# US 10,865,224 B2 · Purification of biological molecules > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,224 B2 - Document ID: US10865224B2 - Status: Granted - Kind: B2 - Application number: 15/654,876 - Filed: 2017-07-20 - Published: 2017-11-09 - Granted: 2020-12-15 - Earliest claimed: 2012-06-29 - Assignees: EMD Millipore Corporation - Applicants: EMD Millipore Corporation - Inventors: Alex Xenopoulos; Michael Phillips; Wilson Moya; Jad Jaber; Mikhail Kozlov; Ajish Potty; Matthew T. Stone; William Cataldo; Christopher Gillespie - Web page: https://patentgrep.com/patent/US10865224B2 ## CPC - C07K 1/36: by a combination of two or more processes of different types - B01D 15/125: {Pre-filtration} - B01D 15/1871: {placed in series} - B01D 15/362: {Cation-exchange} - B01D 15/363: {Anion-exchange} - B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography} - B01D 15/3847: {Multimodal interactions} - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis} - C12M 43/00: {Combinations of bioreactors or fermenters with other apparatus} - C12M 47/12: {Purification (C12M47/04 takes precedence)} - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - 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) - 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) - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to improved processes and systems for purification of biological molecules, where the processes can be performed in a continuous manner. ## Claims ### Claim 1 A continuous process for the purification of a target molecule comprising the steps of: a) providing a sample comprising the target molecule and one or more impurities; b) adding at least one precipitant to the sample and removing one or more impurities, thereby to recover a clarified sample; c) subjecting the clarified sample from step (b) to a bind and elute chromatography step comprising at least two separation units, thereby to obtain an eluate comprising the target molecule; and d) subjecting the eluate to flow-through purification comprising use of two or more media, one of which is activated carbon and the other(s) are selected from anion-exchange chromatography media and cation-exchange chromatography media; wherein at least two steps are performed concurrently for at least a portion of their duration, wherein the process comprises only one bind and elute chromatography step, and wherein the process is a continuous process, wherein the process comprises use of one or more surge tanks and/or one or more static mixers, wherein the flow-through purification is carried out as: activated carbon followed by anion-exchange chromatography media followed by cation-exchange chromatography media, and wherein the in-line static mixer and/or surge tank is used between anion-exchange chromatography media and cation-exchange chromatography media to change pH. ### Claim 2 (depends on 1) The process of claim 1, comprising a virus inactivation step between steps (c) and (d). ### Claim 3 (depends on 2) The process of claim 2, wherein the virus inactivation step comprises use of a virus inactivating agent selected from acid, detergent, solvent and temperature change. ### Claim 4 (depends on 2) The process of claim 2, wherein virus inactivation step comprises use of one or more in-line static mixers. ### Claim 5 (depends on 2) The process of claim 2, wherein virus inactivation comprises use of one or more surge tanks. ### Claim 6 (depends on 1) The process of claim 1, wherein the target molecule is an antibody. ### Claim 7 (depends on 6) The process of claim 6, wherein the antibody is selected from a monoclonal antibody or a polyclonal antibody. ### Claim 8 (depends on 1) The process of claim 1, wherein the precipitant in step (b) is a stimulus responsive polymer. ### Claim 9 (depends on 8) The process of claim 8, wherein the stimulus responsive polymer is a modified polyallylamine polymer. ### Claim 10 (depends on 1) The process of claim 1, wherein the precipitant in step (b) is selected from the group consisting of an acid, caprylic acid, a flocculant and a salt. ### Claim 11 (depends on 1) The process of claim 1, wherein removing impurities in step (b) comprises use of one or more depth filters. ### Claim 12 (depends on 1) The process of claim 1, wherein removing impurities in step (b) comprises use of centrifugation. ### Claim 13 (depends on 1) The process of claim 1, wherein the bind and elute chromatography step in (c) employs continuous multi-column chromatography. ### Claim 14 (depends on 1) The process of claim 1, wherein the bind and elute chromatography step in (c) is selected from the group consisting of affinity chromatography, cation exchange chromatography and mixed-mode chromatography. ### Claim 15 (depends on 1) The process of claim 1, wherein the bind and elute chromatography step in (c) employs Protein A affinity chromatography. ### Claim 16 (depends on 15) The process of claim 15, wherein Protein A affinity chromatography employs a Protein A ligand coupled to a matrix selected from the group consisting of rigid hydrophilic polyvinylether polymer, controlled pore glass and agarose. ### Claim 17 (depends on 1) The process of claim 1, wherein the sample in step (a) is a cell culture. ### Claim 18 (depends on 17) The process of claim 17, wherein the cell culture is provided in a bioreactor. ### Claim 19 (depends on 18) The process of claim 18, wherein the bioreactor is a single use bioreactor. ### Claim 20 (depends on 17) The process of claim 17, wherein the cell culture is provided in a vessel other than a bioreactor. ### Claim 21 (depends on 1) The process of claim 1, wherein the precipitant in step (b) is added to a bioreactor comprising a cell culture. ### Claim 22 (depends on 21) The process of claim 21, wherein the precipitant is added using a static mixer. ### Claim 23 (depends on 1) The process of claim 1, wherein the precipitant in step (b) is added to a vessel other than a bioreactor which comprises the sample comprising the target molecule. ### Claim 24 (depends on 1) The process of claim 1, wherein the flow-through purification in step (d) further comprises use of a virus filtration membrane. ### Claim 25 (depends on 1) The process of claim 1, wherein the cation exchange chromatography media is in the form of a membrane, a bead or a fiber. ### Claim 26 (depends on 1) The process of claim 1, wherein the process comprises use of one or more surge tanks and does not employ any pool tanks between process steps. ### Claim 27 (depends on 1) The process of claim 1, further comprising a formulation step. ### Claim 28 (depends on 27) The process of claim 27, wherein formulation comprises diafiltration, concentration and sterile filtration. ### Claim 29 (depends on 28) The process of claim 28, further concentration comprises tangential flow filtration.