# US 10,370,456 B2 · Process for concentration of antibodies and therapeutic products thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,456 B2 - Document ID: US10370456B2 - Status: Granted - Kind: B2 - Application number: 14/257,907 - Filed: 2014-04-21 - Published: 2014-12-18 - Granted: 2019-08-06 - Earliest claimed: 2004-09-09 - Assignees: Genentech, Inc.; Novartis AG - Applicants: Genentech, Inc.; Novartis AG - Inventors: Charles M. Winter - Web page: https://patentgrep.com/patent/US10370456B2 ## CPC - C07K 16/4291: {against IgE} - A61K 39/39591: {Stabilisation, fragmentation} - B01D 61/146: {comprising multiple ultrafiltration steps} - B01D 61/16: Feed pretreatment - C07K 1/34: by filtration, ultrafiltration or reverse osmosis - C07K 16/065: {Purification, fragmentation} - B01D 2311/04: Specific process operations in the feed stream; Feed pretreatment - B01D 2311/16: Flow or flux control - C07K 2317/21: from primates, e.g. man - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) ## Abstract The present disclosure provides a process for concentrating proteins including an ultrafiltering, a diafiltering, and a second ultrafiltering sequence, at elevated temperatures, such as above about 30° C. The disclosure also includes a process for preparing highly concentrated antibody compositions, and highly concentrated antibody products. ## Claims ### Claim 1 A process for preparing highly concentrated monoclonal antibody in a pharmaceutical formulation comprising: a) concentrating a first purified antibody preparation by a first ultrafiltering to provide a second antibody preparation comprising the retentate of the first ultrafiltration in a first buffer, wherein the first purified antibody preparation has an antibody concentration of from about 0.1 to about 10 grams per liter, wherein the second antibody preparation has an antibody concentration of from about 10 to about 50 grams per liter, and wherein the first purified antibody preparation has been purified with ion-exchange chromatography prior to step a); b) exchanging the first buffer of the second antibody preparation generated in step a) into a second buffer by a diafiltering to provide a diafiltered intermediate antibody preparation comprising the retentate of the diafiltration, wherein the second buffer comprises excipients desired in the pharmaceutical formulation; c) concentrating the diafiltered intermediate antibody preparation generated in step b) by a second ultrafiltering to provide a third antibody preparation comprising the retentate of the second ultrafiltration, wherein the third antibody preparation is substantially free from aggregates and wherein the third antibody preparation has an antibody concentration of from about 150 to about 200 grams per liter; and d) formulating the third antibody preparation generated in step c) into the pharmaceutical formulation suitable for therapeutic administration; wherein the first ultrafiltering, second ultrafiltering, and the diafiltering are accomplished at 35° C. to about 50° C., and wherein the first and the second ultrafiltering are accomplished with an ultra-filter membrane having a nominal pore size of about 5 to about 50 kilo Daltons. ### Claim 2 (depends on 1) The process of claim 1 wherein one or more of the first ultrafiltering, the second ultrafiltering, and the diafiltering are accomplished at about 45° C. ### Claim 3 (depends on 1) The process of claim 1 wherein one or more of the first ultrafiltering, the second ultrafiltering, and the diafiltering are accomplished at 45° C. plus or minus 5° C. ### Claim 4 (depends on 1) The process of claim 1 wherein the first purified antibody preparation has an antibody concentration of about 1 to about 5 grams per liter. ### Claim 5 (depends on 1) The process of claim 1 wherein the second antibody preparation has an antibody concentration of about 20 to about 40 grams per liter. ### Claim 6 (depends on 1) The process of claim 1 wherein the third antibody preparation has an antibody concentration of from about 170 to about 200 grams per liter. ### Claim 7 (depends on 1) The process of claim 1 wherein the intermediate antibody preparation has an antibody concentration of about 25 to about 35 grams per liter and the third antibody preparation has an antibody concentration of from about 170 to about 200 grams per liter. ### Claim 8 (depends on 1) The process of claim 1 wherein the antibody preparation comprises an anti-IgE antibody. ### Claim 9 (depends on 1) The process of claim 1 wherein the process is accomplished in from about 1 to 10 hours. ### Claim 10 (depends on 1) The process of claim 1 wherein the process is accomplished in from about 2 to 5 hours. ### Claim 11 (depends on 1) The process of claim 1 wherein the process is accomplished in about 3 hours. ### Claim 12 (depends on 1) The process of claim 1 wherein the first and the second ultrafiltering are accomplished with an ultra-filter membrane having a nominal pore size of about 10 to about 30 kilo Daltons. ### Claim 13 (depends on 1) The process of claim 1 wherein the antibody in the first purified antibody preparation has an apparent molecular weight of about 100 to about 200 kilo Daltons. ### Claim 14 (depends on 1) The process of claim 1 wherein the antibody in the first purified antibody preparation has an apparent molecular weight of about 150 kilo Daltons. ### Claim 15 (depends on 1) The process of claim 1 wherein the first ultrafiltering concentrates the first purified antibody preparation to provide the second antibody preparation having an antibody concentration of about 30 grams per liter and the second ultrafiltering concentrates the intermediate antibody preparation to provide the third antibody preparation having an antibody concentration of about 170 to about 200 grams per liter. ### Claim 16 (depends on 1) The process of claim 1 wherein the first ultrafiltering and the second ultrafiltering are accomplished with the same ultra-filter membrane. ### Claim 17 (depends on 1) The process of claim 1 wherein the first ultrafiltering and the second ultrafiltering are accomplished with a regenerated cellulose composite ultra-filter membrane. ### Claim 18 (depends on 1) The process of claim 1 wherein the diafiltering accomplishes a buffer exchange at constant volume, constant concentration, or both. ### Claim 19 (depends on 1) The process of claim 1 wherein the diafiltering accomplishes a buffer exchange of from about 5 to about 15 fold volumes. ### Claim 20 (depends on 1) The process of claim 1 wherein the diafiltering accomplishes a buffer exchange of from about 8 fold volumes. ### Claim 21 (depends on 1) The process of claim 1 wherein the first buffer comprises a mixture of aqueous sodium chloride and a TRIS buffer, and the second buffer comprises a mixture of aqueous histidine chloride and arginine chloride. ### Claim 22 (depends on 1) The process of claim 1 wherein the first ultrafiltering, the second ultrafiltering, and the diafiltering are accomplished with tangential flow filtration across an ultra-filter membrane. ### Claim 23 (depends on 1) The process of claim 1 wherein the first ultrafiltering, the second ultrafiltering, and the diafiltering are accomplished with tangential flow filtration across the same ultra-filter membrane. ### Claim 24 (depends on 1) The process of claim 1 wherein the yield of the third antibody preparation is greater than about 70 weight percent based on the weight of antibodies in the first purified antibody preparation. ### Claim 25 (depends on 24) The process of claim 24 wherein the yield of the third antibody preparation is from about 80 to about 100 weight percent based on the weight of antibodies in the first purified antibody preparation. ### Claim 26 (depends on 1) The process of claim 1 wherein the first ultrafiltering has a recirculation rate of from about 0.5 L/min/ft2 to about 5 L/min/ft2. ### Claim 27 (depends on 1) The process of claim 1 wherein the ultrafiltering and diafiltering are accomplished at a transmembrane pressure of from about 10 to about 50 p.s.i. ### Claim 28 (depends on 1) The process of claim 1 wherein there is provided an antibody concentrate with a detectable bio-burden of less than about 100 CFU/mL. ### Claim 29 (depends on 1) The process of claim 1 wherein one or more of steps a), b), and c) are accomplished at a temperature of about 40° C. to about 50° C. ### Claim 30 (depends on 1) The process of claim 1 wherein throughput for step c) is at about 80 g/ft2/hr, about 120 g/ft2/hr, about 135 g/ft2/hr, about 145 g/ft2/hr, about 175 g/ft2/hr, about 180 g/ft2/hr, about 265 g/ft2/hr, about 285 g/ft2/hr, or about 290 g/ft2/hr. ### Claim 31 (depends on 1) The process of claim 1 wherein the yield of the third antibody preparation is greater than about 98 weight percent based on the weight of the antibody in the first purified antibody preparation. ### Claim 32 (depends on 1) The process of claim 1 wherein steps a), b), and c) utilize an ultra-filtration membrane. ### Claim 33 (depends on 1) The process of claim 1 wherein an ultra-filtration membrane utilized in step a) is used in step b) and in step c). ### Claim 34 (depends on 1) The process of claim 1 wherein the third antibody preparation has a detectible bioburden of 1.8 CFU/ml. ### Claim 35 (depends on 1) The process of claim 1 wherein the third antibody preparation has a detectible bioburden of less than 0.13 CFU/ml. ### Claim 36 (depends on 1) The process of claim 1 wherein the second antibody preparation has an antibody concentration of about 20 to about 50 grams per liter. ### Claim 37 (depends on 1) The process of claim 1, wherein step b) comprises more than one diafiltering steps. ### Claim 38 (depends on 37) The process of claim 37, wherein a first diafiltering step accomplishes a buffer exchange of about 4 fold volumes and a second diafiltering step accomplishes a buffer exchange of about 4 fold volumes. ### Claim 39 (depends on 1) The process of claim 1, wherein the aggregated contaminants in the third antibody preparation are less than 5 weight percent. ### Claim 40 (depends on 1) The process of claim 1, wherein the aggregated contaminants in the third antibody preparation are less than 2 weight percent. ### Claim 41 (depends on 1) The process of claim 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody. ### Claim 42 (depends on 1) The process of claim 1, wherein the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab′, F(ab′)2, and Fv fragment. ### Claim 43 (depends on 1) The process of claim 1, wherein the antibody is anti IgE antibody rhuMAb E25 as described in U.S. Pat. No. 6,172,213.