# US 2025/0095773 A1 · Methods and systems for developing chromatography protocols > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0095773 A1 - Document ID: US20250095773A1 - Status: Published application - Kind: A1 - Application number: 18/887,534 - Filed: 2024-09-17 - Published: 2025-03-20 - Earliest claimed: 2023-09-18 - Assignees: Regeneron Pharmaceuticals, Inc. - Applicants: Regeneron Pharmaceuticals, Inc. - Inventors: Nimish GUPTA; Christopher COWAN; John MATTILA - Web page: https://patentgrep.com/patent/US20250095773A1 ## CPC - C07K 1/16: by chromatography - G16B 15/30: Drug targeting using structural data; Docking or binding prediction - B01D 15/3804: {Affinity chromatography} - G01N 30/8658: {Optimising operation parameters} - G01N 30/8693: {Models, e.g. prediction of retention times, method development and validation} - G01N 30/26: Conditioning of the fluid carrier; Flow patterns - G01N 30/50: Conditioning of the sorbent material or stationary liquid - G01N 30/88: Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 - 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) - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY - 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) - 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) ## Abstract A method of purifying a target molecule may include introducing a load including a high molecular weight species concentration (% HMW) to a chromatography apparatus comprising sartobind phenyl chromatography media. A method of generating a chromatography protocol, may include identifying chromatography loading parameters, identifying chromatography performance criteria. The method of generating the chromatography protocol may include selecting combinations of test values of the loading parameters, and conducting a chromatography run for each combination of the set of test values combinations, thereby generating actual performance criteria values corresponding to each combination of the set of test value combinations. ## Claims ### Claim 1 A method of generating a chromatography protocol for a target molecule, the method comprising: identifying chromatography loading parameters; identifying chromatography performance criteria; generating a domain of potential predictive models relating the chromatography loading parameters to the chromatography performance criteria; selecting combinations of test values of the loading parameters, wherein the selected combinations of test values form a set of test value combinations; conducting a chromatography run for each combination of the set of test values combinations, thereby generating actual performance criteria values corresponding to each combination of the set of test value combinations; and ranking each predictive model, of the domain of predictive models, based on a correlation of performance criteria values predicted by the model to the actual performance criteria values; wherein the target molecule is an antibody-drug conjugate. ### Claim 2 (depends on 1) The method of claim 1, wherein the chromatography loading parameters include a loading buffer salt concentration, a chromatography media density, a high molecular weight species content of the load, or a combination thereof. ### Claim 3 (depends on 1) The method of claim 1, wherein the chromatography performance criteria include a high molecular weight species content reduction, yield of a target molecule, or a combination thereof. ### Claim 4 (depends on 1) The method of claim 1, further comprising: selecting a predictive model that has the highest rank; and using the selected model to determine values of the chromatography loading parameters for the chromatography protocol. ### Claim 5 (depends on 4) The method of claim 4, wherein the selected model predicts that the values of the chromatography loading parameters for the chromatography protocol correspond to one or more target performance criteria values. ### Claim 6 (depends on 1) The method of claim 1, further comprising, developing a desirability metric that includes a quantitative relationship between performance criteria. ### Claim 7 (depends on 6) The method of claim 6, wherein the desirability metric is calculated as a composite of two or more performance criteria, and wherein each performance criterion is assigned a weight that contributes to the desirability metric. ### Claim 8 (depends on 7) The method of claim 7, wherein the two or more performance criteria include a high molecular weight species content reduction and a yield of a target molecule, and wherein each performance criterion is equally weighted. ### Claim 9 A method of generating a chromatography protocol for a target molecule, the method comprising: identifying a first chromatography parameter and a second chromatography parameter; identifying a first performance criterion and a second performance criterion; selecting first test values for the first chromatography parameter; selecting second test values for the second chromatography parameter; identifying a chromatography media; generating first performance criterion values, wherein each first performance criterion value corresponds to a combination of a first test value and a second test value; generating second performance criterion values, wherein each second performance criterion value corresponds to a combination of a first test value and a second test value; generating a first pool of multivariate models, wherein each multivariate model of the first pool of multivariate models relates the first and second chromatography parameters to the first performance criterion; generating a second pool of multivariate models, wherein each multivariate model of the second pool of multivariate models relates the first and second chromatography parameters to the second performance criterion; generating first projected performance criterion values using each multivariate model of the first pool of multivariate models, wherein each first projected performance criterion value corresponds to a combination of a first test value and a second test value; generating second projected performance criterion values using each multivariate model of the second pool of multivariate models, wherein each second projected performance criterion value corresponds to a combination of a first test value and a second test value; determining a coefficient of determination for a multivariate model in the first pool of multivariate models; and determining a coefficient of determination for a multivariate model in the second pool of multivariate models; wherein the target molecule is an antibody-drug conjugate. ### Claim 10 (depends on 9) The method of claim 9, wherein identifying the chromatography media comprises: performing a first chromatography run on a first chromatography media to generate a first desirability value corresponding to the first chromatography media; performing a second chromatography run on a second chromatography media to generate a second desirability value corresponding to the second chromatography media; and selecting the chromatography media with the that corresponds to the greatest desirability value. ### Claim 11 (depends on 10) The method of claim 10, wherein the first chromatography run and the second chromatography run are conducted with the same media density and loading buffer composition. ### Claim 12 (depends on 11) The method of claim 11, wherein the first desirability value is calculated based on one or more performance criteria values of the first chromatography run, and the second desirability value is calculated based on one or more performance criteria values of the second chromatography run. ### Claim 13 (depends on 9) The method of claim 9, wherein the first chromatography parameter includes a column loading of a HIC media (g/L), the second chromatography parameter includes a citrate salt concentration of a loading buffer, the first performance criterion is a yield, and the second performance criterion is a quantification of the reduction of an impurity. ### Claim 14 (depends on 9) The method of claim 9, wherein generating the first pool of multivariate models includes: identifying a domain of potential multivariate models; calculating a variance inflation factor for each potential multivariate model of the domain of potential multivariate models; selecting all potential multivariate models with a variance inflation factor less than or equal to a collinearity threshold to generate the first pool of multivariate models. ### Claim 15 A method of purifying a target molecule, the method comprising: introducing a load including a high molecular weight species concentration (% HMW) of approximately 3 percent to approximately 20 percent to a chromatography apparatus comprising sartobind phenyl chromatography media, wherein the load is introduced at a density of approximately 10 grams of load per liter of total volume of the chromatography apparatus to approximately 40 grams per liter, and wherein the load comprises the target molecule and approximately 5 mM to approximately 200 mM of citrate; and passing an eluate comprising the target molecule from the chromatography apparatus, wherein a yield of the target molecule in the eluate is at least approximately 70%, and wherein a difference of the % HMW of load and a % HMW of the eluate is at least approximately 2%; wherein the target molecule is an antibody-drug conjugate. ### Claim 16 (depends on 15) The method of claim 15, wherein the load is introduced at a density of approximately 20 grams per liter to approximately 30 grams per liter. ### Claim 17 (depends on 15) The method of claim 15, wherein the antibody-drug conjugate includes a drug conjugated to an antibody via lysine conjugation. ### Claim 18 (depends on 15) The method of claim 15, wherein: the % HMW of the load is approximately 7% to approximately 15%; the load comprises approximately 110 mM to approximately 175 mM citrate; the yield of the target molecule in the eluate is approximately 75% to approximately 95%; and/or the difference of the % HMW of load and a % HMW of the eluate is approximately 3% to approximately 8%. ### Claim 19 (depends on 15) The method of claim 15, wherein the antibody-drug conjugate includes a cleavable maytansinoid. ### Claim 20 (depends on 15) The method of claim 15, wherein the antibody-drug conjugate includes an IgG 4 antibody.