CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No.

62/691,428, filed on June 28, 2018 and U.S. Provisional Application Serial No. 62/811,710, filed on February 28, 2019. The entire contents of the above-referenced provisional patent applications are incorporated herein by reference.

BACKGROUND

Antibodies are heavily utilized in diagnostic, therapeutic and biological research applications. Antibody stability, however, presents a challenge in the purification and formulation of these proteins. Antibody instability leads to high levels of aggregation in protein formulations, which can have several disadvantages, including changes in protein activity and potentially undesirable immunological responses in patients. There is, accordingly, a longstanding need for improved techniques to enhance the production and purification processes and increase product recovery. The present disclosure addresses this need and provides additional benefits.

SUMMARY

Provided herein are methods for producing an anti-C5 antibody (e.g., ravulizumab).

In one embodiment, the methods described herein comprising culturing mammalian cells (e.g., Chinese Hamster Ovary (CHO) cells) comprising a nucleic acid encoding the anti- C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by one or more (e.g., one, two, three, four, five, six, seven, or eight) steps selected from the group consisting of: a recovery step; purification by Protein A affinity chromatography; a low pH viral inactivation step; purification by cation exchange chromatography (CEX); purification by anion exchange chromatography (AEX); a virus reduction filtration step; a concentration and diafiltration step; and a bulk filtration step.

In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by purification by Protein A affinity chromatography. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by a low pH viral inactivation step. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by purification by CEX. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by purification by AEX. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by a vims reduction filtration step. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g.,

ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by concentration and a

diafiltration/ultrafiltration step.

In another embodiment, the methods described herein comprising culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g.,

ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by one or more (e.g., one, two, three, four, five, six, seven, or eight) steps, including purification by CEX and/or AEX.

In another embodiment, the methods described herein comprising culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g.,

ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by one or more (e.g., one, two, three, four, five, six, seven, or eight) steps, including a single diafiltration/ultrafiltration step (/. _<? ., no more than one diafiltration/ultrafiltration step).

Also provided are methods 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;

c. purification by Protein A affinity chromatography;

d. a low pH viral inactivation step;

e. purification by CEX;

f. purification by AEX;

g. a vims reduction filtration step; and

h. a concentration and diafiltration step.

In another embodiment, the method further comprises a bulk filtration step.

In another embodiment, the method consists of:

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;

c. purification by Protein A affinity chromatography;

d. a low pH viral inactivation step;

e. purification by CEX;

f. purification by AEX;

g. a vims reduction filtration step; and

h. a concentration and diafiltration step.

In another embodiment, the method further comprises a bulk filtration step.

In one embodiment, steps (a)-(h) are performed sequentially in order. For example, in one embodiment, 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, followed by (b) a recovery step, followed by (c) purification by Protein A affinity chromatography, followed by (d) a low pH viral inactivation step, followed by (e) purification by CEX, followed by (f) purification by AEX, followed by (g) a virus reduction filtration step, followed by (h) a concentration and diafiltration step, and optionally followed by a bulk filtration step.

In another embodiment, steps (a)-(h) are performed in any order and/or in any combination. For example, in one embodiment, purification by AEX is performed before purification by CEX. In another embodiment, any and/or all of the purification steps are performed before the low pH viral inactivation and/or vims reduction filtrations step(s).

In another embodiment, the method includes no more than ten steps. In another embodiment, the method includes no more than nine steps. In another embodiment, the method includes no more than eight steps. In another embodiment, the method includes no more than seven steps. In another embodiment, the method includes no more than six steps. In another embodiment, the method includes no more than five steps.

The methods described herein can also include a recovery step that comprises filtering the cell culture production medium. In one embodiment, the cell culture production medium is a commercially available cell culture medium (e.g., from Life Technologies). In another embodiment, the cell culture production medium is not a custom made cell culture production medium. In another embodiment, the cell culture production medium is a protein free and chemically defined cell culture production medium. In another embodiment, the cell culture production medium does not include bovine serum albumin.

In one embodiment, the cell culture medium is filtered by depth filtration. In another embodiment, the cell culture medium is filtered through a depth filtration train (e.g., a two- step depth filtration train), followed by additional filtration (e.g. , through two 0.5/0.2 mhi filters in series) into a container (e.g., a 2,000 L single-use mixing bioprocess container). In another embodiment, the depth filtration train is flushed with WFI and equilibrated with a buffer prior to use. In another embodiment, the equilibration buffer and/or chasing buffer comprises Tris (e.g., 20 mM or about 20 mM) and sodium chloride (e.g., 65 mM or about 65 mM) at a pH of about 7.6 (e.g., pH of 7.4, 7.5, 7.6, 7.7 or 7.8). In another embodiment, the equilibration buffer comprises 20 mM Tris (pH 7.6) and 65 mM sodium chloride. In another embodiment, the cell culture production medium is chased (e.g. , flushed) through the two-step depth filtration train with a buffer, e.g., a buffer comprising 20 mM Tris (pH 7.6) and 65 mM sodium chloride. In another embodiment, the recovery step yields clarified harvest material.

In another embodiment, the processing conditions for the recovery step include one or more (e.g., one, two, three, four, five, six, seven, eight, nine or ten), of the following: a D0HC depth filter load of < 100 L/m ² in the NOR and < 100 L/m ² in the PAR; an A1HC depth filter load of < 200 L/m ² in the NOR and < 200 L/m ² in the PAR; a 0.5/0.2 pm filter load of < 800 L/m ² in the NOR and < 800 L/m ² in the PAR; a harvest load temperature of 18 - 37°C in the NOR and 15 - 37°C in the PAR; a buffer chase volume of 20 - 25 L/m ² in the NOR and 0 - 30 L/m ² in the PAR; a clarified harvest hold time (start of harvest filtration through end of final Pro A cycle load) of < 10 days in the NOR and < 16 days in the PAR; a yield of > 70%; a total filtration time (start through end of harvest filtration, excluding flush and

equilibration) of < 3.3 hours; a bioburden of < 3 CFU/10 mL; and/or an endotoxin concentration of < 5 EU/mL.

In one embodiment, material from a previous step (e.g. , clarified harvest material from the recovery step) is loaded onto a Protein A column through a filter (e.g., 0.5/0.2 pm filter).

In another embodiment, the Protein A affinity chromatography step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight, or nine) buffers, including, but not limited to: (a) sodium hydroxide, (b) tris and sodium chloride, (c) sodium phosphate, sodium chloride, and arginine hydrochloride, (d) sodium acetate, (e) acetic acid, (f) Water For Injection (WFI), and (g) ethanol. In one embodiment, the Protein A affinity

chromatography step includes 0.1 N sodium hydroxide for sanitization. In another embodiment, the Protein A affinity chromatography step includes 20 mM tris and 65 mM sodium chloride at a pH of about 7.6 for equilibration and post- load wash 1. In another embodiment, the Protein A affinity chromatography step includes 50 mM sodium phosphate, 100 mM sodium chloride, and 300 mM arginine hydrochloride at a pH of 6.0 for post-load wash 2. In another embodiment, the Protein A affinity chromatography step includes 20 mM tris and 65 mM sodium chloride at a pH of about 7.6 for post-load wash 3. In another embodiment, the Protein A affinity chromatography step includes 25 mM sodium acetate at a pH of 3.75 for elution. In another embodiment, the Protein A affinity chromatography step includes 100 mM acetic acid for stripping. In another embodiment, the Protein A affinity chromatography step includes WFI for flushing. In another embodiment, the Protein A affinity chromatography step includes 20% ethanol for storage.

In another embodiment, the processing conditions for the Protein A affinity chromatography include one or more (e.g., one, two, three, four, five, six, seven, or eight) of the following:

a. a pre-batch sanitization hold time of 30-60 minutes in the Normal Operating Range

(NOR) and 30-75 minutes in the Proven Acceptable Range (PAR);

b. a post-batch sanitization hold time of 30-60 minutes in the Normal Operating Range

(NOR) and 30-75 minutes in the Proven Acceptable Range (PAR); c. column cycles of < 100 in the Normal Operating Range (NOR) and < 100 in the Proven Acceptable Range (PAR);

d. an eluate hold time (end of filtration through start of low pH acidification) of < 7 days in the Normal Operating Range (NOR) and < 10 days in the Proven Acceptable Range (PAR);

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 endotoxin of < 5 EU/mL.

The methods described herein can also include a low pH viral inactivation step. In one embodiment, the direct vation step comprises treating material from the previous step (e.g., an eluated pool from the Protein A affinity chromatography purification) with acetic acid, confirming low pH (e.g., within a pH of 3.60 to 3.75), increasing the pH, and then filtering out neutralized viral inactivated material. In another embodiment, the low pH viral inactivation step comprises (a) treating material from the previous step (e.g., an eluated pool from the Protein A affinity chromatography purification) with acetic acid (e.g., 1 M acetic acid at a pH range of 3.60-3.70), (b) transferring the pool to a second vessel and incubating it at ambient temperature for a minimum of 60 minutes without mixing and confirming pH range to be within 3.60 to 3.75; (c) increasing to pH 5.0 (e.g., using 1M Tris) and incubating at ambient temperature for a minimum of 60 minutes without mixing; (d) pre-filtering (e.g., 0.5/0.2 pm filter) neutralized viral inactivated material; and storing the filtered product.

In another embodiment, the processing conditions for the low pH viral inactivation step include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) of the following:

a. an acidification pH immediately after titration of 3.60-3.70 in the Normal Operating Range (NOR) and 3.55-3.80 in the Proven Acceptable Range (PAR);

b. an acidification pH immediately after hold time of 3.60-3.75 in the Normal Operating Range (NOR) and 3.55-3.80 in the Proven Acceptable Range (PAR);

c. a hold time at low pH of 60-120 minutes in the Normal Operating Range (NOR) and > 60-360 minutes in the Proven Acceptable Range (PAR);

d. a hold time at neutralized pH prior to 0.5/0.2 pm filtration of 60-120 minutes in the Normal Operating Range (NOR) and > 60 minutes in the Proven Acceptable Range (PAR); e. a filtered neutralized product hold time (end of filtration through end of CEX load) of < 7 days;

f. in the Normal Operating Range (NOR) and < 7 days in the Proven Acceptable Range (PAR);

g. a yield of > 90%;

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

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

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

The methods described herein can also include a cation exchange chromatography (CEX) step. For example, in one embodiment, the methods described herein comprising culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in said cell culture production medium, followed by one or more (e.g., one, two, three, four, five, six, seven, or eight) steps, including purification by CEX and/or AEX.

In one embodiment, material from the previous step (e.g., neutralized filtrate from the low pH viral inactivation step) is loaded onto a cation exchange column (e.g., a POROS HS50 cation exchange column), for example, through a 0.5/0.2 pm filter. In one

embodiment, the CEX step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight, or nine) buffers, including, but not limited to: (a) sodium acetate, (b) sodium chloride, (c) sodium hydroxide, (d) sodium acetate and sodium chloride, and (e) sodium acetate, sodium chloride, and arginine hydrochloride. In another embodiment, the CEX buffer comprises 50 mM Sodium Acetate at a pH of 5.0 for equilibration and post-load wash 1. In another embodiment, the CEX buffer comprises 50 mM sodium acetate and 60 mM sodium chloride at a pH of 4.9 for post-load wash 2. In another embodiment, the CEX buffer comprises 50 mM sodium acetate, 90 mM arginine hydrochloride, and 30 mM sodium chloride at a pH of 5.0 for elution. In another embodiment, the CEX buffer comprises 2.0 M sodium chloride for stripping. In another embodiment, the CEX buffer comprises 1.0 N sodium hydroxide for sanitization. In another embodiment, the CEX buffer comprises 0.1 N sodium hydroxide for storage.

In another embodiment, the processing conditions for the CEX include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or 11) of the following:

a. a load capacity of 22-45 g/L in the Normal Operating Range (NOR) and 15-50 g/L in the Proven Acceptable Range (PAR); b. a temperature of 15 - 25°C in the Normal Operating Range (NOR) 13 - 27°C in the Proven Acceptable Range (PAR);

c. an elution buffer pH of 4.90 - 5.10 in the Normal Operating Range (NOR) and 4.90 - 5.10 in the Proven Acceptable Range (PAR);

d. an elution buffer conductivity of 11.1 - 13.6 mS/cm in the Normal Operating Range (NOR) and 11.1 - 13.6 mS/cm in the Proven Acceptable Range (PAR);

e. an elution flow rate of 150 - 300 cm/hr in the Normal Operating Range (NOR) and 120 - 330 cm/hr in the Proven Acceptable Range (PAR);

f. an eluate hold time (start of eluate collection through end of AEX load adjustment) of < 7 days in the Normal Operating Range (NOR) and < 10 days in the Proven

Acceptable Range (PAR);

g. column cycles of < 100 in the Normal Operating Range (NOR) and < 100 in the Proven Acceptable Range (PAR);

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 eluate volume of 2.3 - 5.0 column volumes.

The methods described herein can also include an AEX step. In one embodiment, material from the previous step (e.g., an eluated pool from the CEX step) is pH adjusted prior to loading on to an AEX column. In another embodiment, material from the previous step (e.g., the eluated pool from the CEX step) is pH adjusted using tris, arginine, and WFI. In another embodiment, material from the previous step (e.g., the eluated pool from the CEX step) is adjusted to a pH of approximately 8.0. In another embodiment, material from the previous step (e.g., the eluated pool from the CEX step) is adjusted to a conductivity of 8.5 mS/cm. In another embodiment, material from the previous step (e.g., the eluated pool from the CEX step) is adjusted to a pH of 8.00 and a conductivity of 8.5 mS/cm with 100 mM Tris at 180 mM Arginine at a pH of 9.0 and WFI.

In another embodiment, the AEX column is a POROS HQ50 AEX column. In another embodiment, the AEX column is a POROS HQ50 AEX column operated in flow through mode. In another embodiment, material from the previous step (e.g., the adjusted eluated pool) is loaded on to an AEX column within 24 hours (e.g., within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours) of the adjustment. In another embodiment, material from the previous step (e.g., the adjusted eluated pool) is loaded on to an AEX column through a filter (e.g., 0.5/0.2 pm filter) and the resulting product is chased (e.g., flushed) from the AEX column, e.g., using buffer filtered through an filter (e.g., 0.5/0.2 pm filter) into a filtrate vessel.

In another embodiment, the AEX step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight, or nine) buffers, including, but not limited to: (a) tris and arginine, (b) WFI, (c) sodium chloride, (d) tris and sodium chloride, and (e) sodium hydroxide. In another embodiment, the AEX step includes the use of 100 mM Tris and 180 mM Arginine at a pH of 9.0 for load pH adjustment. In another embodiment, the AEX step includes the use of WFI for load conductivity adjustment and flush. In another embodiment, the AEX step includes the use of 2 M Sodium Chloride for conditioning. In another embodiment, the AEX step includes the use of 20 mM Tris and 65 mM Sodium Chloride at a pH of 7.6 for equilibration and post-load chase. In another embodiment, the AEX step includes the use of 2 M Sodium Chloride for post-load elution stripping. In another embodiment, the AEX step includes the use of 1.0 N Sodium Hydroxide for sanitization. In another embodiment, the AEX step includes the use of 0.1 N Sodium Hydroxide for storage.

In another embodiment, the processing conditions for the AEX include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or 11) of the following:

a. a load pH of 7.90 - 8.10 in the Normal Operating Range (NOR) and 7.80 - 8.20 in the Proven Acceptable Range (PAR);

b. a load conductivity of 8.0 - 9.0 mS/cm in the Normal Operating Range (NOR) and 7.0 - 10.0 mS/cm in the Proven Acceptable Range (PAR); and

c. a load capacity pH of 25 - 90 g/L in the Normal Operating Range (NOR) and 25 - 100 g/L in the Proven Acceptable Range (PAR);

d. a hold time (AEX load adjustment through start of AEX Load) of < 1 day in the

Normal Operating Range (NOR) and < 4 days in the Proven Acceptable Range (PAR);

e. a product hold time (end of AEX load adjustment through end of UF/DF) of < 4 days in the Normal Operating Range (NOR) and < 6 days in the Proven Acceptable Range (PAR); f. column cycles of < 100 in the Normal Operating Range (NOR) and < 100 in the Proven Acceptable Range (PAR);

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

h. an eluate Endotoxin (post-filtration) of < 5 EU/mL; and a yield of > 67%.

The methods described herein can also include a filtration step to remove viruses or vims-like particles. In one embodiment, the filters are flushed prior to use (e.g., using WFI and buffer). In another embodiment, flow through filtrate from the AEX is filtered through a Viresolve Pro Shield H pre-filter (e.g., 0.5/0.2 pm), followed by filtration through a Viresolve Pro filter (e.g., 20 nm).

In another embodiment, the filtration step to remove viruses or virus-like particles includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight, or nine) buffers, including, but not limited to: (a) tris and sodium chloride and (b) WFI. In another embodiment, the filtration step to remove viruses or virus-like particles includes the use WFI as a pre-use flush. In another embodiment, the filtration step to remove viruses or virus-like particles includes the use of 20 mM Tris and 65 mM Sodium Chloride at a pH of 7.6 for equilibration and post-loading chase.

In another embodiment, the filtration step to remove viruses or virus-like particles includes one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following:

a. a Viresolve Pro Filter Differential Pressure During Load and Chase of 21 - 32 psid in the Normal Operating Range (NOR) and 21 - 35 psid in the Proven Acceptable Range (PAR);

b. a Total Pause Time during Load and Chase of 0 minutes in the Normal Operating Range (NOR) and < 120 minutes in the Proven Acceptable Range (PAR);

c. a chase volume of < 15 L/m ² in the Normal Operating Range (NOR) and < 20 L/m ² in the Proven Acceptable Range (PAR);

d. pass a post-use integrity test;

e. a load concentration of 3.0 - 6.0 g/L in the Normal Operating Range (NOR) and < 6.7 g/L in the Proven Acceptable Range (PAR);

f. a shield H pre-filter load of < 700 L/m ² in the Normal Operating Range (NOR) and < 1200 L/m ² in the Proven Acceptable _Range (PAR);

g. a viresolve pro filter load of < 700 L/m ² in the Normal Operating Range (NOR) and < 700 L/m ² in the Proven Acceptable Range (PAR); h. a product hold time (end of AEX load adjustment through end of UFDF) of < 4 days in the Normal Operating Range (NOR) and < 6 days in the Proven Acceptable Range (PAR);

i. a bioburden (pre-filtration viral filter load) of < 3 CFU/10 mL;

j. an endotoxin (viral filtrate) of < 2 EU/mL;

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

l. a processing time (start of load to end of load) of < 12 hours; and/or a step yield of > 90%.

The methods described herein can also include a concentration and diafiltration step. For example, in one embodiment, the methods described herein comprising 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, followed by one or more (e.g., one, two, three, four, five, six, seven, or eight) steps, including a single diafiltration/ultrafiltration step (/. _<? ., no more than one

diafiltration/ultrafiltration step).

In one embodiment, material from the previous step (e.g., a pool from the virus filtration step) is ultrafiltrated and concentrated. In another embodiment, the pool is further diafiltered. In another embodiment, the concentration of the product is measured and diluted (e.g., to 10.0 g/L or 100 g/L). In another embodiment, material from the previous step (e.g., a pool from the vims filtration) is (a) ultrafiltrated and concentrated, e.g., to 55 g/L using 30 kDa MWCO UF membranes, (b) diafiltered (e.g., with 6 diafiltration volumes) into a formulation buffer and the product concentration is measured and diluted (e.g., to 10.0 g/L or 100 g/L). In another embodiment, the formulation buffer comprises 10 mM sodium phosphate and 150 mM sodium chloride at a pH of 7.0. In another embodiment, the diluted product is filtered (e.g., through a 0.5/0.2 pm filter) and Polysorbate 80 is added to a diluted product pool to achieve a final concentration of 0.02% (w/v) Polysorbate 80.

In another embodiment, the concentration and/or diafiltration steps include the use of one or more (e.g., one, two, three, four, five, six, seven, eight, or nine) buffers, including, but not limited to: (a) WFI, (b) sodium hydroxide, (c) sodium phosphate and 150 mM sodium chloride, and (d) polysorbate 80. In another embodiment, the concentration and/or diafiltration steps include the use of WFI as a flush. In another embodiment, the

concentration and/or diafiltration steps include the use of 0.5 M sodium hydroxide for sanitization. In another embodiment, the concentration and/or diafiltration steps include the use of 10 mM Sodium Phosphate and 150 mM Sodium Chloride at a pH of 7.0 for equilibration, diafiltration, chase, and/or pool dilution. In another embodiment, the concentration and/or diafiltration steps include the use of 0.1 M sodium hydroxide for storage. In another embodiment, the concentration and/or diafiltration steps include the use of 10% (w/v) Polysorbate 80 for excipient.

In another embodiment, the concentration and/or diafiltration steps include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen) of the following:

a. a dilution of within 1% of calculated volume in the Normal Operating Range (NOR) and within 3% of calculated volume in the Proven Acceptable Range (PAR);

b. 10% (w/v) Polysorbate 80 is 0.19 - 0.21% (w/v) of diluted UF/DF product in the Normal Operating Range (NOR) and 0.17 - 0.23% (w/v) of diluted UF/DF product in the Proven Acceptable Range (PAR);

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

d. a diluted UF/DF product concentration of 9.0 - 11.0 mg/mL;

e. passing a pre-use integrity test;

f. a membrane loading of 100 - 500 g/m ² in the Normal Operating Range (NOR) and within 50 - 600 g/m ² in the Proven Acceptable Range (PAR);

g. a feed flux of 240 - 420 LMH in the Normal Operating Range (NOR) and within 180 - 440 LMH in the Proven Acceptable Range (PAR);

h. a transmembrane pressure of 10 - 30 psi in the Normal Operating Range (NOR) and within 8 - 35 psi in the Proven Acceptable Range (PAR);

i. a pressure of 15 - 25°C in the Normal Operating Range (NOR) and within

12 - 30°C in the Proven Acceptable Range (PAR);

j. a fed batch ratio of 1-3 in the Normal Operating Range (NOR) and within 1-5 in the Proven Acceptable Range (PAR);

k. a concentration at end of ultrafiltration target of 13 - 17 g/L in the Normal Operating Range (NOR) and within 12 - 20 g/L in the Proven Acceptable Range (PAR);

l. a diavolume of 5.5 - 7.0 in the Normal Operating Range (NOR) and within 4.5 - 7.0 in the Proven Acceptable Range (PAR); m. an unformulated ultrafiltration and diafiltration retentate hold of < 4 days in the Normal Operating Range (NOR) and within < 6 days in the Proven Acceptable Range (PAR);

n. a product hold (diluted ultrafiltrated/diafiltrated product) of < 7 days in the

Normal Operating Range (NOR) and within < 14 days in the Proven Acceptable Range (PAR);

o. a step yield of > 90%;

p. a processing time (start of initial concentration through end of diafiltration) of < 11.1 hours;

q. a post-use NWP of 75 - 125% of initial;

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

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

In another embodiment, the concentration and/or diafiltration steps include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty) of the following:

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

b. a final concentration target (140 - 160 g/L) (includes 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 NaP0 ₄ (pH 7.4), 25 mM L-Arg (> 20 L/m ² );

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 or < 50 psi (can be increased);

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

n. a final concentration that can be controlled by feed pressure (not TMP or Feed Flow Rate); o. a temperature or 15 - 35°C;

p. a recovery with < lx system hold-up volume (calculation required per CSD); q. a dilution to target 120 g/L with DF/equilibrium buffer;

r. 0.1919 - 0.2393 kg/kg addition of excipient addition buffer (EAB - 50 mM

NaP0 ₄ (pH 7.4), 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 % (expected over 90%);

w. express SHC filterability 120 g/L UL/DL product: < 40 L/m ² ; and

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

An exemplary anti-C5 antibody is ravulizumab (also known as ALXN1210 and antibody BNJ441) comprising the heavy and light chains having the sequences shown in SEQ ID NOs: 14 and 11, respectively, or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain complementarity determining regions (CDRs) or variable regions (VRs) of ravulizumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable (VH) region of ravulizumab having the sequence shown in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of the light chain variable (VL) region of ravulizumab having the sequence shown in SEQ ID NO: 8. In another embodiment, the 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.

In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO:8, respectively.

In another embodiment, the antibody comprises a heavy chain constant region as set forth in SEQ ID NO: 13.

In another embodiment, the antibody comprises a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434, each in EU numbering. In another embodiment, the antibody comprises CDR1, CDR2 and CDR3 heavy chain sequences as set forth in SEQ ID NOs:l9, 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 a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434, each in EU numbering.

In another embodiment, the antibody competes for binding with, and/or binds to the same epitope on C5 as, the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above- mentioned antibodies (e.g., at least about 90%, 95% or 99% variable region identity with SEQ ID NO: 12 and SEQ ID NO:8).

In another embodiment, the antibody binds to human C5 at pH 7.4 and 25°C with an affinity dissociation constant (K _D ) that is in the range 0.1 nM < K _D £ 1 nM. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25°C with a K _D ³ 10 nM. In yet another embodiment, the [(K _D of the antibody or antigen-binding fragment thereof for human C5 at pH 6.0 and at 25°C)/(K _D of the antibody or antigen-binding fragment thereof for human C5 at pH 7.4 and at 25°C)] of the antibody is greater than 25.

In another embodiment, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the BNJ421 antibody (described in PCT/US2015/019225 and US Patent No. 9,079,949). In another embodiment, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the 7086 antibody (see US Patent Nos. 8,241,628 and 8,883,158). In another embodiment, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the 8110 antibody (see US Patent Nos. 8,241,628 and 8,883,158). In another embodiment, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody (see US2016/0176954A1). In another embodiment, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody (see Fukuzawa T. et al., Rep. 2017 Apr

24;7(l):l080). In another embodiment, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the REGN3918 antibody (see US20170355757).

In one embodiment, the anti-C5 antibody produced according to the methods disclosed herein is formulated in a 10 mg/mL solution. In another embodiment, the anti-C5 antibody is formulated in a sterile, preservative-free 10 mg/mL solution e.g., which is suitable for IV administration. In another embodiment, the anti-C5 antibody is supplied in 20 mL single-use vials. In another embodiment, each vial contains 150 mg of ravulizumab in 10 mM sodium phosphate, 150 mM sodium chloride, 0.02% polysorbate 80, and water for injection at a pH of 7.0.

In another embodiment, the anti-C5 antibody produced according to the methods disclosed herein is formulated in a 100 mg/mL solution. In another embodiment, the anti-C5 antibody is formulated in a sterile, preservative-free 100 mg/mL solution e.g., which is suitable for subcutaneous administration. In another embodiment, the anti-C5 antibody is supplied in 2 mL single-use vials. In another embodiment, each vial contains 100 mg/mL of ravulizumab in 50 mM sodium phosphate, 25 mM arginine, 5% sucrose, and 0.05% polysorbate 80, and water for injection at a pH of 7.4.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow diagram of the manufacturing process for ravulizumab.

Figure 2 depicts the process flow and key process parameters (KPP) and key process attributes (KPA) for the inoculum expansion manufacturing process.

Figure 3 depicts the process flow and critical and key process parameters (CPP, KPP), in process controls (IPC) and key process attributes for the cell culture process in the production bioreactor.

Figure 4 depicts the process flow and KPPs and KPAs for step 3 of the ravulizumab manufacturing process.

Figure 5 sets forth an overview of the ravulizumab purification process.

Figure 6 is a flow diagram of the manufacturing process and controls for ravulizumab drug product.

Figure 7 is schematic of the high concentration ultrafiltration/diafiltration (UF/DF) process used in Example 2.

Figure 8 is a plot of the Buffer Chase Volume vs. Concentration (0.2 F Factor).

Figure 9 is a plot of the Buffer Chase Volume vs. Concentration (0.8 F Factor).

Figure 10 sets for the prediction profiled for design of experiment (DOE) 2 using JMP software. DETAILED DESCRIPTION

Methods

Provided herein are methods for producing an antibody that binds to complement component C5 (an“anti-C5 antibody,” e.g., ravulizumab).

As used herein, the terms“purifying” and“separating” are used interchangeably, and refer to the removal of contaminants from a mixture containing a protein of interest (e.g., an anti-C5 antibody).

As used herein, a“mixture” comprises a protein of interest (for which purification is desired) and one or more other components, sometimes, for example, contaminants, i.e., impurities. In one embodiment, a mixture is produced from a host cell that expresses the protein of interest (either naturally or recombinantly). Such mixtures include, for example, cell cultures, cell lysates and clarified bulk (e.g., clarified cell culture supernatant).

As used herein, the term“contaminant” is used in its broadest sense to cover any undesired component or compound within a mixture. In cell cultures, cell lysates or clarified bulk (e.g., cell culture supernatant), contaminants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in a cell culture medium. Host cell contaminant proteins include, without limitation, those naturally or recombinantly produced by the host cell, as well as proteins related to or derived from the protein of interest (e.g., proteolytic fragments) and other process related contaminants (e.g., truncated and aggregated versions of the protein of interest).

As used herein,“washing” or“chasing” refers to passing an appropriate buffer through or over a cation exchange resin or buffer.

As used herein,“eluting” refers to removing a protein of interest (e.g., an anti-C5 antibody) from a resin or column.

As used herein, a“cell culture” refers to cells in a liquid medium that produce a protein of interest (e.g., an anti-C5 antibody). The cells can be from any organism including, for example, bacteria, fungus, mammals or plants. Suitable liquid media include, for example, nutrient media and non-nutrient media. In one embodiment, the cell culture production medium is a commercially available cell culture medium (e.g., from Life

Technologies). In another embodiment, the cell culture production medium is not a custom made cell culture production medium. In another embodiment, the cell culture production medium is a protein free and chemically defined cell culture production medium. In another embodiment, the cell culture production medium does not include bovine serum albumin.

As used herein, the term“clarified bulk” refers to a mixture from which particulate matter (e.g., cells) has been substantially removed. Clarified bulk includes cell culture supernatant, or cell lysate from which cells or cell debris have been substantially removed by, for example, filtration or centrifugation.

As used herein the term“chromatography” refers to the process by which a solute of interest, e.g., an anti-C5 antibody, in a mixture is separated from other solutes in the mixture by percolation of the mixture through an adsorbent, which adsorbs or retains a solute more or less strongly due to properties of the solute, such as, for example, pi, hydrophobicity, size and structure, under particular buffering conditions of the process.

The terms“ion-exchange” and“ion-exchange chromatography” refer to a

chromatographic process in which an ionizable solute of interest (e.g., an anti-C5 antibody in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate conditions of pH and conductivity, such that the solute of interest interacts non-specifically with the charged compound more or less than the solute impurities or contaminants in the mixture. The contaminating solutes in the mixture can be washed from a column of the ion exchange material or are bound to or excluded from the resin, faster or slower than the solute of interest.“Ion-exchange chromatography” specifically includes cation exchange (CEX), anion exchange (AEX) and mixed mode chromatographies (e.g., the combined use of two (or more) retention

mechanisms in a single chromatographic system).

The term“resin” refers to an organic polymer. The polymer may be naturally occurring or synthetic. Resins are often used as solid phase support materials for

chromatography.

In one embodiment, the methods described herein comprising culturing mammalian cells comprising a nucleic acid encoding, for example, an anti-C5 antibody (e.g.,

ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in the cell culture production medium, followed by one or more (e.g., one, two, three, four, five, six, seven or eight) steps selected from the group consisting of: a recovery step;

purification by Protein A affinity chromatography, a low pH viral inactivation step;

purification by CEX, purification by AEX; a vims reduction filtration step, and a

concentration and diafiltration step. Any suitable mammalian cell can be used for the culture step. Exemplary mammalian cells include, but are not limited to murine myeloma cells (NS0), murine hybridomas, Chinese hamster ovary cells (CHO), and PER.C6 human cells. In a particular embodiment, the mammalian cells are CHO cells. In another embodiment, the mammalian cells are not NS0 cells.

In one embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in the cell culture production medium, followed by purification by Protein A affinity chromatography. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in the cell culture production medium, followed by a low pH viral inactivation step. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in the cell culture production medium, followed by purification by CEX. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in the cell culture production medium, followed by purification by AEX. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g., ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in sthe cell culture production medium, followed by a vims reduction filtration step. In another embodiment, the method comprises culturing mammalian cells comprising a nucleic acid encoding the anti-C5 antibody (e.g.,

ravulizumab) in a cell culture production medium, such that the anti-C5 antibody is produced in the cell culture production medium, followed by concentration and diafiltration step.

Also provided are methods of producing an anti-C5 antibody, wherein the method comprises 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; a recovery step; purification by Protein A affinity chromatography; a low pH viral inactivation step; purification by CEX; purification by AEX; a vims reduction filtration step; and a concentration and diafiltration step. In another embodiment, the method consists of 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; a recovery step; purification by Protein A affinity chromatography; a low pH viral inactivation step; purification by CEX; purification by AEX; a virus reduction filtration step; and a concentration and diafiltration step.

In one embodiment, purification steps are performed sequentially in the order described. In another embodiment, the purification steps are performed in any order and/or in any combination. For example, in one embodiment, purification by AEX is performed before purification by CEX. In another embodiment, any and/or all of the purification steps are performed before the low pH viral inactivation and/or vims reduction filtrations step(s).

In another embodiment, the method includes no more than ten steps. In another embodiment, the method includes no more than nine steps. In another embodiment, the method includes no more than eight steps.

Recovery Step

The methods described herein can include a recovery step that comprises

centrifugation and/or filtering the cell culture production medium. In one embodiment, the cell culture medium is centrifuged. In another embodiment, the cell culture medium is filtered through depth filtration. In another embodiment, the cell culture medium is centrifuged and depth filtered. Depth filters are filters that use a porous filtration medium to retain particles throughout the medium, rather than just on the surface of the medium. These filters are commonly used when the fluid to be filtered contains a high load of particles because, relative to other types of filters, they can retain a large mass of particles before becoming clogged (Shukla, A. & Kandula, J., BioPharm International, 21:34-45, 2008).

Depth filtration is widely used for the clarification of cell culture. Cell culture systems can contain yeast, bacterial and other contaminant cells, and, hence, an efficient clarification stage is vital to separate the cells and other colloidal matter to produce a particle free cell system. Most depth filters used in pharmaceutical processes, such as cell system harvesting are composed of cellulose fibers and filter aids. The direct flow design of depth filters provides a financially suitable solution of trapping contaminants within the filter channel while ensuring the maximum recovery rate of the product. The other advantages of this system includes its low power costs, since the pumps utilized during depth filtration require minimal power input due to the low pressure within the system. Depth filtration is also flexible in terms of being able to scale up or down while outputting a high yield (>95%). For cell culture applications, depth filtration trains (e.g., two, three, four or more stage filter systems) are often used and result in more efficient processing.

In one embodiment, depth filtration is used. In another embodiment, a two-step depth filtration train is used. In another embodiment, the depth filtration train is flushed with Water For Injection (WFI) and equilibrated with a buffer prior to use. In another embodiment, the cell culture production medium is chased (e.g., flushed) through the two-step depth filtration train with a buffer. In another embodiment, the equilibration buffer and/or chasing buffer comprises Tris (e.g., 20 mM or about 20 mM), about pH 7.6 (e.g., pH of 7.4, 7.5, 7.6, 7.7. or 7.8), and sodium chloride (e.g., 65 mM or about 65 mM).

In another embodiment, additional filtration is performed after the depth filtration. In another embodiment, the additional filtration is performed through one or more 0.5/0.2 pm filters (e.g., one, two, three or four 0.5/0.2 mhi filters). In another embodiment, additional filtration is performed through two 0.5/0.2 mhi filters in series into a container. In one embodiment, filtration is performed into a bioprocess container (e.g., a 2,000 L single-use mixing bioprocess container). In another embodiment, the recovery step yields clarified harvest material.

In another embodiment, the processing conditions for the recovery step include one or more (e.g., one, two, three, four, five, six, seven, eight, nine or ten), of the following: a D0HC depth filter load of < 100 L/m ² in the Normal Operating Range (NOR) and < 100 L/m ² in the Proven Acceptable Range (PAR); an A1HC depth filter load of < 200 L/m ² in the NOR and < 200 L/m ² in the PAR; a 0.5/0.2 mhi filter load of < 800 L/m ² in the NOR and < 800 L/m ² in the PAR; a harvest load temperature of 18 - 37°C in the NOR and 15 - 37°C in the PAR; a buffer chase volume of 20 - 25 L/m ² in the NOR and 0 - 30 L/m ² in the PAR; a clarified harvest hold time (start of harvest filtration through end of final Pro A cycle load) of < 10 days in the NOR and < 16 days in the PAR; a yield of > 70%; a total filtration time (start through end of harvest filtration, excluding flush and equilibration) of < 3.3 hours; a bioburden of < 3 CFU/10 mL; and/or an endotoxin concentration of < 5 EU/mL.

Protein A Affinity Chromatography Step

The methods described herein can include a Protein A affinity chromatography step. “Protein A affinity chromatography” refers to the separation or purification of substances and/or particles using protein A, where the protein A is generally immobilized on a solid phase. Protein A is a 40-60 kD cell wall protein originally found in Staphylococcus aureas. The binding of antibodies to protein A resin is highly specific. Protein A binds with high affinity to the Fc region of immunoglobulins. It binds with high affinity to human IgGl and IgG2 as well as mouse IgG2a and IgG2b. Protein A binds with moderate affinity to human IgM, IgA and IgE, as well as to mouse IgG3 and IgGl. A protein comprising a CH2/CH3 region may be reversibly bound to, or adsorbed by, the protein A.

Protein A resins are known in the art and suitable for use in the invention.

Non-limiting examples of commercially available Protein A resins include MabSelect ^® , MabSelect Xtra ^® , MabSelect SuRe ^® , rProtein A Sepharose ^® FF, rmpProtein A Sepharose ^® FF, Protein A Sepharose ^® CL-4B and nProtein A Sepharose ^® 4 FF (all commercially available from GE Healthcare); ProSep ^® A, ProSep ^® -vA High Capacity, ProSep ^® -vA Ultra and ProSep ^® -Va Ultra Plus (all commercially available from Millipore); Poros ^® A and Mabcapture ^® A (both commercially available from Poros); IPA-300, IPA-400 and IPA-500 (all commercially available from RepliGen Corp.); Affi-gel ^® protein A and Affi-prep ^® protein A (both commercially available from Bio-Rad); Protein A Ceramic Hyper D F (commercially available from Pall Corporation); Ultralink Immobilized protein A and Agarose protein A (both commercially available from PIERCE); and Protein A Cellthru 300 and Protein A Ultraflow (both commercially available from Sterogen Bioseparations). In a particular embodiment, the Protein A affinity chromatography is MabSelect SuRe ^® Protein A affinity chromatography. In another embodiment, the Protein A affinity chromatography is not rmp Protein A chromatography.

In one embodiment, the Protein A affinity chromatography step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight or nine) buffers, including, but not limited to: (a) sodium hydroxide, (b) tris and sodium chloride, (c) sodium phosphate, sodium chloride, and arginine hydrochloride, (d) sodium acetate, (e) acetic acid, (f) Water For Injection (WFI), and (g) ethanol. In one embodiment, the Protein A affinity

chromatography step includes 0.1 N sodium hydroxide for sanitization. In another embodiment, the Protein A affinity chromatography step includes 20 mM Tris (pH 7.6) and 65 mM sodium chloride for equilibration and post- load wash 1. In another embodiment, the Protein A affinity chromatography step includes 50 mM sodium phosphate (pH 6.0), 100 mM sodium chloride, and 300 mM arginine hydrochloride for post- load wash 2. In another embodiment, the Protein A affinity chromatography step includes 20 mM Tris (pH 7.6) and 65 mM sodium chloride for post-load wash 3. In another embodiment, the Protein A affinity chromatography step includes 25 mM sodium acetate (pH 3.75) for elution. In another embodiment, the Protein A affinity chromatography step includes 100 mM acetic acid for stripping. In another embodiment, the Protein A affinity chromatography step includes WFI for flushing. In another embodiment, the Protein A affinity chromatography step includes 20% ethanol for storage. In another embodiment, clarified harvest material from the recovery step is loaded onto a Protein A column through a filter (e.g., 0.5/0.2 mhi filter).

In another embodiment, the processing conditions for the Protein A affinity chromatography include one or more (e.g., one, two, three, four, five, six, seven or eight) of the following: a pre-batch sanitization hold time of 30-60 minutes in the NOR and 30-75 minutes in the PAR; a post-batch sanitization hold time of 30-60 minutes in the NOR and 30-75 minutes in the PAR; column cycles of < 100 in the NOR and < 100 in the PAR; an elution hold time (end of filtration through start of low pH acidification) of < 7 days in the NOR and < 10 days in the PAR; a step yield of > 70%; an eluate pre-filtration bioburden of < 50 CFU/10 mL; an eluate post-filtration bioburden of < 3 CFU/10 mL; and/or an eluate post-filtration endotoxin concentration of < 5 EU/mL.

Low pH Viral Inactivation Step

Source materials (e.g., cell lines, cellular debris) and viruses introduced during antibody production can present viral contamination risks, which can have potential consequences with serious clinical and economic implications. Direct exposure of process intermediates to pH extremes has been used for viral clearance in biopharmaceutical manufacturing. Studies have proven that low pH treatment (e.g., pH 3.0-3.75) of monoclonal antibodies following affinity chromatography, for example, is effective against enveloped viruses (Brorson, K. et al, Biotechnol. Bioeng., 82:321-9, 2003). In general, exposure to pH extremes during manufacture of monoclonal antibodies can provide effective and robust viral reduction (e.g., > 4.0 loglO reduction). Accordingly, the methods described herein can also include a low pH viral inactivation step.

In one embodiment, the method includes subjecting the material from the previous step (e.g., an eluted pool from the Protein A affinity chromatography purification; a“pool” is the combined fractions from a chromatography step, e.g., the fractions that contain the elution fractions) to low pH conditions. In one embodiment, the low pH is a pH of 3.0, 3.1, 3.2,

3.25, 3.3, 3.4, 3.5, 3.6, 3.7 or 3.75). In another embodiment, the low pH is within a range of 3.0-3.75. In another embodiment, the low pH is within a range of 3.60 to 3.75. In another embodiment, the method includes treating the material from the previous step with acetic acid. In another embodiment, the method includes increasing the pH after a low pH has been confirmed and then filtering out neutralized viral inactivated material.

In another embodiment, the low pH viral inactivation step comprises (a) treating material from the previous step (e.g., an eluted pool from the Protein A affinity

chromatography purification) with acetic acid (e.g., 1 M acetic acid at a pH range of 3.60-3.70), (b) transferring it to a second vessel and incubating it at ambient temperature (e.g., 20°C, 2l°C, 23°C, 24°C or 25°C) for a minimum of 60 minutes (e.g., at least 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 minutes) without mixing and confirming the pH range to be within 3.60 to 3.75; (c) increasing to pH 5.0 (e.g., using 1 M Tris) and incubating at ambient temperature for a minimum of 60 minutes (e.g., at least 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 minutes) without mixing; (d) pre-filtering (e.g., 0.5/0.2 mhi filter) neutralized viral inactivated material, and storing the filtered product.

In another embodiment, the processing conditions for the low pH viral inactivation step include one or more (e.g., one, two, three, four, five, six, seven, eight, nine or ten) of the following: an acidification pH immediately after titration of 3.60-3.70 in the NOR and 3.55-3.80 in the PAR; an acidification pH immediately after hold time of 3.60-3.75 in the NOR and 3.55-3.80 in the PAR; a hold time at low pH of 60-120 minutes in the NOR and > 60-360 minutes in the PAR; a hold time at neutralized pH prior to 0.5/0.2 mhi filtration of 60-120 minutes in the NOR and > 60 minutes in the PAR; a filtered neutralized product hold time (end of filtration through end of CEX load) of < 7 days in the NOR and < 7 days in the PAR; a yield of > 90%; a neutralized pre-filtration pool bioburden of < 50 CFU/10 mL; a neutralized post-filtration pool bioburden of < 3 CFU/10 mL; and/or a neutralized post- filtration pool endotoxin concentration of < 5 EU/mL.

Cation Exchange Chromatography (CEX)

The methods described herein can also include a CEX step. CEX is a form of ion exchange chromatography (IEX), which is used to separate molecules based on their net surface charge. CEX, more specifically, uses a resin modified with negatively charged functional groups. They can be strong acidic ligands such as sulphopropyl, sulfoethyl and sulfoisobutyl groups or weak acidic ligand, such as carboxyl group. CEX has been applied for purification processes for many monoclonal antibodies with pi values ranging from neutral to basic. Most humanized IgGl and IgG2 subclasses are perfect candidates for CEX, in which the antibody is bound to the resin during the loading step and eluted through either increasing conductivity or increasing pH in the elution buffer. The most negatively charged, process-related impurities such as DNA, some host cell protein, leached Protein A and endotoxin are removed in the load and wash fractions. CEX can provide separation power to reduce antibody variants from the target antibody product such as deamidated products, oxidized species and N-terminal truncated forms, as well as high molecular weight species.

A“cation exchange resin” or“CEX resin” refers to a solid phase that is negatively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Any negatively charged ligand attached to the solid phase suitable to form the CEX resin can be used, e.g., a carboxylate, sulfonate and others. Commercially available CEX resins include, but are not limited to, for example, those having a sulfonate based group (e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose ^® Fast Flow, SP Sepharose ^® High Performance from GE Healthcare, Toyopearl ^® SP-650S and SP-650M from Tosoh, Macro-Prep ^® High S from BioRad, Ceramic HyperD ^® S, Trisacryl M and LS SP and Spherodex LS SP from Pall Technologies); a sulfoethyl based group (e.g. , Fractogel ^® SE, from EMD, Poros ^® S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, Poros ^® HS-20 and HS 50 from

Applied Biosystems); a sulfoisobutyl based group (e.g., Fractogel ^® EMD SO.sub.3.sup.- from EMD); a sulfoxyethyl based group (e.g., SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g., CM Sepharose ^® Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., Macro-Prep CM from BioRad, Ceramic HyperD CM,

Trisacryl M CM, Trisacryl LS CM, from Pall Technologies, Matrex ^® Cellufine ^® C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, Toyopearl ^® CM-650S, CM-650M and CM-650C from Tosoh); sulfonic and carboxylic acid based groups (e.g., BAKERBOND ^® Carboxy-Sulfon from J. T. Baker); a carboxylic acid based group (e.g., WP CBX from J. T. Baker, DOWEX ^® MAC-3 from Dow Liquid Separations, Amberlite Weak Cation Exchangers, DOWEX ^® Weak Cation Exchanger, and Diaion ^® Weak Cation Exchangers from Sigma- Aldrich and Fractogel ^® EMD COO from EMD); a sulfonic acid based group (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX ^® Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP Sulfonic from J. T. Baker, Sartobind ^® S membrane from Sartorius, Amberlite Strong Cation Exchangers, DOWEX ^® Strong Cation and Diaion ^® Strong Cation Exchanger from Sigma- Aldrich); and a orthophosphate based group (e.g. , Pl 1 from Whatman). In a particular embodiment POROS ^® HS50 cation exchange column is used.

In one embodiment, material from a previous step (e.g., neutralized filtrate from the low pH viral inactivation step) is loaded onto a cation exchange column (e.g., a POROS ^® HS50 cation exchange column), for example, through a 0.5/0.2 mhi filter. In one

embodiment, the CEX step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight or nine) buffers, including, but not limited to: (a) sodium acetate, (b) sodium chloride, (c) sodium hydroxide, (d) sodium acetate and sodium chloride, and (e) sodium acetate, sodium chloride, and arginine hydrochloride. In another embodiment, the CEX buffer comprises 50 mM sodium acetate (pH 5.0) for equilibration and post-load wash 1. In another embodiment, the CEX buffer comprises 50 mM sodium acetate (pH 4.9) and 60 mM sodium chloride for post-load wash 2. In another embodiment, the CEX buffer comprises 50 mM sodium acetate (pH 5.0), 90 mM arginine hydrochloride, and 30 mM sodium chloride for elution. In another embodiment, the CEX buffer comprises 2.0 M sodium chloride for stripping. In another embodiment, the CEX buffer comprises 1.0 N sodium hydroxide for sanitization. In another embodiment, the CEX buffer comprises 0.1 N sodium hydroxide for storage.

In another embodiment, the processing conditions for the CEX include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or 11) of the following: a load capacity of 22-45 g/L in the NOR and 15-50 g/L in the PAR; a temperature of 15 - 25°C in the NOR and 13 - 27°C in the PAR; an elution buffer pH of 4.90 - 5.10 in the NOR and 4.90 - 5.10 in the PAR; an elution buffer conductivity of 11.1 - 13.6 mS/cm in the NOR and 11.1 - 13.6 mS/cm in the PAR; an elution flow rate of 150 - 300 cm/hr in the NOR and 120 - 330 cm/hr in the PAR; an eluate hold time (start of eluate collection through end of A EX load adjustment) of < 7 days in the NOR and < 10 days in the PAR; column cycles of

< 100 in the NOR and < 100 in the PAR; an eluate post-filtration bioburden of

< 3 CFU/10 mL; an eluate post- filtration endotoxin of < 5 EU/mL; a step yield of > 58%; and/or an elution volume of 2.3 - 5.0 column volumes.

Anion Exchange Chromatography (AEX)

The methods described herein can also include an AEX step. AEX uses a positively charged group (weakly basic such as diethylamino ethyl, DEAE or dimethylamino ethyl, DMAE; or strongly basic such as quaternary amino ethyl, Q or trimethylammonium ethyl, TMAE or quaternary aminoethyl, QAE) immobilized to the resin. It is a powerful tool to remove process-related impurities such as host cell proteins, DNA, endotoxins and leached Protein A, product-related impurities such as dimer/aggregate, endogenous retrovirus and adventitious viruses such as parvovirus, pseudorabies virus (Curtis, S. et al., Biotechnol. Bioeng., 84: 179-86, 2003; Norling, L. et al., J. Chromatogr. A, 1069:79-89, 2005; and Zhou, J. et al., J. Chromatogr. A, 1134:66-73, 2006). A EX can be used either in flow-through mode or in bind and elute mode, depending on the pi of the antibody and impurities to be removed. For antibodies having a pi above 7.5, which includes most humanized IgGl and IgG2 antibodies, flow-through mode can be a better choice to remove impurities. In flow-through mode, the impurities bind to the resin and the product of interest flows through. The column loading capacity can be quite high since the binding sites on the resin are occupied only by the impurities. For antibodies having a pi in the acidic to neutral range, which includes most humanized IgG4 antibodies, bind and elute modes can be used to remove process-related and product-related impurities from the product of interest.

A EX in flow-through mode has been widely used as a polishing step in monoclonal antibody purification processes designed with two or three unit operations to remove residual impurities such as host cell protein, DNA, leached Protein A and a variety of viruses. The operating pH is normally 8 to 8.2, with a conductivity of up to 10 mS/cm in the product load and equilibration and wash buffers. Conditions are chosen such that the product does not bind to the column, while acidic impurities such as nucleic acid and host cell proteins do. Depending on the resin, loading conditions and charge variant profile of the antibody product, the amount of product loaded can reach one hundred grams per liter of resin without compromising product quality (Fahrner, R. et al., Biotechnol. Genet. Eng. Rev., 18:301-27, 2001). In general, the amount of product loaded in a flow-through mode depends on the impurity species and levels to be removed. A lower level of impurity in the product results in a higher amount of product loaded.

Exemplary anion exchange resins include, but are not limited to, quaternary amine resins or“Q-resins” (e.g., Q-Sepharose ^® , QAE Sephadex ^® ); diethylaminoethane (DEAE) resins (e.g., DEAE-Trisacryl ^® , DEAE Sepharose ^® , benzoylated naphthoylated DEAE, diethylaminoethyl Sephacel ^® ); Amberjet ^® resins; Amberlyst ^® resins; Amberlite ^® resins (e.g., Amberlite ^® IRA-67, Amberlite ^® strongly basic, Amberlite ^® weakly basic), cholestyramine resin, ProPac ^® resins (e.g., ProPac ^® SAX-10, ProPac ^® WAX-10, ProPac ^® WCX-10); TSK-GEL resins (e.g., TSKgel ^® DEAE-NPR; TSKgel ^® DEAE-5PW); and Acclaim ^® resins. In one embodiment, the AEX column is a POROS ^® HQ50 AEX column operated, for example, in flow-through mode.

In one embodiment, material from a previous step (e.g., an eluted pool from the CEX step) is pH adjusted prior to loading on to an AEX column. In another embodiment, material from the previous step (e.g., an eluted pool from the CEX step) is pH adjusted using Tris, arginine and WFI. In another embodiment, material from the previous step (e.g., an eluted pool from the CEX step) is adjusted to a pH of approximately 8.0. In another embodiment, material from the previous step (e.g., an eluted pool from the CEX step) is adjusted to a conductivity of 8.5 mS/cm. In another embodiment, material from the previous step (e.g., an eluted pool from the CEX step) is adjusted to a pH of 8.00 and a conductivity of 8.5 mS/cm with 100 mM Tris (pH 9.0), 180 mM arginine and WFI. In another embodiment, material from the previous step (e.g., an eluted pool from the CEX step) is loaded on to an AEX column within 24 hours (e.g., within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours) of the adjustment. In another embodiment, material from the previous step (e.g., an eluted pool from the CEX step) is loaded on to an AEX column through a filter (e.g., 0.5/0.2 mhi filter) and the resulting product is chased (e.g., flushed) from the AEX column, e.g., using buffer filtered through an filter (e.g., 0.5/0.2 mhi filter) into a filtrate vessel.

In another embodiment, the AEX step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight or nine) buffers, including, but not limited to: (a) Tris and arginine, (b) WFI, (c) sodium chloride, (d) Tris and sodium chloride, and (e) sodium hydroxide. In another embodiment, the AEX step includes the use of 100 mM Tris (pH 9.0) and 180 mM arginine for load pH adjustment. In another embodiment, the AEX step includes the use of WFI for load conductivity adjustment and flush. In another embodiment, the AEX step includes the use of 2 M sodium chloride for conditioning. In another embodiment, the AEX step includes the use of 20 mM Tris (pH 7.6) and 65 mM sodium chloride for equilibration and post- load chase. In another embodiment, the AEX step includes the use of 2 M sodium chloride for post-load elution stripping. In another embodiment, the AEX step includes the use of 1.0 N sodium hydroxide for sanitization. In another embodiment, the AEX step includes the use of 0.1 N sodium hydroxide for storage. In another embodiment, the processing conditions for the AEX include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or 11) of the following: a load pH of 7.90 - 8.10 in the NOR and 7.80 - 8.20 in the PAR; a load conductivity of 8.0 - 9.0 mS/cm in the NOR and 7.0 - 10.0 mS/cm in the PAR; a load capacity pH of 25 - 90 g/L in the NOR and 25 - 100 g/L in the PAR; a hold time (AEX load adjustment through start of AEX Load) of < 1 day in the NOR and < 4 days in the PAR; a product hold time (end of AEX load adjustment through end of UF/DF) of < 4 days in the NOR and < 6 days in the PAR; column cycles of < 100 in the NOR and < 100 in the PAR; an eluate bioburden (post-filtration) of < 3 CFU/10 mL; and/or an eluate endotoxin concentration (post-filtration) of < 5 EU/mL; and a yield of > 67%.

Viral Filtration

Many purification processes for biopharmaceuticals use virus-reduction filtration as an part of an overall strategy for viral clearance (Caballero, S. et al., Biologicals, 42:79-85, 2014; and Marques, B. et al , Biotechnol. Prog., 25:483-91, 2009). Virus-reduction filters can provide robust and effective removal of large and medium sized viruses. Such filters also can effectively remove very small viruses (e.g., parvoviruses) with pore sizes <20 nm.

Typical vims filtration membranes are made from hydrophilic polyethersulfone (PES), hydrophilic polyvinylidene (PVDF) and regenerated cellulose. According to the size distribution of viruses that are removed, virus filters can be categorized into retrovirus filters and parvovirus filters. Exemplary virus filters include, but are not limited to, Planova ^® 15N, Planova ^® 20N, Planova ^® 35N, Planova BioEX ^® , Viresolve ^® NFP, Viresolve ^® NFR,

Viresolve ^® Pro, Ultipor ^® DV 20, Ultipor ^® DV 50, and Virosart ^® CPV. Parvoviruses have a diameter of 18-26 nm, and a typical mAh has a hydrodynamic diameter of 8 12 nm.

The methods described herein can also include a filtration step to remove viruses or vims-like particles. In one embodiment, the filters are flushed prior to use (e.g., using WFI and buffer). In another embodiment, flow-through filtrate from the AEX, for example, is filtered through a Viresolve ^® Pro Shield H pre-filter (e.g., 0.5/0.2 pm), followed by filtration through a Viresolve ^® Pro filter (e.g., 20 nm).

In another embodiment, the filtration step to remove viruses or virus-like particles includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight or nine) buffers, including, but not limited to: (a) Tris and sodium chloride and (b) WFI. In another embodiment, the filtration step to remove vimses or virus-like particles includes the use WFI as a pre-use flush. In another embodiment, the filtration step to remove viruses or virus-like particles includes the use of 20 mM Tris (pH 7.6) and 65 mM sodium chloride for equilibration and post-loading chase.

In another embodiment, the filtration step to remove viruses or virus-like particles includes one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve) of the following: a Viresolve ^® Pro filter differential pressure during load and chase of 21 - 32 psid in the NOR and 21 - 35 psid in the PAR; a Total Pause Time during load and chase of 0 minutes in the NOR and < 120 minutes in the PAR; a chase volume of < 15 L/m ² in the NOR and < 20 L/m ² in the PAR; pass a post-use integrity test; a load concentration of 3.0 - 6.0 g/L in the NOR and < 6.7 g/L in the PAR; a shield H pre-filter load of < 700 L/m ² in the NOR and < 1200 L/m ² in the PAR; a Viresolve ^® Pro filter load of < 700 L/m ² in the NOR and < 700 L/m ² in the PAR; a product hold time (end of AEX load adjustment through end of UF/DF) of < 4 days in the NOR and < 6 days in the PAR; a bioburden (pre-filtration viral filter load) of < 3 CFU/10 mL; an endotoxin concentration (viral filtrate) of < 2 EU/mL; pass a pre-use integrity test; and/or a processing time (start of load to end of load) of < 12 hours; and/or a step yield of > 90%.

Concentration and Dialfiltration

The methods described herein can also include a concentration and diafiltration step. In one embodiment, material from the previous step (e.g., a pool from the virus filtration step) is ultrafiltrated and concentrated.

Diafiltration is a technique that uses ultrafiltration membranes to completely remove, replace, or lower the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. The process selectively utilizes permeable (porous) membrane filters to separate the components of solutions and suspensions based on their molecular size. An ultrafiltration membrane retains molecules that are larger than the pores of the membrane while smaller molecules such as salts, solvents and water, which are 100% permeable, freely pass through the membrane.

Ultrafiltration is a pressure-driven membrane process that is widely used for protein concentration and buffer exchange. Ultrafiltration is a size-based separation where species larger than the membrane pores are retained and smaller species pass through freely.

Separation is achieved through differences in the filtration rates of different components across the membrane under a given pressure driving force (van Reis, R. & Zydney, A. Protein ultrafiltration. In: Flickinger MC, Drew SW, editors. Encyclopedia of Bioprocess

Technology-Fermentation, Biocatalysis and Bioseparation. John Wiley & Sons; 1999. pp. 2197-2214). Buffer exchange is achieved using a diafiltration mode in which buffer of the final desired composition is added to the retentate system at the same rate in which filtrate is removed, thus maintaining a constant retentate volume.

In one embodiment, material from the previous step (e.g., a pool from the virus filtration step) is diafiltered. In another embodiment, the resulting concentrated product is measured and diluted (e.g., to 10.0 g/L). In another embodiment, material from the previous step (e.g., a pool from the vims filtration step) is (a) ultrafiltrated and concentrated, e.g., to 55 g/L using a 30 kD molecular weight cut-off (MWCO) UF membrane, (b) diafiltered (e.g., with 6x diafiltration volumes) into a formulation buffer and the resulting concentrated product is measured and diluted (e.g., to 10.0 g/L). In another embodiment, the formulation buffer comprises 10 mM sodium phosphate (pH 7.0) and 150 mM sodium chlordide. In another embodiment, the diluted product is filtered (e.g., through a 0.5/0.2 mhi filter) and Polysorbate 80 is added to a diluted product pool to achieve a final concentration of

0.02% (w/v) Polysorbate 80.

In another embodiment, the concentration and diafiltration step includes the use of one or more (e.g., one, two, three, four, five, six, seven, eight or nine) buffers, including, but not limited to: (a) WFI, (b) sodium hydroxide, (c) sodium phosphate and 150 mM sodium chloride, and (d) polysorbate 80. In another embodiment, the concentration and diafiltration step includes the use of WFI as a flush. In another embodiment, the concentration and diafiltration step includes the use of 0.5 M sodium hydroxide for sanitization. In another embodiment, the concentration and diafiltration step includes the use of 10 mM sodium phosphate (pH 7.0) and 150 mM sodium chloride for equilibration, diafiltration, chase and/or pool dilution. In another embodiment, the concentration and diafiltration step includes the use of 0.1 M sodium hydroxide for storage. In another embodiment, the concentration and diafiltration step includes the use of 10% (w/v) Polysorbate 80 for excipient.

In another embodiment, the concentration and diafiltration step includes one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen or nineteen) of the following: a dilution of within 1% of calculated volume in the NOR and within 3% of calculated volume in the PAR; 10% (w/v) Polysorbate 80 is 0.19 - 0.21% (w/v) of diluted UF/DF product in the NOR and

0.17 - 0.23% (w/v) of diluted UF/DF product in the PAR; an un-formulated drug substance pH of 6.5-7.5; a diluted UF/DF product concentration of 9.0 - 11.0 mg/mL; passing a pre-use integrity test; a membrane loading of 100 - 500 g/m ² in the NOR and within 50 - 600 g/m ² in the PAR; a feed flux of 240 - 420 LMH in the NOR and within 180 - 440 LMH in the PAR; a transmembrane pressure of 10 - 30 psi in the NOR and within 8 - 35 psi in the PAR; a pressure of 15 - 25°C in the NOR and within 12 - 30°C in the PAR; a fed batch ratio of 1-3 in the NOR and within 1-5 in the PAR; a concentration at end of ultrafiltration target of 13 - 17 g/L in the NOR and within 12 - 20 g/L in the PAR; a diavolume of 5.5 - 7.0 in the NOR and within 4.5 - 7.0 in the PAR; an unformulated UF/DF retentate hold of < 4 days in the NOR and within < 6 days in the PAR; a product hold (diluted UF/DF product) of < 7 days in the NOR and within < 14 days in the PAR; a step yield of > 90%; a processing time (start of initial concentration through end of diafiltration) of < 11.1 hours; a post-use normalized water permeability (NWP) flux of 75 - 125% of initial; a diluted UF/DF pre-filtration pool bioburden of < 10 CFU/10 mL; and/or a diluted UF/DF post-filtration pool bioburden of

< 3 CFU/10 mL; and/or a diluted UF/DF post-filtration pool endotoxin concentration of

< 2 EU/mL.

In another embodiment, the concentration and/or diafiltration steps include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty) of the following: an initial concentration target of 40 - 60 g/L; a final concentration target (140 - 160 g/L) (includes 1.07 recovery factor); a diavolume of 4.5 - 7.5, with a target of 6.0; an undiluted

ultrafiltrated/diafiltrated product hold of < 24 hours); a diluted ultrafiltrated/diafiltrated product hold of < 24 hours); use of a Millipore Pellicon 3 Ultracel C screen 30 kDa MWCO filter; a flush WFI > 20 L/m ² ; an equilibrium of 50 mM NaPCL (pH 7.4), 25 mM L-Arg (> 20 L/m ² ); a membrane load of < 600 L/m ² ; a target feed flow rate for all product steps of 360 LMH; a target transmembrane pressure for all product steps of 15 psi; a feed pressure or

< 50 psi (can be increased); a diafiltration buffer that is the same as equilibrium; a final concentration that can be controlled by feed pressure (not TMP or Feed Flow Rate); a temperature of 15 - 35°C; a recovery with < lx system hold-up volume (calculation required per CSD); a dilution to target 120 g/L with DF/equilibrium buffer; 0.1919 - 0.2393 kg/kg addition of excipient addition buffer (EAB - 50 mM NaPCL (pH 7.4), 25 mM L-Arg, 30 % Sucrose 0.30 % (w/v), PS 80) to 120 g/L UF/DF product for final formulation; membrane re-use up to 20 cycles; sanitization with 0.5 M NaOH, storage with 0.1 M NaOH; a yield of > 60 % (expected over 90%); express SHC filterability 120 g/L UF/DF product: < 40 L/m ² ; and express SHC filterability BDS of < 3045 L/m ² .

Bulk Filtration

The methods described herein can also include a bulk filtration step. In one embodiment, material from a previous step (e.g., from the ultrafiltration and diafiltration step) is bulk filtered.

Anti-C5 Antibodies

The term“antibody” describes a polypeptide comprising at least one antigen binding site (e.g., VH/VL region or Fv, or CDR). Antibodies include known forms of antibodies, including, but not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), immunoadhesins, antibody-immunoadhesin chimeras, humanized, human, chimeric, single-chain, camelid, synthetic, recombinant, hybrid, mutated, grafted, or in vitro generated antibodies. The antibody can be a full-length antibody or an antibody fragment. The antibody can be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. The antibody also can be a Fab, Fab’2, ScFv, SMIP, Affibody ^® , nanobody, or a single domain antibody. The antibody also can be of any of the following isotypes: IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, IgE or chimeric versions thereof. The antibody can be a naturally occurring antibody or an antibody that has been altered by a protein engineering technique (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). An antibody can include, for example, one or more variant amino acids (compared to a naturally occurring antibody), which changes a property (e.g., a functional property) of the antibody. Numerous such alterations are known in the art that affect, e.g., half-life, effector function, and/or immune responses to the antibody in a patient. The term antibody also includes artificial or engineered polypeptide constructs that comprise at least one antibody-derived antigen binding site.

An exemplary anti-C5 antibody is ravulizumab comprising heavy and light chains having the sequences shown in SEQ ID NOs:l4 and 11, respectively, or antigen binding fragments and variants thereof. Ravulizumab is described in PCT/US2015/019225 and US Patent No:9,079,949, the teachings of which are hereby incorporated by reference. Ravulizumab selectively binds to human complement protein C5, inhibiting its cleavage to C5a and C5b during complement activation. This inhibition prevents the release of the pro-inflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9 while preserving the proximal or early components of complement activation (e.g., C3 and C3b) essential for the opsonization of microorganisms and clearance of immune complexes.

In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of ravulizumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ravulizumab having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of the VL region of ravulizumab having the sequence set forth in SEQ ID NO:8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs:l9, 18 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs:4, 5 and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

In other embodiments, the anti-C5 antibody produced according to the methods disclosed herein is formulated in a 10 mg/mL solution. In another embodiment, the anti-C5 antibody is formulated in a sterile, preservative-free 10 mg/mL solution e.g., which is suitable for IV administration. In another embodiment, the anti-C5 antibody is supplied in 20 mL single-use vials. In another embodiment, each vial contains 150 mg of ravulizumab in 10 mM sodium phosphate, 150 mM sodium chloride, 0.02% polysorbate 80, and water for injection at a pH of 7.0.

In other embodiments, the anti-C5 antibody produced according to the methods disclosed herein is formulated in a 100 mg/mL solution. In another embodiment, the anti-C5 antibody is formulated in a sterile, preservative-free 100 mg/mL solution e.g., which is suitable for subcutaneous administration. In another embodiment, the anti-C5 antibody is supplied in 2 mL single-use vials. In another embodiment, each vial contains 100 mg/mL of ravulizumab in 50 mM sodium phosphate, 25 mM arginine, 5% sucrose, and 0.05% polysorbate 80, and water for injection at a pH of 7.4.

The exact boundaries of CDRs have been defined differently according to different methods. In some embodiments, the positions of the CDRs or framework regions within a light or heavy chain variable domain can be as defined by Rabat et al. [(1991)“Sequences of Proteins of Immunological Interest.” NIH Publication No. 91-3242, U.S. Department of Health and Human Services, Bethesda, MD]. In such cases, the CDRs can be referred to as “Kabat CDRs” (e.g.,“Rabat LCDR2” or“Rabat HCDR1”). In some embodiments, the positions of the CDRs of a light or heavy chain variable region can be as defined by Chothia, C. et al. (Nature, 342:877-83, 1989). Accordingly, these regions can be referred to as “Chothia CDRs” (e.g.,“Chothia LCDR2” or“Chothia HCDR3”). In some embodiments, the positions of the CDRs of the light and heavy chain variable regions can be as defined by a Rabat-Chothia combined definition. In such embodiments, these regions can be referred to as“combined Rabat-Chothia CDRs” (Thomas, T. et al , Mol. Immunol. , 33:1389-401, 1996) exemplifies the identification of CDR boundaries according to Rabat and Chothia definitions.

In some embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR1 comprising or consisting of GHIFSNYWIQ (SEQ ID NO: 19). In some embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR2 comprising or consisting of EILPGSGHTEYTENFRD (SEQ ID NO: 18). In some embodiments, an anti-C5 antibody described herein comprises a heavy chain variable region comprising

QVQLVQSGAEVRRPGASVRVSCRASGHIFSNYWIQWVRQAPGQGLEWMGEILPGS GHTEYTENFRDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDV W GQGTLVT V S S (SEQ ID NO: 12).

In some embodiments, an anti-C5 antibody described herein comprises a light chain variable region comprising

DIQMTQSPSSLS AS VGDRVTITCGASENIY G ALNWY QQRPGRAPRLLIY GATNLADG VPSRFS GS GS GTDFTLTIS S LQPEDF ATY Y CQN VLNTPLTFGQGTRVEIR (SEQ ID NO:8).

Another exemplary anti-C5 antibody is antibody BNJ421 comprising heavy and light chains having the sequences shown in SEQ ID NOs:20 and 11, respectively, or antigen binding fragments and variants thereof. BNJ421 is described in PCT/US2015/019225 and US Patent No. 9,079,949, the teachings of which are hereby incorporated by reference. In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of BNJ421. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of BNJ421 having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of the VL region of BNJ421 having the sequence set forth in SEQ ID NO:8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: l9, 18 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs:4, 5 and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO:8, respectively.

Another exemplary anti-C5 antibody is the 7086 antibody described in US Patent Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 7086 antibody. In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 21, 22 and 23, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 24, 25 and 26, respectively. In another embodiment, the antibody or antigen binding fragment thereof comprises the VH region of the 7086 antibody having the sequence set forth in SEQ ID NO:27, and the VL region of the 7086 antibody having the sequence set forth in SEQ ID NO:28.

Another exemplary anti-C5 antibody is the 8110 antibody also described in US Patent Nos. 8,241,628 and 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 8110 antibody. In another embodiment, the antibody, or antigen binding fragment thereof, comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 29, 30 and 31, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 32, 33 and 34, respectively. In another embodiment, the antibody comprises the VH region of the 8110 antibody having the sequence set forth in SEQ ID NO:35, and the VL region of the 8110 antibody having the sequence set forth in SEQ ID NO:36.

Another exemplary anti-C5 antibody is the 305LO5 antibody described in

US2016/0176954A1. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody. In another embodiment, the antibody, or antigen binding fragment thereof, comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs:37, 38 and 39, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs:40, 41 and 42, respectively. In another embodiment, the antibody comprises the VH region of the 305LO5 antibody having the sequence set forth in SEQ ID NO:43, and the VL region of the 305LO5 antibody having the sequence set forth in SEQ ID NO:44. Another exemplary anti-C5 antibody is the SKY59 antibody (Fukuzawa, T. el al, Sci. Rep. , 7:1080, 2017). In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody. In another embodiment, the antibody, or antigen binding fragment thereof, comprises a heavy chain comprising SEQ ID NO:45 and a light chain comprising SEQ ID NO:46.

Another exemplary anti-C5 antibody is the REGN3918 antibody (also known as H4H12166PP) described in US20170355757. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO:47 and a light chain variable region comprising SEQ ID NO:48. In another embodiment, the antibody comprises a heavy chain comprising SEQ ID NO:49 and a light chain comprising SEQ ID NO:50.

An anti-C5 antibody described herein can, in some embodiments, comprise a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn) with greater affinity than that of the native human Fc constant region from which the variant human Fc constant region was derived. The Fc constant region can comprise, for example, one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant human Fc constant region was derived. The substitutions, for example, can increase the binding affinity of an IgG antibody containing the variant Fc constant region to FcRn at pH 6.0, while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining pH dependence of the interaction) are known in the art and exemplified in the working examples (PCT/US2015/019225 and US Patent No. 9,079,949 the disclosures of each of which are incorporated herein by reference in their entirety).

Substitutions that enhance the binding affinity of an antibody Fc constant region for FcRn are known in the art and include, e.g., (1) the M252Y/S254T/T256E triple substitution (Dall’Acqua, W. et al, J. Biol. Chem., 281: 23514-24, 2006); (2) the M428L or

T250Q/M428L substitutions (Hinton, P. et al, J. Biol. Chem., 279:6213-6, 2004; Hinton, P. et al, J. Immunol., 176:346-56, 2006); and (3) the N434A or T307/E380A/N434A substitutions (Petkova, S. et al, Int. Immunol., 18:1759-69, 2006). Additional substitution pairings, e.g., P257I/Q311I, P257I/N434H, and D376V/N434H, have also been described (Datta-Mannan, A. et al, J. Biol. Chem., 282:1709-17, 2007). The entire teachings of each of the cited references are hereby incorporated by reference. In some embodiments, the variant constant region has a substitution at EU amino acid residue 255 for valine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 309 for asparagine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 312 for isoleucine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 386.

In some embodiments, the variant Fc constant region comprises no more than 30 (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four, three or two) amino acid substitutions, insertions or deletions relative to the native constant region from which it was derived. In some embodiments, the variant Fc constant region comprises one or more amino acid substitutions selected from the group consisting of: M252Y, S254T, T256E, N434S, M428L, V259I, T250I and V308F. In some embodiments, the variant human Fc constant region comprises a methionine at position 428 and an asparagine at position 434, each in EU numbering. In some embodiments, the variant Fc constant region comprises a 428L/434S double substitution as described in, e.g., U.S. Patent No. 8,088,376 the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments the precise location of these mutations may be shifted from the native human Fc constant region position due to antibody engineering. The 428L/434S double substitution when used in a IgG2/4 chimeric Fc, for example, may correspond to 429L and 435S as in the M429L and N435S variants found in ravulizumab and described in US Patent Number 9,079,949 the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the variant constant region comprises a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382,

384, 385, 386, 387, 389, 424, 428, 433, 434 or 436 (EU numbering) relative to the native human Fc constant region. In some embodiments, the substitution is selected from the group consisting of: methionine for glycine at position 237; alanine for proline at position 238; lysine for serine at position 239; isoleucine for lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan or tyrosine for threonine at position 250; phenylalanine, tryptophan or tyrosine for methionine at position 252; threonine for serine at position 254; glutamic acid for arginine at position 255; aspartic acid, glutamic acid or glutamine for threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine or valine for proline at position 257; histidine for glutamic acid at position 258; alanine for aspartic acid at position 265; phenylalanine for aspartic acid at position 270; alanine or glutamic acid for asparagine at position 286; histidine for threonine at position 289; alanine for asparagine at position 297; glycine for serine at position 298; alanine for valine at position 303; alanine for valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine for threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine or threonine for valine at position 308; alanine, aspartic acid, glutamic acid, proline or arginine for leucine or valine at position 309; alanine, histidine or isoleucine for glutamine at position 311; alanine or histidine for aspartic acid at position 3l2;lysine or arginine for leucine at position 314; alanine or histidine for asparagine at position 315; alanine for lysine at position 317; glycine for asparagine at position 325; valine for isoleucine at position 332; leucine for lysine at position 334; histidine for lysine at position 360; alanine for aspartic acid at position 376; alanine for glutamic acid at position 380; alanine for glutamic acid at position 382; alanine for asparagine or serine at position 384; aspartic acid or histidine for glycine at position 385; proline for glutamine at position 386; glutamic acid for proline at position 387; alanine or serine for asparagine at position 389; alanine for serine at position 424; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan or tyrosine for methionine at position 428; lysine for histidine at position 433; alanine, phenylalanine, histidine, serine, tryptophan or tyrosine for asparagine at position 434; and histidine for tyrosine or phenylalanine at position 436, all in EU numbering.

Exemplary anti-C5 antibodies comprise a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and/or a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:l l. Alternatively, the anti-C5 antibodies can comprise a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:20 and/or a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11.

In one embodiment, the antibody binds to C5 at pH 7.4 and 25°C (and, otherwise, under physiologic conditions) with an affinity dissociation constant (K _D ) that is at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95 or 0.975) nM. In some embodiments, the KD of the anti-C5 antibody, or antigen binding fragment thereof, is no greater than 1 (e.g., no greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2) nM.

In other embodiments, the [(K _D of the antibody for C5 at pH 6.0 at 25°C)/(K _D of the antibody for C5 at pH 7.4 at 25°C)] is greater than 21 (e.g., greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,

160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450,

500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500 or 8000).

The following examples are merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES

EXAMPLE 1: RAVULIZUMAB MANUFACTURING PROCESS

A vial of the Working Cell Bank (WCB) containing 3A5-50D6 cells of Chinese hamster ovary (CHO) origin was thawed and cultures were progressively expanded using growth medium through a series of cell culture steps using shake flasks, rocker bioreactors, and seed expansion bioreactors prior to inoculation into the production bioreactor. Upon completion of the cell culture steps, cells and cell debris were removed by a series of depth filters. The clarified harvest was filtered through a 0.5/0.2 mhi filter prior to purification.

The downstream ravulizumab drug substance manufacturing process includes three chromatography steps (MabSelect SuRe ^™ Protein A affinity chromatography, POROS ^® HS50 cation exchange (CEX) chromatography, and POROS ^® HQ50 anion exchange (AEX) chromatography), a low pH vims inactivation step, and a virus filtration (20 nm) step.

Following the final concentration/diafiltration and formulation step, the formulated diluted ultrafiltration/diafiltration (UF/DF) pool was 0.5/0.2 mhi filtered into containers and ravulizumab drug substance was stored at 2 - 8°C pending batch disposition, long term storage and shipment to the drug product manufacturing facility. A flow diagram of the manufacturing process for ravulizumab drug substance is set forth in Figure 1. Throughout the manufacturing process, single use consumables including bioreactor bags, intermediate hold vessels and filters were utilized.

The criticality of all parameters and attributes of the drug substance manufacturing process has been determined to employ a comprehensive control strategy for ravulizumab that ensures consistent potency and safety of the drug substance. Upon transfer to the manufacturing site, a facility specific risk assessment was completed to align with site policy/definitions. There was no change in the criticality assigned to any parameter or attribute from the control strategy.

Process characterization studies, driven by risk assessments, were completed to determine a proven acceptable range (PAR) for each parameter identified within the manufacturing process that has the potential to impact product quality. A normal operating range (NOR) that is within or equal to the PAR was specified for routine operation within the manufacturing process description. During routine processing, any excursion outside of the NOR would have prompted the initiation of a deviation and subsequent investigation.

Following investigation, material would have been dispositioned accordingly.

Ravulizumab was manufactured in CHO cells at the 2,000 L bioreactor scale. The cells used to inoculate the 2,000 L production bioreactor originated from a single WCB vial. Multiple drug substance batches, however, could have been produced from a single vial of WCB by utilizing rollback cultures. Each drug substance batch yielded approximately 300 L at a concentration of 10 g/L ravulizumab. Antibody expression and step yields are factors in the ravulizumab drug substance manufacturing process that affect the individual batch yield. A unique identifying batch number was assigned for each unit operation. In the event of reprocessing at the viral filtration or bulk filtration steps, a new unique identifying batch number was assigned.

1. Cell Culture and Primary Recovery

This section describes the cell culture and primary recovery (harvesting) process for the manufacture of ravulizumab drug substance. The cell culture and harvesting process comprised three discrete steps (inoculum expansion, cell culture in production bioreactor, and primary recovery) as summarized in Figure 1 and detailed further in Figures 2 - 4.

a. Step 1 : Inoculum Expansion

The purpose of the inoculum expansion process step was to expand the WCB to a sufficient cell mass to inoculate the 2,000 L production bioreactor. The temperature of the culture conditions during the inoculum expansion step was maintained at a set point of 36.5°C. Figure 2 outlines the process flow and key process parameters (KPP) and key process attributes (KPA) for the inoculum expansion manufacturing process. Table 1 includes the KPP and KPA for the inoculum expansion.

A vial of the WCB was removed from vapor phase of liquid nitrogen storage and transferred to a biosafety cabinet in the production suite. The cells were thawed, washed to remove the cryopreservation medium, and re-suspended and cultured in a 250 mL shake flask with inoculum media (CD-CHO AGT medium supplemented with 25 mM I, -methionine sulfoximine (MSX)). The flask culture was incubated under 10% CO2 and agitation for up to 6 days until the criteria described in Table 1 were met prior to seeding into a 1 L shake flask. This initiates the shake flask phase of the inoculum expansion.

The cells were expanded in inoculum media incubated under 10% CO2 and agitation for up to 6 days until the criteria described in Table 1 were met prior to seeding into the rocker bioreactor phase of inoculum expansion. The 1 L shake flask culture could have been used to inoculate a backup culture. If the backup culture was not needed, it was discarded at the 20 L rocker bioreactor expansion.

The cells were further expanded in inoculum media in rocker bioreactors with increasing volumes (2 L to 20 L) prior to transitioning from rocker bioreactors to a 50 L seed bioreactor. Each rocker bioreactor was incubated with 5% CO2 and rocked for up to 6 days until the criteria described in Table 1 were met prior to proceeding into the next rocker bioreactor of increased size or seeding into the seed bioreactor phase of inoculum expansion. If required, the 20 L rocker bioreactor culture could have been used to inoculate a rollback 2 L culture.

The cells were expanded in expansion media (CD-CHO AGT medium without MSX supplementation) in single use bioreactors (SUB) with increasing volumes (50 L to 500 L) prior to transitioning to Step 2 of the process. Following inoculation, each SUB was sparged with air and oxygen as required with agitation for up to 6 days until the criteria described in Table 1 was met prior to proceeding to the next SUB of increased size or proceeding to Step 2 of the process. A dissolved oxygen set point of 30% and pH of set point of 6.95 was maintained. pH was controlled using sodium carbonate as base and CO2 as acid. Antifoam (animal-origin free) could have been added to the seed bioreactor if necessary to control foaming. Table 1: Processing Conditions for Inoculum Expansion

allowed and do not constitute an excursion

b. Step 2: Cell Culture in Production Bioreactor

The purpose of this step was to produce the ravulizumab antibody. Figure 3 outlines the process flow and critical process parameters (CPPs) and KPPs, in process controls (IPC) and KPAs for the cell culture process in the production bioreactor. Copper sulfate pentahydrate was added to the 2,000 L production bioreactor at a concentration of 20 mM. The 500 L SUB cell culture was inoculated into production media (CD-CHO AGT medium supplemented with 0.34 g/kg L-cysteine hydrochloride

monohydrate and 0.27 g/kg L-tyrosine). The pH of the bioreactor was controlled by use of CO2 for acidic control and 1 M sodium carbonate for base control. The pH was maintained at a target of 6.95 and the temperature was maintained at a target of 36.5°C. To minimize foaming, antifoam could have been added when there was excessive foam. The dissolved oxygen was controlled at a set point of 30% by sparging air and oxygen as required. The culture was supplemented with glucose as needed. Efficient Feed C+ was added to the production bioreactor on Day 4, Day 6 and Day 8.

The production bioreactor phase of the process continued until the harvest criteria specified in Table 3 was met prior to proceeding to Step 3 of the process. Samples were taken for bioburden, mycoplasma, in vitro vims assay, and murine minute virus assay by quantitative polymerase chain reaction (q-PCR).

Table 2: Critical Processing Conditions for Cell Culture in Production Bioreactor

¹ Range applicable after DO drops to set point in the first few days of the culture (typically 0- 3 days). DO excursions down to 0% or up to 100% for up to 1 hour is acceptable. In addition, DO excursions to < 5% for up to 24 hours during growth phase (days 0 - 6) was acceptable.

Table 3: Key Processing Conditions for Cell Culture in Production Bioreactor

¹ pH excursions up to 7.60 for a maximum of 3 hours were acceptable during stationary phase (days 7-10), pH excursions for a maximum of 3 hours were acceptable during stationary phase (days 7-10) and death phase of the cell culture (day 11-14).

² pCCL excursions over 250 mmHg for purpose of pH control were acceptable.

c. Step 3: Primary Recovery: Clarification and Post-Harvest 0.5/0.2 pm

Filtration

The primary recovery step separates the ravulizumab antibody in the cell culture broth from the cells and cellular debris. The depth filtration train was flushed and equilibrated prior to use. The cell culture broth was filtered and chased through a two-step depth filtration train in series immediately followed by filtration through two 0.5/0.2 pm filters in series into a jacketed 2,000 L single-use mixing bioprocess container.

Figure 4 outlines the process flow and KPPs and KPAs for the ravulizumab Step 3 of the manufacturing process. Buffers used in the primary recovery step are presented in Table 4. Table 5 includes the KPPs and KPAs for the primary recovery step.

Table 4: Buffers used for Primary Recovery

Table 5: Key Processing Conditions for Primary Recovery

¹ Excluding flush and equilibration

2. Purification and Modification Reactions

The purification process was designed to purify ravulizumab by removal of process and product related impurities from the clarified harvest using orthogonal purification steps, followed by concentration and formulation into bulk drug substance (BDS). Figure 5 provides an overview of the ravulizumab purification process. The temperature during the entire purification process was maintained at ambient temperature.

The purification process started with the capture of the ravulizumab antibody by MabSelect SuRe Protein A affinity chromatography. This pool was then treated with a low pH viral inactivation hold, followed by further purification by POROS HS50 CEX chromatography and POROS HQ50 AEX chromatography steps. This pool was then filtered through a Viresolve Pro ^® 20 nm virus reduction filtration step. This filtrate was concentrated and diafiltered into the drug product formulation buffer. Finally, polysorbate 80 was added to complete formulation of the product. The resulting material was 0.5/0.2 mhi filtered into bags for storage at 2 - 8°C prior to shipment for drug product manufacturing.

Throughout the process 0.5/0.2 mhi filtration was used as described in Figure 5.

Process intermediates were sampled for bioburden and endotoxin at the end of their respective in process holds, just prior to filtration. Limits for microbial monitoring were included in Table 7, Table 9, Table 12, Table 15, Table 18, Table 21 and Table 23. a. Step 4: MabSelect SuRe Protein A Affinity Chromatography

The purpose of this step was the primary capture of the ravulizumab antibody.

Protein A chromatography is an affinity chromatography step in which the resin selectively binds to the FC portion of ravulizumab, allowing impurities to flow through the packed column. The bound product is then eluted from the resin by decreasing the pH with the elution buffer. Buffers used for this step are listed in Table 6.

Before first use of each batch and after every cycle, the column is cleaned/sanitized and equilibrated. Following column packing, the column performance was verified to achieve number of theoretical plates > 1000 N/m (KPA) and an asymmetry factor of 0.8- 1.6 (KPA).

The clarified harvest material from Step 3 served as the load for the MabSelect SuRe column. The material was loaded onto the column through a 0.5/0.2 mhi filter. Impurities were removed with wash buffers. The product was eluted from the column using elution buffer. This step was normally operated in three cycles per batch and the eluates were pooled for further processing. The pool was stored at ambient temperature prior to the start of the next process step.

Table 7 summarizes the KPPs and KPAs for the MabSelect SuRe chromatography step. The column load was calculated based on titer by Protein A HPLC of the clarified harvest. The step yield was based on the concentration measurement (based on ProA titer) from the clarified harvest and the concentration measured by A280 from the MabSelect SuRe Protein A Affinity pool. After each cycle, the column was stripped, flushed, and

cleaned/sanitized.

Table 6: Buffers used for the MabSelect SuRe Protein A Affinity Chromatography

Table 7: Key Processing Conditions for the MabSelect SuRe Protein A Affinity Chromatography

b. Step 5: Low pH Hold Viral Inactivation

The purpose of this step was to inactivate potential enveloped viruses in the process stream by low pH treatment. The Protein A pool was treated with 1 M acetic acid to a pH range of 3.60 to 3.70. Following the pH adjustment, the pool was transferred to a second vessel and incubated at ambient temperature for a minimum of 60 minutes without mixing during hold. At the completion of the incubation, the pH was measured again following the incubation to be within 3.60 to 3.75. Following the hold, the pH was increased to pH 5.0 using 1 M Tris and incubated at ambient temperature for a minimum of 60 minutes without mixing during the hold to allow consistent precipitate formation that is subsequently removed by filtration. The neutralized viral inactivated material was pre-filtered and then 0.5/0.2 mhi filtered and stored at ambient temperature until the initiation of Step 6.

Table 8 summarizes the CPPs and acceptance criteria. Table 9 summarizes the KPPs and KPAs for the low pH virus inactivation step. The step yield was based on the concentration measurement (A ₂₈₀ ) from the MabSelect SuRe Protein A Affinity pool and the concentration measure by A ₂₈₀ from the neutralized viral inactivated material.

Table 8: Critical Processing Conditions for the Low pH Virus Inactivation

Table 9: Key Processing Conditions for the Low pH Virus Inactivation

c. Step 6: POROS HS50 Cation Exchange Chromatography (CEX)

The purpose of this step is to remove high-molecular-weight impurities as well as other process-related impurities and potential viruses from the process stream. The in-process material from the viral inactivation step was purified using a chromatography column in which the product binds to POROS HS50 CEX resin. The bound product was then eluted from the resin by increasing ionic strength with the elution buffer. This step was normally operated in one cycle.

Before use and after every batch the column was sanitized. Buffers used for this step are listed in Table 10. Following column packing, the column performance was verified to achieve number of theoretical plates > 1000 N/m (KPA) and an asymmetry factor of 0.8- 1.6 (KPA).

For each cycle load, the neutralized filtrate from the low pH hold, served as the load for the cation exchange column. The material was loaded onto the column through a

0.5/0.2 mhi filter. The product was eluted from the column using elution buffer. The single eluate was collected through an inline 0.5/0.2 mhi filter and stored at ambient temperature until the initiation of Step 7.

Table 11 summarizes the CPP and acceptance criteria and Table 12 summarizes the KPPs and KPAs for the CEX step. The column load was calculated based on the A ₂ so of the filtrate from Step 5. The step yield was based on the A ₂ so measurement from the Low pH filtrate and CEX filtered eluate. After each cycle, the column was stripped, cleaned/sanitized, flushed and stored in final storage solution. Table 10: Buffers used for the POROS HS50 CEX Chromatography

d. Step 7: POROS HQ50 Anion Exchange Chromatography (AEX) The purpose of this step was to further remove high-molecular-weight impurities, as well as other process related impurities and potential viruses from the process stream. The adjusted CEX eluate was further purified using a chromatography column packed with POROS HQ50 anion exchange resin and operated in flow-through mode. The step is normally operated in one cycle.

The CEX pool was adjusted to a pH of 8.00 and conductivity of 8.5 mS/cm with

100 mM Tris (pH 9.0), 180 mM arginine and WFI. Processing over the chromatography column was initiated within 24 hours of the load adjustment. Before use, the column was sanitized, conditioned and equilibrated. Buffers used for this step are listed in Table 13. Following column packing, the column performance was verified to achieve number of theoretical plates > 1000 N/m (KPA) and an asymmetry factor of 0.8-1.6 (KPA). The adjusted material was loaded onto the column through a 0.5/0.2 mhi filter. The product was chased from the column using buffer filtered through an inline 0.5/0.2 mhi into the filtrate vessel.

Table 14 summarizes the CPP and acceptance criteria and Table 15 summarizes the KPPs and KPAs for the AEX step. The adjusted material column load was calculated based on the A ₂₈ o of the filtrate from the CEX step. The expected step yield was based on the A ₂₈₀ measurement from the CEX and AEX pools. After each cycle, the column was stripped, sanitized and stored.

Table 14: Critical Processing Conditions for the POROS HQ50 AEX Chromatography

e. Step 8: Viral Filtration (20 nm)

The purpose of this step was to remove potential viruses or vims-like particles from the process stream on the basis of size. Viral reduction was accomplished by filtration of the AFX flow-through filtrate through a Viresolve Pro Shield H pre-filter followed by filtration through a Viresolve Pro filter (20 nm).

Prior to use, the 20 nm filters were integrity tested and were flushed using WFI and buffer. The material was loaded onto the filter followed by a flush with 20 mM Tris (pH 7.6), 65 mM sodium chloride to minimize product loss. The Viresolve filtrate was 0.5/0.2 mhi filtered. The virus reduction filter was post-use integrity tested.

Table 16 includes the buffers used at this step. The filters were integrity tested. In the event that it was determined reprocessing was justified, the material could have been reprocessed once. Reprocessing was not conducted due to bioburden above the action limits. Table 17 summarizes the CPPs and the acceptance criteria and Table 18 summarizes the KPPs and KPAs for this virus filtration step. The step yield was based on the A280 measurements from the AEX filtrate and Viresolve filtrate and was stored at ambient conditions.

Table 16: Buffers used for the Vims Filtration

Table 17: Critical Processing Conditions for the Vims Filtration

Table 18: Key Processing Conditions for the Virus Filtration

f. Step 9: Ultrafiltration/Diafiltration (30 kDa) and Formulation

The purpose of this UF/DF step was to concentrate the process stream to its specified concentration and to exchange the in process buffer with the formulation buffer (10 mM sodium phosphate (pH 7.0), 150 mM sodium chloride) and then complete formulation by the addition of polysorbate 80.

Before use, product dedicated UF membranes were flushed, integrity tested and sanitized. The membranes were then equilibrated prior to loading of the viral filtrate. The buffers used in this step are outlined in Table 19.

The pool from the vims reduction filtration step was concentrated to a target of 15 g/L using 30 kDa MWCO UF membranes. The concentrated pool was then diafiltered with

6 diafiltration volumes into formulation buffer (10 mM sodium phosphate (pH 7.0), 150 mM sodium chloride). The UF membranes were flushed with formulation buffer to enhance product recovery. The product concentration was measured and diluted to 10.0 g/L. The diluted material was 0.5/0.2 mhi filtered. Polysorbate 80 was added to the diluted pool to achieve a final concentration of 0.02% (w/v) Polysorbate 80.

Table 20 summarizes the critical processing conditions and acceptance criteria and Table 21 summarizes the KPPs and KPAs for the UF/DF step. The step yield was based on A280 measurements post-UF/DF and the viral filtration filtrate.

Table 19: Buffers used for the UF/DF Formulation

Table 20: Critical Processing Conditions for the UF/DF Formulation

Table 21: Key Processing Conditions for the UF/DF Formulation

g. Step 10: Final Filtration, BDS Fill, Storage, and Transportation

The formulated BDS from Step 9 was filtered through 0.5/0.2 mhi filter into bioprocess bags. The expected percent yield was > 90% based on protein mass post-UF/DF and post- fill. After filling was complete, the final filter must have passed filter integrity testing. In the event that that it was determined reprocessing was required, the product could have been pooled into an identical UF/DF retentate vessel and the product could have been reprocessed once into BDS. Reprocessing was not conducted due to bioburden over-action limits. Table 22 summarizes the IPC and acceptance criteria and Table 23 summarizes the KPAs for the drug substance fill step.

Table 22: Critical Processing Conditions for the BDS Fill

Table 23: Key Processing Conditions for the BDS Fill

The BDS was labeled and stored at 2 - 8°C, and protected from light. The bioprocess containers were securely wrapped and stored in a sealed secondary plastic containment to add protection for the bioprocess container and minimize environmental variations in humidity. Following BDS release for forward processing, the BDS was shipped at 2 - 8°C for drug product manufacturing using an active temperature controlled shipper via a combination of surface road transport and air freight shipping.

3. Drug Product Manufacturing

Ravulizumab drug substance is supplied as an aseptically filled liquid product at a concentration of 10 mg/mL in 100 L disposable bags. The drug product manufacturing begins with pooling of the drug substance through a single 0.22 pm bioburden reduction filter into the compounding vessel. Once the pooling process is complete, the drug substance is sterile filtered and aseptically filled into sterile, depyrogenated vials using an automated filling machine (Filling Line 1). The aseptically filled vials are then stoppered and capped. There are no reprocessing steps or procedures allowed in the ravulizumab drug product manufacturing process. A flow diagram of the manufacturing process and controls for ravulizumab drug product is set forth in Figure 6. a. Step 1 : Equipment and Component Preparation

Wet component preparation activities were carried out in an ISO 8 (Grade D, Class 100,000) classified area. Dry preparation component activities, including the autoclaving of components, were conducted in an ISO 7 (Grade C, Class 10,000) classified area. Following preparation, sterile wrapped components were stored in ISO 6 (Grade B, Class 1,000) classified area.

Prior to each drug product manufacturing campaign, the stoppering bowl, tracks, pick/place heads, pusher pins and the surge vessel were cleaned out of place (COP) and sterilized in the autoclave.

The Bioburden Filtration and Sterile Filtration Manifold with incorporated filters and the Pooling Manifold were supplied pre- sterilized (gamma irradiated) and were disposable (single use). The sterilizing filters were integrity tested both pre- and post-use, and post-usage testing was carried out on the second filter. The first filter could have been tested post-use if there was a test issue with the second filter. The transfer line between the sterilizing filters was in an ISO 7 (Grade C, Class 10,000) classified area and the surge tank in ISO 5 (Grade A, Class 100) was composed of single use disposable tubing manifold supplied pre- sterilized.

The primary product-contact packaging components include the 30 mL glass vial and the rubber stopper. Prior to the start of filling operations, drug product container closure system components were processed and prepared for use as described below. Cleaning, sterilization and depyrogenation parameters are summarized in Table 24.

Vials were washed in an inverted state through a series of rinsing stations in the vial washer (ISO 7 (Grade C, Class 10,000)). Stations include internal and external rinses with recycled WFI, followed by internal and external rinses with fresh WFI. Filtered clean dry air was used to dry the internal and external vial surfaces prior to reversion and placement on the infeed belt of the depyrogenation tunnel. Vials were transferred to the heating zone, where electrically heated laminar air flowed continuously to depyrogenate the vials. Downstream of the heating zone, the vials entered a cooling zone where the glass temperature was progressively lowered to less than 25 °C. Depyrogenated vials were discharged into an ISO 5 (Grade A, Class 100) environment onto an accumulation table that feeds the filling machine. Bags of stoppers were supplied ready for sterilization and autoclaved on site. The sterilized stoppers were transferred to the filling machine through a transfer port into the stoppering bowl.

The seals were supplied ready for sterilization, autoclaved, brought into the capping and crimping area and loaded into the capping machine. The filling assembly, needle connectors, needles and surge tank were autoclaved and transferred into the ISO 5 (Grade A, Class 100) fill cabinet.

Table 24: Cleaning, Sterilization and Depyrogenation Apparatus and Parameters Monitored in Equipment and Component Preparation

b. Step 2: Pooling and Stirring

The drug substance was removed from 2 - 8°C storage and transferred to the pooling room (ISO 7, Grade C, Class 10,000). Up to two drug substance batches could have been pooled up to the validated maximum batch size into a single drug product batch.

Each bag was connected to a single use pooling manifold, and the drug substance was pumped one bag at a time by a peristaltic pump through a single 0.22 mhi bioburden reduction filter into the 1,000 L stainless steel (316 grade) temperature controlled jacketed

compounding tank. The transfer of the drug substance into the tank via peristaltic pump must have been completed within 12 hours of the removal of the drug substance from 2 - 8°C storage. The pooled drug substance was mixed at 60 RPM for 30 to 90 minutes and verified that temperature is 2 - 8°C before the mixer was turned off.

The drug substance could have been held in the compounding tank for up to 24 hours, at 2 - 8°C, before beginning sterile filtration and transfer to the filling machine. The process parameters and in-process control for the Pooling and Stirring step are presented in Table 25. Table 25: In-Process Controls for the Pooling/Stirring Step

c. Step 3: Sterile Filtration

Sterile filtration occurred under a closed system in an ISO 7 (Grade C, Class 10,000) classified area. When filling was ready to begin, a sample of the pooled drug substance was taken from the vessel for pre-filtration bioburden and endotoxin testing.

The drug substance was sterile filtered through two sequential hydrophilic 10” absolute 0.22 mhi Durapore polyvinylidene fluoride (PVDF) (KVGLG1TTT1) sterilizing grade filters each with a filtration area of 0.73 m ² . Pressure transfer (Nitrogen vessel set-point target pressure of 0.5 (range 0.2 - 1.3 Bar)) moved the product from the

compounding tank, through the two 0.22 pm filters arranged in series in the compounding room, through irradiated tubing in the ISO 6 (Grade B, Class 1,000) area and into the aseptic filling room (Grade A (RABS)/B (Room)). Both filters were integrity tested wetted with WFI pre-filtration. The second filter, closest to the filling line, was considered the final sterile product filter. This filter was bubble point tested, wetted with WFI, post-filtration. If the second filter failed to meet the integrity test criteria, the first filter was integrity tested with WFI post-use. The process parameters and in-process control for the Sterile Filtration step are presented in Table 26.

The post-use flushing procedure was as follows: Post-sterile filtration, a blowdown with process nitrogen was performed post each filter on the sterile filtration manifold until product was visually removed through to the manifold and onto the Filling Line surge tank.

Following this product blowdown, a WFI flush was performed on both filters. Sterilizing filter 1 was initially flushed with 10 L of WFI and then both filters were flushed with 36 L of WFI resulting in a total flush volume of 46 L of WFI. A post-use filter integrity test was then performed on the second filter.

Table 26: In-Process Controls for the Sterile Filtration Step

d. Step 4: Aseptic Filling

All product contact equipment used in the filling process was single use disposable.

The sterile filtered drug substance was aseptically filled into sterile, depyrogenated vials using an automated filling machine (Grade A RABS LAF in a Grade B room). Each vial was filled to 32.00 ± 0.96 g based on weight (1.008 g = 1.00 mL). Stoppers were then aseptically inserted into the filled vials by the filling machine. 100% in-process monitoring of fill weight was performed throughout the filling step to ensure that the vial fill weight was effectively monitored and controlled. In the event a vial was found outside of the fill weight limits, it was rejected.

During the filling operations, particulates and microbiological monitoring was performed for the ISO 5 environment (Grade A, Class 100 LAF) along with the system controls (e.g., temperature, differential pressure). Additional environmental and personnel monitoring were performed during filling as appropriate as per established procedures. The process parameters and in-process control for the Aseptic Filling step are presented in

Table 27.

Table 27: In-Process Controls for the Aseptic Filling Step

e. Step 5: Capping

The filled and stoppered vials were conveyed to the capping machine for capping in an ISO 5 (Grade A, Class 100) environment. Vials exited the capping machine into the ISO 8 (Grade D, Class 100,000) environment. The vials had a batch number printed on the seal by the inkjet printer. Filled, sealed and coded vials were loaded into polypropylene boxes. The process parameters and in-process control for the Capping step were presented in Table 28.

Table 28: In-Process Controls for the Capping Step

The entire Aseptic Filling step, including capping, must have been completed within 24 hours of the start of sterile filtration. The sealed vials were 100% visually inspected for particulates, seal defects, glass and minor defects. Defective vials were removed.

4. Storage and Shipping

The ravulizumab drug product was stored at 2 - 8°C. The drug product unlabeled vials were packed in a secondary container and transported under temperature controlled conditions for packaging and labeling. 5. Labeling and Secondary Packaging

Vials were labeled using a fully automated labeling machine in a dedicated production room. Prior to secondary packaging, labels, cartons and package inserts (PI) were inspected. Unlabeled vials were removed from 2 - 8°C storage and allowed to warm to ambient temperature for the remainder of the labeling and secondary packaging process.

During labeling, a label was applied to the vial and the labeled vial was inserted into a unit carton along with a PI and the carton was closed. Finished unit cartons were sampled at regular intervals and visually and/or electronically inspected. Finished unit cartons were packed into corrugated shippers for subsequent storage at 2 - 8°C and shipment to distributors. 6. Ravulizumab Drug Product

Ravulizumab is supplied as a sterile aqueous solution for intravenous administration containing at a concentration of 10 mg/mL in 10 mM sodium phosphate, 150 mM sodium chloride, 0.02% (w/v) Polysorbate 80 in a stoppered 30 mL glass vial. The quantitative and qualitative composition of the ravulizumab drug product are presented in Table 29. Excipients are tested to the United States Pharmacopeia (USP), European Pharmacopoeia (Ph. Eur.), and/or Japanese Pharmacopeia (JP). The ravulizumab vial content is based on the extractable volume.

Table 29: Ravulizumab Drug Product (10 mg/mL) Composition

The release and stability specification for ravulizumab drug product is presented in Table 30.

Table 30: Ravulizumab Drug Product Specification

¹ Release testing only

² Stability testing only; to be performed in lieu of sterility testing

EXAMPLE 2: UF/DF RECOVERY STRATEGY FOR HIGH CONCENTRATION

MONOCLONAL ANTIBODY AT MANUFACTURING SCALE

Ultrafiltration/Diafiltration (UF/DF) is a rapid and efficient method for separation and purification of biomolecules. For the production of highly concentrated monoclonal antibody intermediate solutions in the course of downstream processing, UF is the industry standard in manufacturing scale. UF/DF can be used to concentrate and desalt sample solutions ranging in volume from 10 mL to thousands of liters.

Key challenges associated with UFDF are achieving high end concentrations and reducing both process time and aggregate formation, particularly for therapeutic proteins intended for subcutaneous administration· UF process yields are affected by the system design and the recovery procedure.

The parameters affecting UF/DF recovery were identified by way of the present experiments as (1) recovery chase volume, (2) target bulk concentration, and (3) a ratio referred to herein as“F-factor”. F-factor is defined as ratio of system hold-up volume to total retentate volume. Recovery chase is defined as the volume of buffer added to chase remaining product held up in system. System hold-up volumes vary at different

manufacturing facilities. This may end up with a more diluted or concentrated product if hold up volume is not considered in the final formulation UF/DF. Table 31 defines key nomenclature.

Table 31: Nomenclature

A full factorial design of experiment (DOE) study was designed using JMP software around these three parameters. From this study a specific chase volume for a range of F-factors was defined which can be transferred to any manufacturing facility irrespective of the hold-up volume. The objectives of this study were to: (1) determine a specific chase volume for a range of F-factors that can be transferred to any manufacturing facility, irrespective of the hold-up volume and (2) decrease process time by minimizing the number of in-process A ₂₈ o measurements by developing a robust recovery method. Figure 7 shows the UF/DF schematic used for this study. 12 UF/DF runs were generated in a random order, which included two center point runs as shown in Table 32. The recovery chase was performed as dictated by the DOE design using DF buffer and recirculated for 10 minutes. Weights and concentrations were used to calculate the volume of chase to add to retentate product to target 120 g/L. If the combined product concentration was > 125 g/L, dilution was performed using DF buffer to target 120 g/L. A target concentration of 100 g/L was achieved by final excipient addition buffer (EAB).

The critical processing parameters (CPPs) for the 100 mg/mL final UF/DF and formulation were as follows: UFDF product time until excipient addition buffer (EAB) addition (end of final concentration until start of EAB addition) < 16 hours; and EAB addition: 0.1919 - 0.2393 kg EAB/kg diluted UFDF product.

The key processing parameters (KPP) were as follows:

Pre-use membrane integrity test;

Membrane load (g/m ² );

Feed flow rate (LMH);

TMP (psi);

Temperature (C);

Fed-batch ratio;

Initial concentration target (40 - 60 g/L);

Final concentration target (140 - 160 g/L);

Diavolumes (4.5 - 7.5);

Undiluted UF/DF product hold (< 24 hours); and

Diluted UF/DF product hold (< 24 hours).

The specific unit operation details were as follows:

Filter: Millipore Pellicon 3 Ultracel C screen 30 kDa MWCO;

Flush: WFI > 20 L/m ² ;

Equilibrium: 50 mM NaPCL (pH 7.4), 25 mM L-Arg (> 20 L/m ² );

Membrane load: < 600 L/m ² ;

Initial concentration: 40 - 60 g/L;

Feed flow rate (all product steps): target 360 LMH;

Transmembrane pressure (all product steps): target 15 psi;

Feed pressure: < 50 psi (can be increased);

DF Buffer: same as equilibrium;

Diavolumes: 4.5 - 7.5 (Target 6.0);

Final concentration to 140 - 160 g/L (includes 1.07 recovery factor);

Final concentration may be controlled by feed pressure (not TMP or Feed Flow Rate);

Temperature: 15 - 35°C;

Recovery with < lx system hold-up volume (calculation required per CSD); Dilution to target 120 g/L with DF/equilibrium buffer; 0.1919 - 0.2393 kg/kg addition of excipient addition buffer (EAB - 50 mM NaP0 ₄ (pH 7.4), 25 mM L-Arg, 30 % Sucrose 0.30 % (w/v), PS 80) to 120 g/L UF/DF product for final formulation;

Membrane re-use: Up to 20 cycles;

Sanitization: 0.5 M NaOH;

Storage: 0.1 M NaOH;

Yield: > 60 % (expected over 90%);

Express SHC filterability 120 g/L UF/DF product: < 40 L/m ² ; and

Express SHC filterability BDS: < 3045 L/m ² (please“right-size” filter to minimize product loss).

Additional experiments were performed to determine the change in buffer chase concentration with incremental buffer volume additions for an UF/DF evaluation at

0.2 F-factor and target bulk concentration of 150 g/L. The buffer volume and concentration data is shown in Table 33 and Figure 8. A linear regression was fit to the data and the slope and y-intercept was calculated.

Table 33: Buffer Chase Volume / Concentration Data (0.2 F Factor)

The target concentration for multivariate runs with 0.8 F-factor and buffer chase volumes 0.4 and l.lx were 140 and 160 g/L. An average of buffer chase concentration (C _c ) and combined retentate and chase concentration (C _a ) was performed to provide the theoretical concentrations at 150 g/L. The individual data points and averaged data used to calculate are shown in Table 34 and Table 35. The averaged 0.8 F-factor and DOE Experiment 11 data was used to construct a cohesive data set of buffer chase volumes related to buffer chase concentration as shown in Table 36 and Figure 9. Table 34: Concentration Data for 0.8 F Factor and 0.4X Chase

Table 35: Concentration Data for 0.8 F Factor and 1.1X Chase

Table 36: Buffer Chase Volume / Concentration Data (0.8 F Factor)

The slopes and y-intercept from Figure 8 and Figure 9 were averaged to provide a linear expression that could be applied across an F Factor range of 0.2-0.8 (see Table 37).

Table 37: Averaged Slopes and Y-Intercepts

The process recovery was defined as the yield after combining the retentate and buffer chase products. The recovery data from the multivariate study is provided in Table 32. These data indicate that the F-factor and the chase volume are significant factors impacting process recovery. Figure 9 shows the prediction profiler where DOE Experiment 02 was used an example. F-factors evaluated at 0.2 - 0.3 with a target bulk concentration of 140 and 160 g/L, and chase volumes 0.4-1. lx provided 87.2-91.7% yield. These data indicate that F-factors within 0.2-0.3 allow for a broader recovery chase strategy irrespective of the retentate product concentration. As the F-factor increases to ~0.9, however, the retentate concentration and buffer chase volume become significant to process recovery (Table 38).

Table 38: Recovery DOE Data

A model was developed using this data set and Equation 1 for predicting the acceptable chase volume range for a given F-factor to ensure > 120 g/L and 90% process recovery. F-factors were evaluated from 0.1-0.9, buffer chase volumes from 0.1-1.6x, and retentate concentration maintained at 140 g/L to account for the worst case concentration result. The theoretical diluted UF/DF product concentrations for each F-factor and buffer chase volume is provided in Table 38. F factor and buffer chase volume conditions that resulted in > 120 g/L are in bold and values < 120 g/L are underlined to clearly identify acceptable and non-acceptable conditions. Results from the study were used to group the F-factors into three levels and provide corresponding buffer chase volume ranges for ease of batch record implementation. Manufacturing control strategy was implemented from this study.

Table 39: Theoretical Diluted UF/DF Product Concentrations

In conclusion, the F Factor and buffer chase volume UF/DF parameters were classified as KPPs. Table 35 was used to group the F-factors into three levels and provide corresponding buffer chase volume ranges for ease of batch record implementation at CMOs. This eliminated the A ₂₈ o measurements of individual UF/DF product and Chase. Eliminating the A ₂₈ o measurements at these two steps decreased the total UF/DF processing time, which helps in maintaining the product stability. SEQUENCE SUMMARY