant region set forth in SEQ ID NO:13;

(c) comprises a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14 and a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:11;

(d) binds to human C5 at pH 7.4 and 25° C. with an affinity dissociation constant (KD) that is in the range 0.1 nM≤KD≤1 nM;

(e) binds to human C5 at pH 6.0 and 25° C. with a K D ≥10 nM; and/or

(f) is ravulizumab.

3. The method of claim 1 , wherein the depth filter is a two-step depth filtration train.

4. The method of claim 1 , wherein processing conditions for the recovery step include one or more of the following:

a. a D0HC depth filter load of ≤100 L/m 2 in the Normal Operating Range and ≤100 L/m 2 in the Proven Acceptable Range;

b. an A1HC depth filter load of ≤200 L/m 2 in the Normal Operating Range and ≤200 L/m 2 in the Proven Acceptable Range;

c. a 0.5/0.2 μm filter load of ≤800 L/m 2 in the Normal Operating Range and ≤800 L/m 2 in the Proven Acceptable Range;

d. a harvest load temperature of 18°-37° C. in the Normal Operating Range and 15-37° C. in the Proven Acceptable Range;

e. a buffer chase volume of 20-25 L/m 2 in the Normal Operating Range and 0-30 L/m 2 in the Proven Acceptable Range;

f. a clarified harvest hold time of ≤10 Days in the Normal Operating Range and ≤16 Days in the Proven Acceptable Range;

g. a yield of ≥70%;

h. a total filtration time of <3.3 hours;

i. a bioburden of <3 CFU/10 mL; and/or

j. an endotoxin of <5 EU/mL.

5. The method of claim 1 , wherein the Protein A affinity chromatography is Protein A affinity chromatography with modified resin, and wherein the Protein A affinity chromatography with modified resin comprises one or more buffers selected from the group consisting of:

a. 0.1 N sodium hydroxide for sanitization;

b. 20 mM Tris and 65 mM sodium chloride at a pH of 7.6 for equilibration and Post-Load Wash 1;

c. 50 mM sodium phosphate, 100 mM sodium chloride, and 300 mM arginine hydrochloride at a pH of 6.0 for Post-Load Wash 2;

d. 20 mM Tris and 65 mM sodium chloride at a pH of 7.6 for Post-Load Wash 3;

e. 25 mM sodium acetate at a pH of 3.75 for elution;

f. 100 mM acetic acid for stripping;

g. Water For Injection (WFI) for flushing; and/or

h. 20% ethanol for storage.

6. The method of claim 1 , wherein processing conditions for the Protein A affinity chromatography include one or more of the following:

a. a pre-batch sanitization hold time of 30-60 minutes in the Normal Operating Range and 30-75 minutes in the Proven Acceptable Range;

b. a post-batch sanitization hold time of 30-60 minutes in the Normal Operating Range and 30-75 minutes in the Proven Acceptable Range;

c. column cycles of ≤100 in the Normal Operating Range and ≤100 in the Proven Acceptable Range;

d. an eluate hold time of ≤7 days in the Normal Operating Range and ≤10 days in the Proven Acceptable Range;

e. a step yield of ≥70%;

f. an eluate pre-filtration bioburden of <50 CFU/10 mL;

g. an eluate post-filtration bioburden of <3 CFU/10 mL; and/or

h. an eluate post-filtration endotxin of <5 EU/mL.

7. The method of claim 1 , wherein the low pH viral inactivation step comprises subjecting an eluated pool from the Protein A affinity chromatography purification step to low pH conditions.

8. The method of claim 7 , wherein the low pH is within a range of 3.60-3.70.

9. The method of claim 7 , wherein the method includes treating the eluted pool with acetic acid.

10. The method of claim 1 , wherein processing conditions for the low pH viral inactivation step include one or more of the following:

a. an acidification pH immediately after titration of 3.60-3.70 in the Normal Operating Range and 3.55-3.80 in the Proven Acceptable Range;

b. an acidification pH immediately after hold time of 3.60-3.75 in the Normal Operating Range and 3.55-3.80 in the Proven Acceptable Range;

c. a hold time at low pH of 60-120 minutes in the Normal Operating Range and ≥60-360 minutes in the Proven Acceptable Range;

d. a hold time at neutralized pH prior to 0.5/0.2 μm filtration of 60-120 minutes in the Normal Operating Range and ≥60 minutes in the Proven Acceptable Range;

e. a filtered neutralized product hold time of ≤7 days in the Normal Operating Range and ≤7 days in the Proven Acceptable Range;

f. a yield of ≥90%;

g. a neutralized pre-filtration pool bioburden of <50 CFU/10 mL;

h. a neutralized post-filtration pool bioburden of <3 CFU/10 mL; and/or

i. a neutralized post-filtration pool endotoxin of <5 EU/mL.

11. The method of claim 1 , wherein neutralized filtrate from the low pH viral inactivation step is loaded onto a cation exchange column.

12. The method of claim 11 , wherein the cation exchange column is a POROS HS50 cation exchange column.

13. The method of claim 1 , wherein the cation exchange step comprises using one or more buffers selected from the group consisting of:

a. 50 mM sodium acetate at a pH of 5.0 for equilibration and Post-Load Wash 1;

b. 50 mM sodium acetate and 60 mM sodium chloride at a pH of 4.9 for Post-Load Wash 2;

c. 50 mM sodium acetate, 90 mM arginine hydrochloride, and 30 mM sodium chloride at a pH of 5.0 for elution;

d. 2.0 M sodium chloride for stripping;

e. 1.0 N sodium hydroxide for sanitization; and/or

f. 0.1 N sodium hydroxide for storage.

14. The method of claim 1 , wherein processing conditions for the cation exchange step include one or more of the following:

a. a load capacity of 22-45 g/L in the Normal Operating Range and 15-50 g/L in the Proven Acceptable Range;

b. a temperature of 15-25° C. in the Normal Operating Range 13-27° C. in the Proven Acceptable Range;

c. an elution buffer pH of 4.90-5.10 in the Normal Operating Range and 4.90-5.10 in the Proven Acceptable Range;

d. an elution buffer conductivity of 11.1-13.6 mS/cm in the Normal Operating Range and 11.1-13.6 mS/cm in the Proven Acceptable Range;

e. an elution flow rate of 150-300 cm/hr in the Normal Operating Range and 120-330 cm/hr in the Proven Acceptable Range;

f. an eluate hold time of ≤7 days in the Normal Operating Range and ≤10 days in the Proven Acceptable Range;

g. column cycles of ≤100 in the Normal Operating Range and ≤100 in the Proven Acceptable Range;

h. an eluate post-filtration bioburden of <3 CFU/10 mL;

i. an eluate post-filtration endotoxin of <5 EU/mL;

j. a step yield of ≥58%; and/or

k. an elution volume of 2.3-5.0 column volumes.

15. The method of claim 13 , wherein the pooled eluate from the cation exchange step is adjusted to a pH of 8.00 and a conductivity of 8.5 mS/cm with 100 mM Tris, 180 mM arginine at a pH of 9.0 and Water For Injection.

16. The method of claim 15 , wherein the adjusted pooled eluate from the cation exchange step is loaded on an anion exchange column within 24 hours of the adjustment.

17. The method of claim 1 , wherein the anion exchange step comprises one or more buffers selected from the group consisting of:

a. 100 mM Tris and 180 mM arginine at a pH of 9.0 for load pH adjustment;

b. Water For Injection for load conductivity adjustment and flush;

c. 2 M sodium chloride for conditioning;

d. 20 mM Tris and 65 mM sodium chloride at a pH of 7.6 for equilibration and post-load chase;

e. 2 M sodium chloride for post-load elution stripping;

f. 1.0 N sodium hydroxide for sanitization; and/or

g. 0.1 N sodium hydroxide for storage.

18. The method of claim 1 , wherein processing conditions for the anion exchange step include one or more of the following:

a. a load pH of 7.90-8.10 in the Normal Operating Range and 7.80-8.20 in the Proven Acceptable Range;

b. a load conductivity of 8.0-9.0 mS/cm in the Normal Operating Range and 7.0-10.0 mS/cm in the Proven Acceptable Range;

c. a load capacity pH of 25-90 g/L in the Normal Operating Range and 25-100 g/L in the Proven Acceptable Range;

d. a hold time of ≤1 day in the Normal Operating Range and ≤4 days in the Proven Acceptable Range;

e. a product hold time of ≤4 days in the Normal Operating Range and ≤6 days in the Proven Acceptable Range;

f. column cycles of ≤100 in the Normal Operating Range and ≤100 in the Proven Acceptable Range;

g. a post-filtration eluate bioburden of <3 CFU/10 mL; and/or

h. a post-filtration eluate endotoxin concentration of <5 EU/mL; and

i. a product yield of ≥67%.

19. The method of claim 1 , wherein flow-through filtrate from the anion exchange step is filtered to remove viruses or virus-like particles.

20. The method of claim 19 , wherein the virus filtration step comprises a pre-flush with Water For Injection, and/or equilibration and a post-loading chase using 20 mM Tris (pH 7.6) and 65 mM sodium chloride.

21. The method of claim 1 , wherein processing conditions for the virus filtration step include one or more of the following:

a. a virus filter differential pressure during load and chase of 21-32 psid in the Normal Operating Range and 21-35 psid in the Proven Acceptable Range;

b. a total pause time during load and chase of 0 minutes in the Normal Operating Range and ≤120 minutes in the Proven Acceptable Range;

c. a chase volume of ≤15 L/m 2 in the Normal Operating Range and ≤20 L/m 2 in the Proven Acceptable Range;

d. passing of a post-use integrity test;

e. a load concentration of 3.0-6.0 g/L in the Normal Operating Range and ≤6.7 g/L in the Proven Acceptable Range;

f. a virus filter load of ≤700 L/m 2 in the Normal Operating Range and ≤1200 L/m 2 in the Proven Acceptable Range;

g. a virus filter load of ≤700 L/m 2 in the Normal Operating Range and ≤700 L/m 2 in the Proven Acceptable Range;

h. a product hold time of ≤4 days in the Normal Operating Range and ≤6 days in the Proven Acceptable Range;

i. a pre-filtration bioburden of <3 CFU/10 mL;

j. an endotoxin concentration of <2 EU/mL;

k. passing of a pre-use integrity test; and/or

l. A processing time of ≤12 hours; and/or

m. a step yield of ≥90%.

22. The method of claim 1 , wherein a pool from the virus filtration step is:

(a) concentrated and diafiltered; or

(b) ultrafiltrated and concentrated to 55 g/L using 30 kDa molecular weight cut-off ultrafiltration membranes; diafiltered with 6 diafiltration volumes into a formulation buffer comprising 10 mM sodium phosphate and 150 mM sodium chloride at a pH of 7.0; and measured and diluted to a product concentration of 10.0 g/L.

23. The method of claim 22 , wherein the diluted product is 0.5/0.2 μm filtered and Polysorbate 80 is added to a diluted product pool to achieve a final concentration of 0.02% (w/v) Polysorbate 80.

24. The method of claim 1 , wherein the ultrafiltration and diafiltration steps comprise the use of one or more buffers selected from the group consisting of:

a. Water For Injection as a flush;

b. 0.5 M sodium hydroxide for sanitization;

c. 10 mM sodium phosphate and 150 mM sodium chloride at a pH of 7.0 for equilibration, diafiltration, chase and pool dilution;

d. 0.1 M sodium hydroxide for storage; and/or

e. 10% (w/v) Polysorbate 80 for excipient.

25. The method of claim 1 , wherein processing conditions for the ultrafiltration and diafiltration steps include one or more of the following:

a. a dilution of within 1% of calculated volume in the Normal Operating Range and within 3% of calculated volume in the Proven Acceptable Range;

b. 10% (w/v) Polysorbate 80 is 0.19-0.21% (w/v) of diluted ultrafiltration/diafiltration product in the Normal Operating Range and 0.17-0.23% (w/v) of diluted ultrafiltration/diafiltration product in the Proven Acceptable Range;

c. an un-formulated drug substance pH of 6.5-7.5;

d. a diluted ultrafiltration/diafiltration product concentration of 9.0-11.0 mg/ml;

e. passing of a pre-use integrity test;

f. a membrane loading of 100-500 g/m 2 in the Normal Operating Range and 50-600 g/m 2 in the Proven Acceptable Range;

g. a feed flux of 240-420 LMH in the Normal Operating Range and 180-440 LMH in the Proven Acceptable Range;

h. a transmembrane pressure of 10-30 psi in the Normal Operating Range and within 8-35 psi in the Proven Acceptable Range;

i. a pressure of 15-25° C. in the Normal Operating Range and 12-30° C. in the Proven Acceptable Range;

j. a fed batch ratio of 1-3 in the Normal Operating Range and 1-5 in the Proven Acceptable Range;

k. a concentration at end of ultrafilitration target of 13-17 g/L in the Normal Operating Range and 12-20 g/L in the Proven Acceptable Range;

l. A diavolume of 5.5-7.0 in the Normal Operating Range and 4.5-7.0 in the Proven Acceptable Range;

m. an unformulated ultrafiltration and diafiltration retentate hold of ≤4 days in the Normal Operating Range and ≤6 days in the Proven Acceptable Range;

n. a diluted ultrafiltrated/diafiltrated product hold of ≤7 days in the Normal Operating Range and ≤14 days in the Proven Acceptable Range;

o. a step yield of ≥90%;

p. a processing time from the start of initial concentration through end of diafiltration of ≤11.1 hours;

q. a post-use normalized water permeability (NWP) flux of 75-125% of initial;

r. a diluted ultrafiltrated/diafiltrated pre-filtration pool bioburden of <10 CFU/10 mL; and

S. a diluted ultrafiltrated/diafiltrated post-filtration pool bioburden of <3 CFU/10 mL; and/or a diluted ultrafiltrated/diafiltrated post-filtration pool endotoxin concentration of <2 EU/mL.

26. The method of claim 1 , wherein processing conditions for the ultrafiltration and diafiltration steps include one or more of the following:

a. an initial concentration target of 40-60 g/L;

b. a final concentration target of 140-160 g/L, including a 1.07 recovery factor);

c. a diavolume of 4.5-7.5, with a target of 6.0;

d. an undiluted ultrafiltrated/diafiltrated product hold of ≤24 hours;

e. a diluted ultrafiltrated/diafiltrated product hold of ≤24 hours;

f. use of a Millipore Pellicon 3 Ultracel C screen 30 kDa MWCO filter;

g. a flush WFI≥20 L/m 2 ;

h. an equilibrium of 50 mM NaPO 4 pH 7.4, 25 mM L-Arg;

i. a membrane load of ≤600 L/m 2 ;

j. a target feed flow rate for all product steps of 360 LMH;

k. a target transmembrane pressure for all product steps of 15 psi;

l. A feed pressure of ≤50 psi;

m. a diafiltration buffer that is the same as equilibrium;

n. a final concentration that can be controlled by feed pressure;

o. a temperature of 15-35° C.;

p. a recovery with ≤1×system hold-up volume;

q. a dilution to target 120 g/L with DF/equilibrium buffer;

r. 0.1919-0.2393 kg/kg addition of excipient addition buffer, 25 mM L-Arg, 30% Sucrose 0.30% (w/v), PS 80) to 120 g/L UF/DF product for final formulation;

s. membrane re-use up to 20 cycles;

t. sanitization with 0.5 M NaOH;

u. storage with 0.1 M NaOH;

v. a yield of >60%;

w. express SHC filterability 120 g/L UF/DF product: ≤40 L/m 2 ; and

x. express SHC filterability BDS of ≤3045 L/m 2 .

27. The method of claim 1 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.

Assignments (1)

Continuity (3)

References Cited (400)