# US 10,481,164 B2 · Method for using light scattering in real time to directly monitor and control impurity removal in purification processes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,481,164 B2 - Document ID: US10481164B2 - Status: Granted - Kind: B2 - Application number: 13/850,664 - Filed: 2013-03-26 - Published: 2013-11-14 - Granted: 2019-11-19 - Earliest claimed: 2012-03-26 - Assignees: Amgen Inc. - Applicants: Amgen Inc. - Inventors: Arthur C. Hewig, III; Duke H. Phan; Yinges Yigzaw; Robert Bailey - Web page: https://patentgrep.com/patent/US10481164B2 ## CPC - G01N 33/6854: {Immunoglobulins} - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 16/065: {Purification, fragmentation} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - 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) ## Abstract The invention provides a method for controlling contaminants in biopharmaceutical purification processes by using light scattering and UV absorbance to establish a determinant. The invention makes use of multi-angle light scattering (MALS) and UV as a continuous monitoring system to provide information about the elution peak fractions in real-time instead of conventional pooling methods that rely on a predetermined percent UV peak max value to initiate the pooling process; regardless of product quality. ## Claims ### Claim 1 A method for controlling levels of biological impurities in an elution pool from a cation exchange chromatography resin comprising: loading a mixture containing a recombinant protein and biological impurities on to a cation exchange chromatography resin; eluting by a salt gradient the recombinant protein from the cation exchange chromatography resin; measuring the intensity of light scattered by the eluate with a laser light scattering detector to obtain a light scattering (LS) signal; measuring the absorbance of the eluate with an ultraviolet (UV) absorbance detector to obtain an absorbance signal; calculating a ratio of the LS signal to the absorbance signal for each elution fraction to obtain a LS/UV fraction ratio; monitoring in real time during the elution of the recombinant protein the LS/UV fraction ratio over time for each elution fraction, wherein an increase in the LS/UV fraction ratio over time indicates an increase in the levels of biological impurities in the elution pool; and terminating the elution when an increase in the LS/UV fraction ratio over time is detected. ### Claim 2 (depends on 1) The method according to claim 1, wherein the laser light scattering detector is a single or multiple angle light scattering detector. ### Claim 3 (depends on 1) The method according to claim 1, wherein the light scattering detector and UV absorbance detector are in series or in parallel. ### Claim 4 (depends on 1) The method according to claim 1, wherein the eluate is continuously delivered to the UV absorbance detector, the laser light scattering detector, or both. ### Claim 5 (depends on 1) The method according to claim 1, wherein the entire eluate is delivered to the UV absorbance detector, the laser light scattering detector or both. ### Claim 6 (depends on 4) The method according to claim 4, wherein a sample of the eluate is diverted to the UV absorbance detector, the laser light scattering detector, or both, by a slipstream. ### Claim 7 (depends on 1) The method according to claim 1, further comprising contacting the eluate with a second chromatography resin or processing step; and recovering the recombinant protein from the second chromatography resin or processing step. ### Claim 8 (depends on 7) The method according to claim 7, wherein the second chromatography resin is an affinity resin, an ion exchange resin, a hydrophobic interaction resin, a mixed modal resin, or a hydroxyapatite resin. ### Claim 9 (depends on 1) The method according to claim 1, wherein the recombinant protein is an antibody. ### Claim 10 A method for determining the purity of a recombinant protein in the eluate pool from a cation exchange resin comprising: loading a mixture containing a recombinant protein and biological impurities on to a cation exchange chromatography resin; eluting by a salt gradient the recombinant protein from the cation exchange chromatography resin; measuring the intensity of light scattered by the eluate with a laser light scattering detector to obtain a light scattering (LS) signal; measuring the absorbance of the eluate with a UV absorbance detector to obtain an absorbance signal; calculating a ratio of the LS signal to the absorbance signal for each elution fraction to obtain a LS/UV fraction ratio; monitoring in real time during the elution of the recombinant protein the LS/UV fraction ratio over time for each elution fraction; and detecting a change in the LS/UV fraction ratio over time, wherein an increase in the LS/UV fraction ratio over time indicates an increase of biological impurities in the eluate pool. ### Claim 11 (depends on 10) The method according to claim 10, wherein the laser light scattering detector is a single or multiple angle light scattering detector. ### Claim 12 (depends on 10) The method according to claim 10, wherein the light scattering detector and UV absorbance detector are in series or in parallel. ### Claim 13 (depends on 10) The method according to claim 10, wherein the eluate is continuously delivered to the UV absorbance detector, the laser light scattering detector, or both. ### Claim 14 (depends on 13) The method according to claim 13, wherein the entire eluate is delivered to the UV absorbance detector, the laser light scattering detector or both. ### Claim 15 (depends on 13) The method according to claim 13, wherein a sample of the eluate is diverted to the UV absorbance detector, the laser light scattering detector, or both, by a slipstream. ### Claim 16 (depends on 10) The method according to claim 10, further comprising contacting the eluate with a second chromatography resin or processing step; and recovering the recombinant protein from the second chromatography resin or processing step. ### Claim 17 (depends on 16) The method according to claim 16, wherein the second chromatography resin is an affinity resin, an ion exchange resin, a hydrophobic interaction resin, a mixed modal resin, or a hydroxyapatite resin. ### Claim 18 (depends on 10) The method according to claim 10, wherein the recombinant protein is an antibody. ### Claim 19 (depends on 1) The method according to claim 1, wherein the biological impurities comprise high molecular weight species of the recombinant protein. ### Claim 20 (depends on 10) The method according to claim 10, wherein the biological impurities comprise high molecular weight species of the recombinant protein. ### Claim 21 A method for controlling the separation of an antibody from high molecular weight species of the antibody comprising: loading a mixture containing the antibody and high molecular weight species of the antibody on to a cation exchange chromatography resin; eluting by a salt gradient the antibody from the cation exchange chromatography resin; measuring the intensity of light scattered by the eluate with a laser light scattering detector to obtain a light scattering (LS) signal; measuring the absorbance of the eluate with an ultraviolet (UV) absorbance detector to obtain an absorbance signal; calculating a ratio of the LS signal to the absorbance signal for each elution fraction to obtain a LS/UV fraction ratio; monitoring in real time during the elution of the antibody the LS/UV fraction ratio over time for each elution fraction, wherein an increase in the LS/UV fraction ratio over time indicates an increase in high molecular weight species of the antibody in the eluate; and terminating the elution when an increase in the LS/UV fraction ratio over time is detected. ### Claim 22 (depends on 21) The method of claim 21, wherein the high molecular weight species of the antibody comprise multimers and aggregates of the antibody. ### Claim 23 (depends on 21) The method according to claim 21, wherein the laser light scattering detector is a single or multiple angle light scattering detector. ### Claim 24 (depends on 21) The method according to claim 21, wherein the light scattering detector and UV absorbance detector are in series or in parallel. ### Claim 25 (depends on 21) The method according to claim 21, wherein the eluate is continuously delivered to the UV absorbance detector, the laser light scattering detector, or both. ### Claim 26 (depends on 25) The method according to claim 25, wherein the entire eluate is delivered to the UV absorbance detector, the laser light scattering detector or both. ### Claim 27 (depends on 25) The method according to claim 25, wherein a sample of the eluate is diverted to the UV absorbance detector, the laser light scattering detector, or both, by a slipstream.