Materials and methods for processing proteins

Abstract

This invention provides materials and methods for protein synthesis and analysis. [Solution] A method for processing a protein, comprising: fragmenting the protein to generate polypeptides; adding the polypeptides to a chromatography column; and eluting the polypeptides in an eluate containing mobile phase B solvent. Mobile phase B solvent comprises trifluoroacetic acid (TFA), acetonitrile, and alcohol. The method further comprises constructing a structural map of the protein, the structural map comprising HCDR3 and LCDR3.

Description

Technical Field

[0001] The disclosed subject matter relates to the field of polypeptide analysis.

[0002] Incorporation by reference of electronically submitted materials This application has been filed with an electronic sequence listing. The sequence listing filed as a file named "55406_Seqlisting.txt" was created on October 13, 2021, and is 268,106 bytes in size. The electronic information of the sequence listing is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] Peptide mapping is a valuable technique for combining positional quantitative information with protein tissue distribution information and domain information. In particular, annotated peptide mapping is a useful procedure and has become an extremely important goal in many biomedical and biopharmaceutical research and production efforts.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Proteins are complex macromolecules, and the biological production and characteristics of protein pharmaceuticals ("biologics") pose many stringent analytical challenges that do not occur with small molecule drugs. Biologics tend to have manufacturing challenges such as sequence variations, misfolding, variant glycosylation, and post-translational degradation including aggregation and modification, such as oxidation and deamidation. These challenges can lead to loss of safety and efficacy, so in the biopharmaceutical industry, it is necessary to identify and quantify variant and degraded forms of products at low concentrations and further obtain tertiary structure information.

Means for Solving the Problems

[0005] In one embodiment, a method for processing a protein is described herein, comprising: fragmenting the protein under enzymatic and / or non-enzymatic conditions to produce polypeptides; generating polypeptides; adding the polypeptides to a chromatographic column; and eluting the polypeptides in an eluate containing mobile phase B solvent, wherein mobile phase B solvent comprises trifluoroacetic acid (TFA), acetonitrile, and alcohol. In some embodiments, the protein comprises complementarity-determining regions (CDRs) of the variable regions of an antigen-binding protein. For example, the protein may comprise CDR3 of the heavy chain variable region (HCDR3) and / or CDR3 of the light chain variable region (LCDR3), and the method further comprises constructing a structural map of the protein, the structural map comprising HCDR3 and LCDR3. In some embodiments, mobile phase B comprises 0.05% to 0.09% %TFA.

[0006] In some embodiments, at least 50% of the HCDR3-containing polypeptide and / or at least 50% of the LCDR3-containing polypeptide are eluted from the chromatography column. In some embodiments, mobile phase B contains about 35–45% acetonitrile, 35–45% alcohol, and TFA in water. Exemplary alcohols include, but are not limited to, isopropyl alcohol, propanol, and butyl alcohol.

[0007] In some embodiments, the elution step is performed in a gradient between mobile phase B solvent and polar mobile phase A solvent. In some embodiments, mobile phase A comprises TFA and water. In some embodiments, mobile phase A contains less than 0.1% TFA (e.g., 0.05%, 0.06%, 0.07%, 0.08%, or 0.09% TFA).

[0008] Chromatographic columns used in accordance with the methods described herein include columns containing porous particles having a particle size of about 2 μm to about 7 μm (e.g., 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 7 μm, including a range between any two of the listed values). In some embodiments, the chromatography column contains porous particles having a pore size of about 100 to 500 angstroms each (e.g., about 100, about 200, about 300, about 400, or about 500 angstroms). In some embodiments, the porous particles have a pore size of about 300 angstroms and a particle size of about 3 μm each. In some embodiments, the chromatography column contains divinylbenzene (DVB) resin.

[0009] The chromatography column used in the methods described herein may have a height of at least 10 cm (for example, at least 10 cm, at least 15 cm, at least 20 cm, at least 25 cm, or at least 30 cm).

[0010] In some embodiments, the methods described herein further include performing spectroscopic analysis of the eluted polypeptide.

[0011] In some embodiments, the protein includes a therapeutic protein. Examples of therapeutic proteins include, but are not limited to, antibodies or their antigen-binding fragments, derivatives of antibodies or antibody fragments, or fusion polypeptides. In some embodiments, the therapeutic protein is infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, avagovomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artimomab, aselizumab, altinumab, at Lizumab, atrolimumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, vectumomab, belimumab, benralizumab, vertilimumab, besilesomab, bezlotoxumab, bisilomab, vibatuzumab, vibatuzumab meltansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab meltansine, caplacizumab, capromab pendetide, carlumab, catsumakisomab, CC49, sedelizumab, Sertolizumab pegol, Sitatuzumab bogatox, Sixtumumab, Clazakizumab, Clenoliximab, Cribatuzumab tetraxetan, Conatumumab, Crenezumab, CR6261, Dasetuzumab, Dacrizumab, Darotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Dorozumab, Durigotuzumab, Dupilumab, Eclomeximab, Eculizumab, Edovacomab, Edrecolomab, Ephalizumab, Efungumab, Elotuzumab, Elsilimo Mab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab citucetan, epratuzumab, erenumab, erlizumab, erzumaxomab, etalacizumab, etrolizumab, evolocumab, excivivirumab, fanolesomab, falalimomab, falletuzumab, facinumab, fbta05, felbizumab, fezakinumab, ficratuzumab, figitumumab, framotumab, fontrizumab, foralumab, folavirumab,Fresolimmab, Fluranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gabirimomab, Gemtuzumab Ozogamicin, Gevokizumab, Gilentuximab, Grembatumumab Vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab Chiuxetan, Icurumab, Igobomab, Imsilomab, Imugatuzumab, Intracumab, Indatuximab Tansine, Intetumumab, Inorimmomab Inotuzumab ozogamicin, ipilimumab, iratumumab, itorizumab, ixekizumab, keriximab, rabetuzumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, rivivirumab, rigerizumab, lintuzumab, lirirumab, rorbotuzumab meltansine, lucatumumab, lumiliximab, mapatumumab, masurimomab, mapurimumab, matsuzumab, mepolizumab, meterimumab, milatuzumab, minretumomab Mitsumomab, mogamulizumab, morolimumab, motabizumab, moxetumomab pasdotox, muromonab-cd3, nacolomabutafenatox, namilumab, naptumomab estafenatox, nalnatumumab, natalizumab, nevacumab, necitumumab, nererimomab, nesbakumab, nimotuzumab, nivolumab, nofetumomab merpentan, okalatuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab Omalizumab, Onartuzumab, Oportuzumab Monatox, Olegobomab, Orticumab, Oterixizumab, Oxerumab, Ozanezumab, Ozoralizumab, Padibaximab, Panitumumab, Panobacumab, Pulsatuzumab, Pascolizumab, Pateclizumab, Patrizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexerizumab, Pizilizumab, Pintumomab, Prakurumab, Ponezumab, Priliximab, Pritumumab, PRO 140, quilizumab, lacosumomab, radrezumab, rafivirumab, ramucirumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevilumab, cibrotuzumab, cifalimumab, siltuximab, simtuzumab, ciprizumab,Silkumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamlumab, thresomab, subizumab, tabarmab, tacuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tapritumomab paptox, tefivazumab, terimomab aritox, tenatumomab, teneriximab, teprizumab, teprotumumab, tezeperumab, TGN1412, tremelimumab, tisilimmab, tildrakizumab, tigatuzumab, TNX-650, tralizuma These include tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotsuzumab cermoloukin, tubirumab, ubrituximab, urerumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, bertuzumab, bepalimomab, besenkumab, vizilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanorimumab, zatuximab, diralimumab, and zolimomab aritox. In other embodiments, therapeutic polypeptides include glycoproteins, CD polypeptides, HER receptor polypeptides, cell adhesion polypeptides, growth factor polypeptides, insulin polypeptides, insulin-related polypeptides, coagulation polypeptides, coagulation-related polypeptides, albumin, IgE, blood group antigens, colony-stimulating factors, receptors, neurotrophic factors, interferons, interleukins, viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor α and β, enkephalinases, mouse gonadotropin-related peptides, and DNAs. e, inhibin, activin, integrin, protein A, protein D, rheumatoid factor, immunotoxin, bone morphogenetic protein, superoxide dismutase, surface membrane polypeptide, disintegration promoter, HIV envelope, transport polypeptide, homing receptor, adresin, regulatory polypeptide, immunoadhesin, myostatin, TALL polypeptide, amyloid polypeptide, thymic interstitial lymphocyte generating factor, RANK ligand, c-kit polypeptide, TNF receptor, and angiopoietin, as well as polypeptides selected from the group consisting of these and their bioactive fragments, analogs, or variants.

[0012] In some embodiments, the therapeutic protein includes a BiTE® (bispecific T cell engager) molecule. For example, the therapeutic protein may include a half-life extended (HLE) BiTE® molecule.

[0013] In another embodiment, a chromatography column comprising a polypeptide fragment of a protein and an eluate comprising a mobile phase B solvent comprising trifluoroacetic acid (TFA), acetonitrile, and an alcohol is disclosed herein. In some embodiments, the protein comprises a variable region CDR. In some embodiments, the protein comprises a heavy chain variable region CDR3 and / or a light chain variable region CDR3. In some embodiments, the eluate comprises more HCDR3 and / or more LCDR3 than is bound to the column.

[0014] In some embodiments, mobile phase B of any of the disclosed chromatography columns contains TFA in about 35–45% acetonitrile, 35–45% alcohol, and water. In some embodiments, mobile phase B contains TFA in about 40% acetonitrile, 40% alcohol, and 20% water. Exemplary alcohols include, but are not limited to, isopropyl alcohol, propanol, and butyl alcohol.

[0015] In some embodiments, the eluate of the chromatography column is a gradient of mobile phase B solvent and polar mobile phase A solvent. In some embodiments, the mobile phase comprises TFA and water. In some embodiments, mobile phase A contains less than 0.1% TFA (e.g., 0.05%, 0.06%, 0.07%, 0.08%, or 0.09% TFA).

[0016] In some embodiments, the chromatography column includes porous particles having a particle size of about 2 μm to about 7 μm (e.g., 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 7 μm, including a range between any two of the listed values). In some embodiments, the chromatography column includes porous particles having pore sizes of about 100 to 500 angstroms each (e.g., about 100, about 200, about 300, about 400, or about 500 angstroms). In some embodiments, the porous particles each have a pore size of about 300 angstroms. In some embodiments, the chromatography column includes fully porous particles having a pore size of about 300 angstroms and a particle size of about 5 μm.

[0017] Any of the disclosed chromatography columns has a height of at least 10 cm (e.g., at least 10 cm, at least 15 cm, at least 20 cm, at least 25 cm, or at least 30 cm).

[0018] The use of the singular form includes the plural form unless otherwise specified. The use of "or" means "and / or" unless otherwise specified. The use of the term "contains" and other forms such as "contains" and "includes" is not restrictive. Terms such as "element" or "component" include both elements and components containing one unit and elements and components containing two or more subunits unless otherwise specified. The use of the term "part" can include a part or all of a part. For example, when a numerical range such as 1 to 5 is mentioned, all intermediate values, e.g., 1, 2, 3, 4 and 5, and their fractions, e.g., 1.5, 2.2, 3.4 and 4.1 are explicitly included.

[0019] When "approximately" or "~" modifies a quantity (e.g., "approximately" 3 mM), it means that there may be fluctuations around the modified quantity. These fluctuations can be caused by various means, such as typical measurement and processing procedures, careless errors, and the purity of the components.

[0020] The terms “contains” and “includes” are intended to mean that the method includes the enumerated elements but does not exclude other unenumerated elements. The terms “essentially consisting of” and “essentially consisting of” include the enumerated elements and exclude unenumerated elements that would alter the fundamental nature of the method, but do not exclude other unenumerated elements. The terms “consisting of” and “comprising” exclude substantial method steps when used to define a method. Embodiments defined by each of these transitional terms are included within the scope of this disclosure. [Modes for carrying out the invention]

[0021] Peptide mapping is widely used in the biopharmaceutical industry, with applications ranging from advanced characterization methods to important and routine release multi-attribute assays that replace some conventional methods in release specifications.

[0022] The CDR regions of monoclonal antibodies (such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) are known to influence the overall efficacy of a drug through their antigen-antibody-mediated binding properties. The impact of any post-translational modifications (PTMs) within this region on the efficacy and safety of the product must be carefully evaluated. As described in the examples herein, the recovery rates of CDR-containing peptides from several therapeutic proteins were examined using peptide mapping assays with conventional column chemicals. These findings indicated that the recovery rates of some CDR-containing polypeptides using conventional column chemicals were poor, resulting in challenges in identifying and quantifying post-translational modifications (PTMs) of CDR-containing peptides. A peptide mapping method, usable under both reducing and non-reducing conditions, is described herein, which improves the recovery rates of these peptides and significantly enhances the quantification and identification of previously unmeasured PTMs. PTMs include, but are not limited to, site-directed glycosylation, isomerization, covalent bonding, oxidation, deamidation, hydroxylation, glycation, amino acid substitution (sequence mutations), and / or truncation.

[0023] In one embodiment, a method for processing a protein is described herein, comprising fragmenting the protein to produce polypeptides, adding the polypeptides to a chromatographic column, and eluting the polypeptides in an eluate containing mobile phase B solvent comprising trifluoroacetic acid (TFA), acetonitrile, and an alcohol. In some embodiments, the protein is reduced. The term “reduced protein” (and similar terms) as used herein means a protein in which at least one of its interchain or intrachain disulfide bonds is broken. Such disulfide bonds may be formed between reduced thiol groups, for example, available groups on a cysteine ​​residue. In other embodiments, the protein is not reduced. The term “not reduced protein” (and similar terms) as used herein means a protein in which at least one of its interchain or intrachain disulfide bonds remains unchanged. In some embodiments, the method includes reducing the protein.

[0024] The methods disclosed herein may include fragmenting the protein to be analyzed in a sample, thereby generating at least two polypeptide fragments of the protein. Any suitable method for fragmenting the protein can be used, provided that at least two polypeptide fragments of the protein are generated. For example, the protein may be cleaved by a protease or a chemical and / or fragmented by thermal decomposition. In some embodiments, at least three polypeptide fragments of the protein are generated. In some embodiments, at least four, five, six, seven, eight, nine, or ten fragments are generated.

[0025] In some embodiments, the protein is cleaved by a protease. Any suitable protease can be used as long as such protease cleaves the protein into at least two polypeptide fragments. Exemplary proteases include, but are not limited to, trypsin, neutrophil elastase, endoproteinase Glu-C, endoproteinase Arg-C, pepsin, chymotrypsin, chymotrypsin B, Lys-N protease, Lys-C protease, Glu-C protease, Asp-N protease, pancreatic peptidase, carboxypeptidase A, carboxypeptidase B, proteinase K, and thermolysin. In some embodiments, the protein is cleaved by two or more proteases.

[0026] In some embodiments, the protein and protease are combined in a protein:protease ratio (w / w) of 10:1, 20:1, 25:1, 50:1, or 100:1. In some embodiments, the ratio is 20:1. In some embodiments, the protease used is at a concentration of approximately 100 ng / ml to 1 mg / ml, or approximately 100 ng / ml to 500 μg / ml, or approximately 100 ng / ml to 100 μg / ml, or approximately 1 μg / ml to 1 mg / ml, or approximately 1 μg / ml to 500 μg / ml, or approximately 1 μg / ml to 100 μg / ml, or approximately 10 μg / mg to 1 mg / ml, or approximately 10 μg / mg to 500 μg / ml, or approximately 10 μg / mg to 100 μg / ml. In some embodiments, the fragmentation step is carried out for about 10 minutes to about 48 hours, or about 30 minutes to about 48 hours, or about 30 minutes to about 24 hours, or about 30 minutes to about 16 hours, or about 1 hour to about 48 hours, or about 1 hour to about 24 hours, or about 1 hour to about 16 hours, or about 1 to about 8 hours, or about 1 to about 6 hours, or about 1 to about 4 hours. In some embodiments, the fragmentation step is carried out at a temperature of about 20°C to about 45°C, or about 20°C to about 40°C, or about 22°C to about 40°C, or about 25°C to about 37°C. In some embodiments, the fragmentation step is carried out at about 37°C. Those skilled in the art can select appropriate conditions (buffer, incubation time, amount, volume of protease, etc.) as in vitro protease digestion is understood in the art.

[0027] In some embodiments, fragmenting a protein into polypeptide fragments is achieved chemically, preferably using a chemical that cleaves the protein in a site-specific manner. Such chemicals include cyanogen bromide (CNBr; carbononitridic bromide) that cleaves the C-terminus of methionine residues; 2-nitro-5-thiocyanobenzoate (NTCB) that cleaves the N-terminus of cysteine residues; asparagine-glycine dipeptide that can be cleaved using hydroxylamine; formic acid that cleaves the asparagine-proline (Asp-Pro) peptide bond and BNPS-skatole (3-bromo-3-methyl-2-(2-nitrophenyl)sulfanylindole) that cleaves the C-terminus of tryptophan residues. Those skilled in the art understand how to select appropriate variables including, for example, the concentration of the polypeptide, the concentration of the chemical, the incubation time and temperature. See, for example, (Crimmins et al., 2001; Li et al., 2001; Tanabe et al., 2014) as well.

[0028] Chromatography Chromatography is a method of separating polypeptide fragments in a mobile phase that is processed through a structure that holds a stationary phase. For example, a chromatography column is a structure that holds a stationary phase during chromatography. Since polypeptide fragments differ in size and composition, each fragment has a unique partition coefficient. Due to the different partition coefficients, the polypeptides are differentially retained on the stationary phase. Exemplary chromatography methods include, but are not limited to, gas chromatography, liquid chromatography, high performance liquid chromatography, ultra-high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, expanded bed adsorption chromatography, reverse phase chromatography, and hydrophobic interaction chromatography.

[0029] The chromatographic methods described herein include the steps of adding a polypeptide to a chromatographic column and eluting the polypeptide in an eluate containing a mobile phase B solvent comprising trifluoroacetic acid (TFA), acetonitrile, and an alcohol.

[0030] In some embodiments, mobile phase B contains TFA in about 30% acetonitrile (e.g., about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%, including a range between any two listed values, e.g., 30-40%, 30-45%, 35-40%, or 35-45%). Mobile phase B may contain less than 0.1% TFA (e.g., 0.09%, 0.08%, 0.07%, 0.06%, or 0.05% TFA). In some embodiments, mobile phase B contains TFA in about 30% alcohol (e.g., about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%, including a range between any two listed values, e.g., 30-40%, 30-45%, 35-40%, or 35-45%). Mobile phase B may contain less than 0.1% TFA (e.g., 0.09%, 0.08%, 0.07%, 0.06%, or 0.05% TFA).

[0031] In some embodiments, mobile phase B is about 30% acetonitrile (e.g., about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45%), and within a range between any two of the listed values, e.g., 30-40%, 30-45%, 35-40%, or 35-45%). Including); approximately 30% alcohol (e.g., approximately 31%, 32%, 33%, 34%, 35%, 36%, 37%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%, including a range between any two listed values, e.g., 30-40%, 30-45%, 35-40%, or 35-45%); and TFA in water. Mobile phase B may contain less than 0.1% TFA (e.g., 0.09%, 0.08%, 0.07%, 0.06%, or 0.05% TFA). In some embodiments, mobile phase B comprises about 30% acetonitrile (e.g., about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45%, including a range between any two listed values, e.g., 30-40%, 30-45%, 35-40%, or 35-45%); and about 35-45% alcohol; and TFA in water. In some embodiments, mobile phase B contains about 35–45% acetonitrile; about 30% alcohol (e.g., about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45%, including a range between any two listed values, e.g., 30–40%, 30–45%, 35–40%, or 35–45%); and TFA in water. Mobile phase B may contain less than 0.1% TFA (e.g., 0.09%, 0.08%, 0.07%, 0.06%, or 0.05% TFA).

[0032] In some embodiments, mobile phase B contains about 30% acetonitrile, about 30% alcohol, and TFA in water. In some embodiments, mobile phase B contains about 35% acetonitrile, about 35% alcohol, and TFA in water. In some embodiments, mobile phase B contains about 35-45% acetonitrile, about 35-45% alcohol, and TFA in water. In some embodiments, mobile phase B contains less than 0.1% TFA (e.g., 0.09%, 0.08%, 0.07%, 0.06%, 0.05% TFA). In some embodiments, mobile phase B contains 0.05-0.09% %TFA. In some embodiments, mobile phase B contains 40% isopropanol, 40% acetonitrile, 20% water, and 0.05% TFA.

[0033] Examples of alcohols in mobile phase B include, but are not limited to, isopropyl alcohol, propanol, and butyl alcohol.

[0034] In some embodiments, the elution step is performed in a gradient between mobile phase B solvent and polar mobile phase A solvent. In some embodiments, mobile phase A comprises trifluoroacetic acid (TFA) and water. In some embodiments, mobile phase A contains less than 0.1% TFA (e.g., 0.09%, 0.08%, 0.07%, 0.06%, 0.05% TFA). In some embodiments, mobile phase A contains about 0.05% to 0.09% % TFA. In some embodiments, mobile phase A is water and 0.05% TFA.

[0035] In some embodiments, the chromatography column used in the methods described herein is a polymer-based column or a silica-based column. In some embodiments, the chromatography column contains a divinylbenzene (DVB) resin. In some embodiments, the chromatography column contains polystyrene and a divinylbenzene (DVB) resin. An example of an available column containing a DVB resin is the PLPR-S column (AGILENT), which is not conventionally recommended or used for peptide mapping applications. Another choice of chromatography column for the methods described herein is a graphite-carbon column.

[0036] It has been observed herein that pore size can affect peptide recovery, as conventional C8 and C18 columns with a pore size of approximately 1.7 μm (Examples 1-2) yielded lower CDR3 peptide recovery rates than columns with larger pore sizes of 3-5 μm (Examples 3-4). In some embodiments, the chromatography column comprises porous particles having a particle size of at least 2 μm. In some embodiments, the chromatography column comprises porous particles having a particle size of at least approximately 2 μm, or approximately 3 μm, or approximately 4 μm, or approximately 5 μm, or approximately 6 μm, or approximately 7 μm, or approximately 8 μm, or approximately 9 μm, or approximately 10 μm, and including a range between any two of the listed values. In some embodiments, the chromatography column comprises porous particles having a particle size of approximately 2-5 μm, approximately 2-7 μm, approximately 3-5 μm, or approximately 3-7 μm.

[0037] In some embodiments, the chromatography column includes porous particles having a pore size of at least about 100 angstroms (e.g., about 100, about 200, about 300, about 400, or about 500 angstroms, and including a range between any two of the listed values, e.g., 100 to 500 angstroms), such as fully porous particles or surface porous particles. In some embodiments, the chromatography column includes fully porous particles having a pore size of 300 angstroms and a particle size of about 5 μm. The term “fully porous particles” as used herein refers to particles having a porous core and a porous shell. The term “surface porous particles” as used herein refers to particles having a solid core and a porous shell.

[0038] In some embodiments, the protein is an antibody and comprises a heavy chain variable region CDR3 (HCDR3) and / or a light chain variable region CDR3 (LCDR3). The amount of unmodified H-CDR3-containing polypeptide and / or unmodified LCDR3-containing polypeptide eluted from a chromatography column can be identified using the methods described herein. The term “unmodified” as used herein with respect to HCDR3-containing polypeptide and / or CDR3-containing polypeptide eluted from a chromatography column refers to an HCDR3 or LCDR3-containing polypeptide without post-translational modification (PTM).

[0039] Advantageously, the methods described herein allow for the easy recovery of hydrophobic peptides such as CDR3 peptides (e.g., HCDR3 and / or LCDR3 peptides). Conventional peptide mapping methods may not recover enough HCDR3 and / or LCDR3 to enable peptide mapping of the HCDR3 and / or LCDR3 regions (each, respectively). For example, if less than 10% of the peptide is recovered from the chromatography column, the detection of that peptide may be limited, and the quantification of modified peptides may be inaccurate. Conventional methods have yielded recovery rates of approximately 1-2% for the CDR3 peptide of some proteins. Therefore, conventional peptide mapping may yield coverage of approximately 85%, 90%, or 95% or less of the light or heavy chain, and the CDR3 region may be excluded from coverage (see Examples 1-2). However, according to the methods of some embodiments herein, at least 10%, 15%, 20%, 25%, or 30% of the CDR3 peptide can be recovered. Therefore, the method described herein allows for the recovery of HCDR3 and / or LCDR3 peptides to create a peptide map containing coverage of HCDR3 and / or LCDR3 (each). The peptide map can cover all or substantially the protein. For example, the method described herein can provide coverage of at least 96%, 97%, 98%, 99%, 99.5%, or 100% of a protein (see Examples 3-4). In some embodiments, the method provides coverage of at least 96%, 97%, 98%, 99%, or 99.5% of a light chain polypeptide, heavy chain polypeptide, or light and heavy chain polypeptide. In some embodiments, the method provides 100% coverage of a light chain polypeptide, heavy chain polypeptide, or light and heavy chain polypeptide.

[0040] In some embodiments, at least 50% and / or at least 50% of the HCDR3-containing polypeptide eluted from the chromatography column are unmodified (e.g., lacking post-translational modifications (PTMs)). In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the HCDR3-containing polypeptide eluted from the chromatography column are unmodified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the LCDR3-containing polypeptide eluted from the chromatography column is unmodified.

[0041] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of both the HCDR3-containing polypeptide and the LCDR3-containing polypeptide eluted from the chromatography column are unmodified.

[0042] The chromatography methods described herein allow for the modification of various parameters. These parameters include protein load, operating temperature, conductivity of the protein loaded onto the column, bed height (height of the chromatography column), linear velocity, and pH. For example, the protein load may be approximately 10–200 g / L, 50–200 g / L, 55–85 g / L, 60–80 g / L, 65–75 g / L, 100–200 g / L, 100–150 g / L, 125–175 g / L, 150–200 g / L, or 90–140 g / L. The on-column operating temperature may be approximately 15°C–25°C or 18°C–22°C. The height of the chromatography column may be approximately 10 cm to 35 cm, or approximately 20 cm to 30 cm, or approximately 23 cm to 27 cm (for example, heights of approximately 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, 30 cm, 31 cm, 32 cm, 33 cm, 34 cm, or 35 cm, including the range between any two values ​​listed). The linear velocity may be approximately 10 cm / hour to approximately 250 cm / hour, or approximately 120 cm / hour to approximately 220 cm / hour, approximately 125 cm / hour to approximately 165 cm / hour, or approximately 180 cm / hour to approximately 210 cm / hour. The pH may be approximately 5 to approximately 9, approximately 5 to approximately 7, approximately 7 to approximately 9, approximately 6 to approximately 8, approximately 5.5 to approximately 8.5, approximately 6.5 to approximately 8.5, approximately 5 to approximately 6, approximately 8 to approximately 9, approximately 7 to approximately 8, approximately 7 to approximately 7.5, or approximately 7.5 to approximately 8. In various cases, the pH is ±2 pH units of the pI of the target protein, or ±1 pH unit of the pI of the target protein, or ±0.5 pH units of the pI of the target protein. The sample solution and / or formulation used in chromatography may have a pH as described herein.The conductivity of the protein loaded onto the column may be approximately 10-50 mS / cm, 10-20 mS / cm, 15-25 mS / cm, 10-30 mS / cm, 10-40 mS / cm, 20-50 mS / cm, 30-50 mS / cm, 40-50 mS / cm, 20-30 mS / cm, 30-40 mS / cm, or 15-30 mS / cm.

[0043] The exposure time of mobile phase B to the chromatography column is at least 15 minutes. In some embodiments, the exposure time of mobile phase B to the chromatography column is in the range of 15 minutes to 3 hours, 6 hours, 12 hours, or 24 hours. In some embodiments, the exposure time of washing to the column is approximately 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 49 minutes, 50 minutes, 51 minutes. , 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 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, 23 hours, 24 hours or more, and include a range between any two of the listed values. In some embodiments, peptide mapping is performed for about 5 hours or less, for example, 2-3 hours, 2-4 hours, 2-5 hours, 3-4 hours or 3-5 hours.

[0044] spectroscopy In some embodiments, the methods described herein further include the step of performing a spectroscopic analysis of the eluted polypeptide. Examples of spectroscopic analysis include, but are not limited to, mass spectrometry (Rubakhin and Sweedler, 2010), ultraviolet spectroscopy, visible light spectroscopy, fluorescence spectroscopy, ultraviolet-visible light spectroscopy, and infrared spectroscopy.

[0045] The underlying principle of mass spectrometry (MS) involves ionizing a chemical substance to generate charged molecules or molecular fragments, and then measuring their mass-to-charge ratio. An exemplary MS procedure involves loading the sample into an MS instrument and vaporizing it, ionizing the components of the sample by one of various methods that cause the formation of positively charged particles (for example, by bombarding them with an electron beam), then accelerating these positive ions with a magnetic field, calculating the mass-to-charge ratio (m / z) of the particles based on the details of the ions' motion as they pass through the electromagnetic field, and detecting the ions separated according to the m / z ratio.

[0046] An exemplary MS instrument has three modules: an ion source that converts sample molecules in the gas phase into ions (or, in the case of electrospray ionization, moves ions present in solution into the gas phase); a mass spectrometer that separates the ions by their mass-to-charge ratio by applying an electromagnetic field; and a detector that measures numerical values ​​of indicator quantities, thereby providing data for calculating the abundance of each ion present.

[0047] MS techniques have both qualitative and quantitative applications, including identifying unknown compounds, determining the isotopic composition of elements within molecules, and determining the structure of compounds by observing their fragmentation. Examples include gas chromatography-mass spectrometry (GC / MS or GC-MS), liquid chromatography-mass spectrometry (LC / MS or LC-MS), ion mobility spectroscopy / mass spectrometry (IMS / MS or IMMS), matrix-assisted laser desorption / ion source (MALDI-TOF) consisting of a TOF analyzer; electrospray ionization mass spectrometry (ESI-MS), inductively coupled plasma mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization mass spectrometry (TIMS), and spark source mass spectrometry (SSMS).

[0048] Therapeutic protein In some embodiments, the protein processed in any of the methods described herein is a therapeutic protein. In exemplary embodiments, the therapeutic protein is an antibody. As used herein, the term “antibody” refers to a protein having the conventional immunoglobulin form, comprising a heavy chain and a light chain, and comprising a variable region and a constant region. For example, an antibody may be IgG, which is a “Y-type” structure of two identical pairs of polypeptide chains, each pair having one “light” chain (typically with a molecular weight of about 25 kDa) and one “heavy” chain (typically with a molecular weight of about 50–70 kDa). Antibodies have a variable region and a constant region. In the IgG form, the variable region generally consists of about 100–110 or more amino acids and includes three complementarity-determining regions (CDRs), which are primarily involved in antigen recognition and substantially different among other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region consists of the N-terminal regions of each light and heavy chain, while the constant region consists of the C-terminal regions of each heavy and light chain. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4) th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0049] The general structure and properties of antibody CDRs have been described in the art. Briefly, in the antibody backbone, CDRs are embedded within the framework of the variable regions of the heavy and light chains, where they constitute regions that play a significant role in antigen binding and recognition. The variable region typically contains three heavy or light chain CDRs (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within the framework region (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, op. cit.).

[0050] Antibodies may include any constant region known in the art. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha, or epsilon, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG includes, but is not limited to, several subclasses such as IgG1, IgG2, IgG3, and IgG4. IgM includes, but is not limited to, subclasses such as IgM1 and IgM2. Embodiments of this disclosure include all such classes or isotypes of antibodies. Light chain constant regions may be, for example, kappa or lambda light chain constant regions, e.g., human kappa or human lambda light chain constant regions. Heavy chain constant regions may be, for example, alpha, delta, epsilon, gamma, or mu heavy chain constant regions, e.g., human alpha, human delta, human epsilon, human gamma, or human mu heavy chain constant regions. Therefore, in the exemplary embodiment, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody, and includes one of IgG1, IgG2, IgG3, or IgG4.

[0051] Antibodies can be monoclonal or polyclonal antibodies. In some embodiments, antibodies contain sequences substantially similar to naturally occurring antibodies produced by mammals, such as mice, rabbits, goats, horses, chickens, hamsters, and humans. In this respect, antibodies can be considered mammalian antibodies, such as mouse antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, and human antibodies. In certain embodiments, antibodies are human antibodies. In certain embodiments, antibodies are chimeric antibodies or humanized antibodies. The term "chimeric antibody" refers to an antibody that contains domains derived from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain derived from one species and a variable domain derived from a second species, or more generally, a stretch of amino acid sequences derived from at least two species. A chimeric antibody may also contain domains from two or more different antibodies within the same species. When used in relation to antibodies, the term "humanization" refers to an antibody of non-human origin that has at least a CDR region and has been manipulated to have a structure and immune function more similar to a true human antibody than to the original source antibody. For example, humanization may include grafting a CDR from a non-human antibody, such as a mouse antibody, onto a human antibody. Humanization may also include selecting amino acid substitutions to further resemble a human sequence in a non-human sequence.

[0052] Antibodies can be fragmented into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. In exemplary embodiments of this disclosure, the therapeutic protein is an antigen-binding fragment or an antibody. As used herein, the term “antigen-binding antibody fragment” refers to the portion of an antibody that can bind to an antigen, and is also known as “antigen-binding fragment” or “antigen-binding moiety.” In exemplary examples, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0053] In various embodiments, therapeutic proteins are antibody protein products. As used herein, the term “antibody protein product” refers in various examples to one of several antibody substitutes that are based on the structure of an antibody but are not found in nature. In some embodiments, antibody protein products have a molecular weight in the range of at least about 12 to 150 kDa. In certain embodiments, antibody protein products have a valency (n) in the range of monomer (n=1) to dimer (n=2), trimer (n=3), and tetramer (n=4), if not of a higher valency. In some embodiments, antibody protein products are products based on the complete antibody structure and / or products that mimic antibody fragments that retain complete antigen-binding ability, such as scFv, Fab, and VHH / VH (described below). The smallest antigen-binding antibody fragment that retains a complete antigen-binding site is the Fv fragment, which consists entirely of a variable (V) region. A soluble and mobile amino acid peptide linker is used to stabilize the molecule by linking the V region to the scFv (single-chain variable fragment) fragment, or by adding a constant (C) domain to the V region to form a Fab fragment [antigen-binding fragment]. Both scFv and Fab fragments can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody forms such as diabodies, triabodies, and tetrabodies or minibodies (mini-Abs), which include different forms consisting of disulfide-bonded scFv (ds-scFv), single-chain Fab (scFab), and scFv linked to an oligomeric domain. The smallest fragment is the VHH / VH of camelid heavy-chain Ab and single-domain Ab (sdAb). The most frequently used building block for creating novel antibody forms is the single-chain variable (V)-domain antibody fragment (scFv), which contains V domains (VH domain and VL domain) derived from the heavy and light chains, linked by a peptide linker of approximately 15 amino acid residues. Peptibodies, or peptide-Fc fusions, are yet another antibody protein product. The structure of a peptide body consists of a bioactive peptide grafted onto an Fc domain. Peptibodies have been well described in the art.For example, see Shimamoto et al., mAbs 4(5):586-591 (2012).

[0054] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, and bispecific or trispecific antibodies. Bispecific antibodies can be classified into five main classes: BsIgG, IgG adducts, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0055] In exemplary embodiments, the therapeutic protein is a bispecific T cell engager (BiTE®) molecule, which is an artificial bispecific monoclonal antibody. A standard BiTE® molecule is a fusion protein containing two scFvs of different antibodies. One binds to CD3, and the other binds to the target antigen. BiTE® molecules are known in the art. See, for example, Huehls et al., Immuno Cell Biol 93(3):290-296(2015); Rossi et al., MAbs 6(2):381-91(2014); Ross et al., PLoS One 12(8):e0183390.

[0056] In exemplary embodiments, the therapeutic protein is a chimeric antigen receptor (CAR). A chimeric antigen receptor is a genetically engineered fusion protein composed of multiple domains of other naturally occurring molecules typically expressed by immune cells. In some embodiments, a CAR includes an extracellular antigen-binding or antigen-recognition domain, a signaling domain, and a costimulatory domain. CARs have been described in the Art. See, for example, Maus et al., Clin Cancer Res 22(8):1875-1884(2016); Dotti et al., Immuno Rev(2014)257(1):10.1111 / imr.12131; Lee et al., Clin Cancer Res(2012):18(10):2780-2790; and June and Sadelain, NEJM 379:64-73(2018).

[0057] Exemplary therapeutic proteins include, but are not limited to, CD proteins, growth factors, growth factor receptor proteins (e.g., HER receptor family proteins), cell adhesion molecules (e.g., LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and αv / β3 integrins), hormones (e.g., insulin), coagulation factors, coagulation-related proteins, colony-stimulating factors and their receptors, as well as other receptors and receptor-related proteins or ligands for these receptors, and viral antigens.

[0058] Examples of therapeutic proteins include any one of the CD proteins, such as CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD3 7, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD4 9e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66 d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD 90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, C D115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132 , CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD150, CD151, CD152, CD153,CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and CD182 are examples.

[0059] Examples of growth factors include, for instance, vascular endothelial growth factor ("VEGF"), growth hormone, thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone-releasing factor (GHRF), parathyroid hormone (PTH), Müllerian duct inhibitor (MIS), human macrophage inflammatory protein (MIP-I-α), erythropoietin (EPO), nerve growth factor (NGF), such as NGF-β, and platelet-derived growth factor. Examples of growth factor receptors include (PDGF), fibroblast growth factor (FGF) including aFGF and bFGF, epidermal growth factor (EGF), particularly transforming growth factors (TGF) including TGF-α and TGF-β, for example TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5, insulin-like growth factors I and II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and bone induction factors. In some embodiments, therapeutic proteins are insulin or insulin-related proteins, such as insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins. Exemplary growth factor receptors include any receptor for any of the above growth factors. In various embodiments, growth factor receptors include HER receptor family proteins (e.g., HER2, HER3, HER4, and EGF receptors), VEGF receptors, TSH receptors, FSH receptors, LH receptors, GHRF receptors, PTH receptors, MIS receptors, MIP-1-α receptors, EPO receptors, NGF receptors, PDGF receptors, FGF receptors, EGF receptors (EGFR), TGF receptors, or insulin receptors.

[0060] Exemplary coagulation proteins and coagulation-related proteins include, for example, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin and thrombopoietin; other blood and serum proteins such as, but not limited to, albumin, IgE and blood group antigens; colony-stimulating factors and their receptors, particularly M-CSF, GM-CSF and G-CSF, and their receptors such as the CSF-1 receptor (c-fms); receptors and receptor-related proteins such as the flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor, such as c-Kit and other receptors. Receptor ligands, such as OX40L, the ligand for the OX40 receptor. Neurotrophic factors, such as bone-derived neurotrophic factor (BDNF) and neurotrophins-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A chain, relaxin B chain, and prorelaxin; interferons and interferon receptors, such as interferon-α, -β, and -γ, and their receptors. Interleukins and interleukin receptors, such as IL-I to IL-33 and IL-I to IL-33 receptors, especially the IL-8 receptor. Viral antigens, such as HIV enveloped virus antigens. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactants, tumor necrosis factor α and β, enkephalinases, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptides, DNases, inhibin, and activin.Integrins, proteins A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, denaturation inhibitors (DAFs), HIV envelope, transport proteins, homing receptors, adrecins, regulatory proteins, immunoadhesins, antibodies. Further exemplary therapeutic proteins include, for example, myostatin, TALL protein, e.g., TALL-I, amyloid protein, e.g., amyloid-beta protein, but not limited to, thymic-interstitial lymphocyte generating factor ("TSLP"), RANK ligand ("RANKL or OPGL"), c-kit, TNF receptors, e.g., TNF receptor type 1, TRAIL-R2, angiopoietin, and any of the aforementioned bioactive fragments, analogs, or variants.

[0061] In exemplary embodiments, therapeutic proteins include Activase® (alteplase); alirocumab, Aranesp® (darbepoetin-alpha), Epogen® (epoetin-alpha or erythropoietin); Avonex® (interferon β-1a); Bexxar® (tocitumomab); Betaseron® (interferon β); vococizumab (an anti-PCSK9 monoclonal antibody called L1L3, see U.S. Patent No. 8080243); Campath® (aremtuzumab); Dynepo® (epoetin delta); Velcade® (bortezomib); MLN0002 (anti-α4β7) mAb);MLN1202 (anti-CCR2 chemokine receptor mAb);Enbrel® (etanercept);Eprex® (epoetin alfa);Erbitux® (cetuximab);Evolocumab;Genotropin® (somatropin);Herceptin® (trastuzumab);Humatrope® (somatropin [rDNA-derived] injection);Humira® (adalimumab);Infergen® (interferon alpha-1);Natrecor® (nesiritide);Kineret® (anakinra), Leukine® (sargamostim);Lymph oCide(registered trademark) (epratuzumab); Benlysta(trademark) (belimumab); Metalyse(registered trademark) (tenecteplase); Mircera(registered trademark) (methoxypolyethylene glycol epoetin beta); Mylotarg(registered trademark) (gemtuzumab ozogamicin); Raptiva(registered trademark) (efalizumab); Cimzia(registered trademark) (certolizumab pegol); Soliris(trademark) (eculizumab); pexerizumab (anti-C5 complement); MEDI-524 (Numax(registered trademark)); Lucentis(registered trademark) (ranibizumab); edrecolomab (Panorex(registered trademark)); Trabio(registered trademark) (reldelimumab); TheraCim hR3 (nimotuzumab); Omnitarg (pertuzumab, 2C4);Osidem(registered trademark)(IDM-I); OvaRex(registered trademark)(B43.13); Nuvion(registered trademark)(vizilizumab); Cantuzumab meltansine (huC242-DMl); NeoRecormon(registered trademark)(epoetin beta); Neumega(registered trademark)(oprelbequin); Neulasta(registered trademark)(PEGylated filgastrim, PEGylated G-CSF, PEGylated hu-Met-G-CSF); Neupogen(registered trademark)(filgrastim); Orthoclone OKT3(registered trademark) (muromonab-CD3), Procrit(registered trademark) (epoetin alfa); Remicade(registered trademark) (infliximab), Reopro(registered trademark) (absiximab), Actemra(registered trademark) (anti-IL6 receptor mAb), Avastin(registered trademark) (bevacizumab), HuMax-CD4(zanorimumab), Rituxan(registered trademark) (rituximab); Tarceva(registered trademark) (erlotinib); Roferon-A(registered trademark) (interferon alfa-2a); Simulect(registered trademark) (vasili Ximab; Stellara (trademark) (ustekinumab); Prexige (registered trademark) (lumiracoxib); Synagis (registered trademark) (palivizumab); 146B7-CHO (anti-IL15 antibody, see U.S. Patent No. 7153507); Tysabri (registered trademark) (natalizumab); Valortim (registered trademark) (MDX-1303, anti-anthrax (B. anthracis) protective antigen mAb); ABthrax (trademark); Vectibix (registered trademark) (panitumumab); Xolair (registered trademark) (omalizumab); ETI211 (anti-MRSA) mAb), IL-1 Trap (Fc portion of human IgG1 and extracellular domains of both IL-I receptor components (type 1 receptor and receptor co-protein)), VEGF Trap (Ig domain of VEGFR1 fused with IgG1 Fc), Zenapax® (daclizumab); Zenapax® (daclizumab), Zevalin® (ibritumomab tiuxetan), Zetia (ezetimibe), Atasicept (TACI-Ig), anti-α4β7 mAb (vedolizumab);Galiximab (anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab); BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); Simponi (trademark) (golimumab); Mapatumumab (human anti-TRAIL receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (saltumumab); M200 (boroxiximab, anti-α5β1 integrin mAb); MDX-010 (ipilimumab, anti-CTLA-4 mAb and VEGFR-1 (IMC-18F1); anti-BR3 mAb; MDX-066 (CDA-I) and MDX-1388, which are anti-C. difficile toxin A and toxin BC mAbs); anti-CD22 dsFv-PE38 conjugate (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibody; anti-TSLP receptor antibody (US Patent No. 8101182); anti-TSLP antibody called A5 (US Patent No. 7982016); (anti-CD3 mAb (NI-0401)); adecatumumab (MT201, anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAb); MDX-1333 (anti-IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti-CTGF idiopathic pulmonary fibrosis stage 1 fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxin 1 mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb; anti-sclerostin antibody (see U.S. Patent No. 8715663 or No. 7592429), anti-sclerostin antibody called Ab-5 (U.S. Patent No. 8715663 or No. 7592429); anti-ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF receptor mAb (CAM-3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNα mAb); anti-IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); anti-IL12 / IL23p40 mAb (briakinumab); anti-IL-23p19 mAb(LY2525623);Anti-IL13 mAb (CAT-354); Anti-IL-17 mAb (AIN457); Anti-IL2Ra mAb (HuMax-TAC); anti-IL5 receptor mAb; anti-integrin receptor mAb (MDX-Ol8, CNTO95); anti-IPIO ulcerative colitis mAb (MDX-1100); anti-LLY antibody; BMS-66513; anti-mannose receptor / hCGβ mAb (MDX-1307); anti-mesothelin dsFv-PE38 conjugate (CAT-5001); anti-PDlmAb (MDX-1 106 (ONO-4538)); anti-PDGFRα antibody (IMC-3G3); anti-TGFβ mAb (GC-1008); anti-TRAIL receptor-2 human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR / Flt-1 mAb; anti-ZP3 This is one of the following known therapeutic agents: mAb (HuMax-ZP3); NVS antibody #1; NVS antibody #2; or amyloid-beta monoclonal antibody.

[0062] Further examples of therapeutic proteins include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, avagovomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artitumomab, aselizumab, artinumab, atorizumab, atrolimumab, tocilizumab, bapineuzumab, basiliximab, and babitsu. Simab, Vectumomab, Belimumab, Benralizumab, Vertilimumab, Besilesomab, Bezlotoxumab, Bisilomab, Vibatuzumab, Vibatuzumab Meltansine, Blinatumomab, Brosozumab, Brentuximab Vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab Meltansine, Caplacizumab, Capromab Pendetide, Carlumab, Katsumakisomab, CC49, Sedelizumab, Certolizumab Pegol, Sitatuzumab Bogatox, Sixtumumab, Clazakizumab, Clenoliximab, Cribatuzumab Tetraxetan, Conatum Mab, crenezumab, cr6261, dasetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorulimomab aritox, dorozizumab, duligotuzumab, dupilumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, erucirimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab citucetan, epratuzumab, erenumab, erlizumab, ertzumakisomab, etalacizumab Etrolizumab, evolocumab, excivivirumab, fanolesomab, falarimomab, falletuzumab, facinumab, fbta05, felbizumab, fezakinumab, ficratuzumab, figitumumab, frambotumab, fontrizumab, foralumab, folavirumab, fresolimmab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab, gabirimomab, gemtuzumab ozogamicin, gevokizumab, gylenetuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibarizumabIbritumomab tiuxetan, iclucumab, igobomab, imusilomab, imugatuzumab, incrumab, indatuximab tansine, intetumumab, inorimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itorizumab, ixekizumab, keriximab, rabetsuzumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, livi Bilmab, rigerizumab, lintuzumab, lirirumab, lorbotuzumab meltansine, lucatumumab, lumiliximab, mapatumumab, masurimomab, mapurilimumab, matsuzumab, mepolizumab, meterimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimmab, motabizumab, moxetumomab pasdotox, muromonab-cd3, nacolomabutafe Natox, Namilumab, Naptumomab, Estafenatox, Narunatumab, Natalizumab, Nevacumab, Necitumumab, Nererimomab, Nesbacumab, Nimotuzumab, Nivolumab, Nofetumomab, Merpentan, Okalatuzumab, Oclerizumab, Odurimomab, Ofatumumab, Oraratuzumab, Orokizumab, Omalizumab, Onarutuzumab, Oporutuzumab, Monatox, O Legovomab, Orticumab, Oterixizumab, Oxerumab, Ozanezumab, Ozoralizumab, Padibaximab, Panitumumab, Panobacumab, Pulsatuzumab, Pascolizumab, Pateclizumab, Patrizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexerizumab, Pizilizumab, Pintumomab, Prakurumab, Ponezumab, Priliximab, Pritumumab, PRO 140, quilizumab, lacosumomab, radrezumab, rafivirumab, ramucirumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevilumab, sibro Tuzumab, cifarimumab, siltuximab, simtuzumab, ciprizumab, silumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamlumab, thresomab, suvizumab, tabarmab, tacutuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tapritumomab paptox, tefivazumab, terimomab aritox,Tenatumomab, teneriximab, teprizumab, teprotumumab, tezeperumab, TGN1412, tremelimumab, tisilimmab, tildrakizumab, tigatuzumab, TNX-650, tralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotsuzumab cermoloykin, tubirumab, ubrituxi Examples of antibodies include mab, urelumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, bertuzumab, bepalimomab, besenkumab, vizilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanorimumab, zatuximab, diralimumab, and zolimomab aritox.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066] In some embodiments, the therapeutic polypeptide is a BiTE® molecule. Blinatumomab (BLINCYTO®) is an example of a CD19-specific BiTE® molecule. Modified BiTE® molecules (e.g., those modified to extend their half-life) can also be used in the manner of this disclosure.

[0067] All identified patents and other publications are expressly incorporated herein by reference, in whole or in relevant parts, for the purpose of describing and disclosing methodologies described in such publications that may be used in connection with the information contained herein, for example.

[0068] The use of the terms “one(a),” “one(an),” and “it,” as well as similar reference subjects, in relation to the description of this disclosure (particularly in relation to the following claims), should be construed as encompassing both singular and plural unless otherwise indicated herein or unless explicitly contradicted by the context. The terms “contains,” “have,” “includes,” and “contains” should be construed as open-ended terms (i.e., “contains, but not limited to”) unless otherwise noted.

[0069] Unless otherwise indicated herein, the descriptions of value ranges are merely abbreviations referring individually to each of the distinct values ​​at each of the ranges and endpoints, and each of the distinct values ​​and endpoints is incorporated herein as if it were individually described herein.

[0070] All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless it is clearly inconsistent with the context. The use of any examples or representative language provided herein (e.g., "etc.") is intended solely to clarify the disclosure and does not impose any limitation on the scope of the disclosure unless otherwise claimed. No language herein should be construed as indicating any unclaimed element as essential to performing the disclosure.

[0071] The following embodiments are provided merely to illustrate the present invention and are not intended to limit its scope. [Examples]

[0072] Example 1 - Conventional peptide mapping under reducing conditions Samples (100-500 mg) of monoclonal antibodies mAb A, mAb B, mAb C, and mAb D were denatured by dilution in a denaturation buffer containing 0.25 M Tris, 7.5 M guanidine-HCl, pH 7.5, and then incubated under reducing conditions in 0.5 M dithiothreitol (DTT) at room temperature for 25 minutes. The reduced samples were then alkylated using 0.5 M sodium iodide acetate / acetic acid and incubated in the dark at room temperature for 20 minutes. The reduced and alkylated samples were then buffer-changed using a size exclusion column to digestion buffer (0.1 M Tris, pH 7.5) to remove previous buffer components. Next, the samples were digested using trypsin endopeptidase in a 1:10 (enzyme:sample) ratio and incubated at 37°C for 30 minutes. The reaction was quenched by adding trifluoroacetic acid to a final concentration of 1% (v / v). The digested samples were analyzed using liquid chromatography-tandem mass spectrometry (MS / MS).

[0073] A liquid chromatography-MS / MS system was constructed using a UPLC / HPLC system connected in-line to a mass spectrometer. Samples (10-50 ug) were injected into a C18 / C8 stationary phase column maintained at 50°C. Separation was achieved by applying a linear gradient of 0%-40% mobile phase B (using 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, respectively) for 210 minutes at a flow rate of 0.1 mL / min. Data acquisition was performed in positive mode, and each peptide was subjected to MS / MS for sequence information. The presence or absence of post-translational modifications (PTMs) of HCDR3 and LCDR3-containing polypeptides was evaluated. The modification rate was calculated by dividing the total area of ​​oxidized peptides by the sum of the total areas of oxidized and unoxidized peptides. The results are shown in Table 1 below.

[0074] [Table 4]

[0075] The results showed that 84.1% of the unmodified (e.g., lacking PTM) light chain and 91.5% of the unmodified heavy chain of mAb A were recovered in the eluate, 96.7% of the unmodified light chain and 81.6% of the unmodified heavy chain of mAb B were recovered in the eluate, 75.7% of the unmodified light chain and 85.0% of the unmodified heavy chain of mAb C were recovered in the eluate, and 97.2% of the unmodified light chain and 93.1% of the unmodified heavy chain of mAb D were recovered in the eluate. The tryptophan in HCDR3 of mAb A had been identified as a site susceptible to oxidation, but peptides containing this tryptophan had not been recovered at all by conventional methods.

[0076] Example 2 - Conventional peptide mapping under non-reducing conditions mAb A of the sample (100-500 mg) was denatured using RapiGest (Waters Corp., Milford, MA), and then digested overnight under denaturation conditions in the presence of NEM (n-ethylmaleimide) with the endopeptidase trypsin. The reaction was quenched by adding trifluoroacetic acid to a final concentration of 1% (v / v). The digested sample was then analyzed by liquid chromatography-tandem mass spectrometry (MS / MS).

[0077] A liquid chromatography-MS / MS system was constructed using a UPLC / HPLC system connected in-line to a mass spectrometer. Samples (10–50 ug) were injected into a Waters Acquity BEH C4 stationary phase column maintained at 50°C. Separation was achieved by applying a linear gradient of 0%–40% mobile phase B (using 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, respectively) over 220 minutes at a flow rate of 0.1 mL / min. Data acquisition was performed in positive mode, and each peptide was subjected to MS / MS. The presence or absence of post-translational modifications (PTMs) in HCDR3 and LCDR3-containing polypeptides was evaluated. Disulfide bonds were identified and confirmed using sequence information and then informatics tools. The results are shown in Table 2 below.

[0078] [Table 5]

[0079] The results showed that 95.5% of the unmodified light chains and 94.8% of the unmodified heavy chains were recovered in the eluate.

[0080] Example 3 - High-yield peptide mapping method under reducing conditions Samples of mAb A, mAb B, mAb C, or mAb D (100-500 mg) were denatured by dilution in a denaturation buffer containing 0.25 M Tris, 7.5 M guanidine-HCl, 0.25 mM EDTA, and pH 7.5, and then reduced by incubation in 0.5 M dithiothreitol (DTT) at room temperature for 25 minutes. The reduced samples were then alkylated using 0.5 M sodium iodide acetate / acetic acid and incubated in the dark at room temperature for 20 minutes. Subsequently, the reduced and alkylated samples were buffer-exchanged using a size exclusion column to digestion buffer (0.1 M Tris, pH 7.5) to remove previous buffer components. Next, the samples were digested using trypsin endopeptidase in a 1:10 (enzyme:sample) ratio and incubated at 37°C for 30 minutes. The reaction was quenched by adding trifluoroacetic acid to a final concentration of 1% (v / v). Next, the digested samples were analyzed using liquid chromatography-tandem mass spectrometry (MS / MS).

[0081] A liquid chromatography MS / MS system was constructed using a UPLC / HPLC system connected in-line to a mass spectrometer. Samples (10-50 ug) were injected into an Agilent PLRP-S column maintained at 50°C. Separation was achieved by applying a linear gradient at a flow rate of 0.2 mL / min over 104 minutes, using water containing 0.05-0.1% formic acid or 0.05-0.1% trifluoroacetic acid (ranging from 0% to 48%) as mobile phase B, and 40% isopropyl alcohol / 40% acetonitrile / 20% water containing 0.05-0.1% formic acid or 0.05-0.1% trifluoroacetic acid. Data acquisition was performed in positive mode, and each peptide was subjected to MS / MS for sequence information. The presence or absence of post-translational modifications (PTMs) in HCDR3 and LCDR3-containing polypeptides was evaluated. The modification rate was calculated by dividing the total area of ​​oxidized peptides by the sum of the total areas of oxidized and unoxidized peptides. The results are shown in Table 3 below.

[0082] [Table 6]

[0083] The results showed that 100% of both the unmodified light and heavy chains of mAb A were recovered in the eluate.

[0084] The results also showed that 96.3% of the unmodified (e.g., lacking PTM) light chain and 98% of the unmodified heavy chain of mAb B were recovered in the eluate, 98.1% of the unmodified light chain and 96.6% of the unmodified heavy chain of mAb C were recovered in the eluate, and 98.1% of the unmodified light chain and 98.0% of the unmodified heavy chain of mAb D were recovered in the eluate.

[0085] Example 4 - High-yield peptide mapping method under non-reducing conditions Samples (100-500 mg) containing different HLE BiTE® molecules (HLE-BiTE® A, HLE-BiTE® B, and HLE-BiTE® C) were denatured using RapiGest, and then digested overnight under denaturation conditions in the presence of NEM (n-ethylmaleimide) using the endopeptidase trypsin. The reaction was quenched by adding trifluoroacetic acid to a final concentration of 1% (v / v). The digested samples were analyzed by liquid chromatography-tandem mass spectrometry (MS / MS).

[0086] The liquid chromatography-MS / MS system consisted of a UPLC / HPLC system connected in-line to a mass spectrometer. Samples (10-50 ug) were injected into Agilent PLRP-S, Acuity C8, or Acuity C4 columns maintained at 50°C, and separation was achieved by applying a linear gradient at a flow rate of 0.2 mL / min for 70 minutes using 5% isopropyl alcohol containing 95% water / 0.1% formic acid and 40% isopropyl alcohol / 40% acetonitrile / 20% water / 0.1% formic acid, with B ranging from 0% to 35%.

[0087] Peptide yield was also evaluated under conventional peptide mapping conditions. Briefly, samples (10-50 ug) were injected into Zorbax SB-C18, Acquity C18, or Acquity C8 columns maintained at 50°C, and a linear gradient of mobile phase B, ranging from 0% to 40% using 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively, was applied for 220 minutes at a flow rate of 0.1 mL / min.

[0088] Data acquisition was performed in positive mode, and each peptide was subjected to MS / MS for sequence information. The presence or absence of post-translational modifications (PTMs) in HCDR3 and LCDR3-containing polypeptides was evaluated. Using the sequence information, disulfide bonds were then identified and confirmed using informatics tools.

[0089] The results shown in Table 4 below for HLE-BiTE®A demonstrate that the method described in this example, using a PLRPS column and mobile phase B containing 40% isopropyl alcohol / 40% acetonitrile / 20% water, successfully identified all target peptides (indicated by "X" in Table 4 below). Similar results were observed for HLE-BiTE®B and HLE-BiTE®C.

[0090] [Table 7]

[0091] Example 5 - Direct comparison of columns including performance overview mAb A samples (100-500 mg) were denatured by dilution in a denaturation buffer containing 0.25 M Tris, 7.5 M guanidine-HCl, 0.25 mM EDTA, and pH 7.5, and then reduced by incubation in 0.5 M dithiothreitol (DTT) at room temperature for 25 minutes. The reduced samples were then alkylated using 0.5 M sodium iodide acetate / acetic acid and incubated in the dark at room temperature for 20 minutes. Subsequently, the reduced and alkylated samples were buffer-changed using a size exclusion column to digestion buffer (0.1 M Tris, pH 7.5) to remove previous buffer components. Next, the samples were digested using trypsin endopeptidase in a 1:10 (enzyme:sample) ratio and incubated at 37°C for 30 minutes. The reaction was quenched by adding trifluoroacetic acid to a final concentration of 1% (v / v). Next, the digested samples were analyzed using liquid chromatography-tandem mass spectrometry (MS / MS).

[0092] The liquid chromatography-MS / MS system consisted of a UPLC / HPLC system connected in-line to a mass spectrometer. Separation was achieved by one of the following methods: (Method 1) The sample (10-50 ug) is injected into a Waters Acquity BEHC130 C18 stationary phase column maintained at 50°C, and a linear gradient of 0%-40% mobile phase B, using 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile as mobile phases A and B respectively, is applied at a flow rate of 0.1 mL / min for 220 minutes; (Method 2) Inject the sample (10-50 ug) into a Waters Acquity BEHC130 C18 stationary phase column maintained at 50°C, and apply a linear gradient of 0%-48% mobile phase B, using 0.1% trifluoroacetic acid in water as mobile phase A and 0.05-0.1% trifluoroacetic acid and 40% isopropyl alcohol / 40% acetonitrile / 20% water as mobile phase B, at a flow rate of 0.1 mL / min for 220 minutes; or (Method 3) The sample (10-50 ug) is injected into an Agilent PLRP-S column maintained at 50°C, and a linear gradient is applied for 220 minutes at a flow rate of 0.1 mL / min using water containing 0.1% trifluoroacetic acid as mobile phase A and 0.05-0.1% trifluoroacetic acid and 40% isopropyl alcohol / 40% acetonitrile / 20% water as mobile phase B.

[0093] Data acquisition was performed in positive mode, and each peptide was subjected to MS / MS for sequence information. The presence or absence of post-translational modifications (PTMs) of HCDR3 and LCDR3-containing polypeptides was evaluated. The percentage of modifications was calculated by dividing the total area of ​​oxidized peptides by the sum of the total areas of oxidized and unoxidized peptides. The results are shown in Table 5 below.

[0094] [Table 8]

[0095] The results showed that 100% of both the unmodified light and heavy chains of mAb A were recovered in the eluate under the conditions of Method 3. Therefore, the data in this example demonstrate that the PLRP-S column of Method 3 was superior to the C18 column of Method 2 when the same conditions were applied (100% vs. 88% for %LC sequence coverage and 100% vs. 95% for %HC sequence coverage, respectively). In addition, using the PLRP-S column with mobile phase B solvent containing TFA, acetonitrile, and alcohol was far superior to conventional methods for processing proteins (i.e., Method 1).

[0096] Example 6 - Further Column and Performance Overview The method described in Example 3 was repeated under reducing conditions using various columns to compare the peptide mapping performance of HLE-BiTE(registered trademark)B. The columns evaluated included PLRP-S, Polaris C18-Ether, Polaris C8-Ether, Peptide HSS T3, CORTECS T3, CORTECS C8, CORTECS phenyl, and CSH C18. The results are shown in Table 6 below.

[0097] [Table 9]

[0098] As shown in Table 5, when using the PLRP-S column, peptide 1 (CDR3 of HLE-BiTE® B) was observed with a higher recovery rate (>20%). A recovery rate of >20% enabled peptide mapping that reliably covered the residue of HLE-BiTE® B CDR3.

Claims

1. A method for processing proteins, The aforementioned protein is fragmented, thereby generating polypeptides. Adding the polypeptide to the chromatography column, The process involves eluting the polypeptide in an eluate containing mobile phase B solvent, wherein mobile phase B solvent is Trifluoroacetic acid (TFA), Acetonitrile, and alcohol Including elution A method that includes this.

2. The method according to claim 1, wherein the protein and polypeptide include complementarity-determining regions (CDRs) of variable regions, such as HCDR3 of the heavy chain variable region and / or LCDR3 of the light chain variable region.

3. The method according to claim 2, further comprising constructing a structural map of the protein, wherein the structural map includes the HCDR3 and the LCDR3.

4. The method according to any one of claims 1 to 3, wherein the mobile phase B contains 0.05% to 0.09% TFA.

5. The method according to any one of claims 2 to 4, wherein at least 50% of the CDR-containing polypeptide, for example, at least 50% of the HCDR3-containing polypeptide and / or at least 50% of the LCDR3-containing polypeptide, are eluted from the chromatography column.

6. The method according to any one of claims 1 to 5, wherein the mobile phase B comprises about 35 to 45% acetonitrile, 35 to 45% alcohol, and the TFA in water.

7. The method according to any one of claims 1 to 6, wherein the alcohol is isopropyl alcohol, propanol, or butyl alcohol.

8. The method according to any one of claims 1 to 7, wherein the elution occurs in the gradient between the mobile phase B solvent and the polar mobile phase A solvent.

9. The method according to claim 8, wherein the mobile phase A comprises TFA and water.

10. The method according to claim 9, wherein the mobile phase A contains less than 0.1% TFA, for example, 0.05% to 0.09% TFA.

11. The method according to any one of claims 1 to 10, wherein the chromatography column comprises porous particles having a particle size of about 2 to 5 μm, 2 to 7 μm, 3 to 5 μm, or 3 to 7 μm.

12. The method according to claim 11, wherein each of the porous particles has a pore size of about 100 to 500 angstroms, for example, about 300 angstroms.

13. The method according to any one of claims 1 to 10, wherein the chromatography column comprises perfectly porous particles having a pore size of about 300 angstroms and a particle size of about 5 μm.

14. The method according to any one of claims 1 to 13, wherein the chromatography column is at least 10 cm high.

15. The method according to any one of claims 1 to 13, wherein the chromatography column is at least 15 cm high.

16. The method according to any one of claims 1 to 13, wherein the chromatography column has a height of at least 25 cm.

17. The method according to any one of claims 1 to 16, wherein the chromatography column comprises a divinylbenzene (DVB) resin.

18. The method according to any one of claims 1 to 17, wherein the protein is in its reduced form.

19. The method according to any one of claims 1 to 17, wherein the protein is in its non-reduced form.

20. The method according to any one of claims 1 to 19, further comprising analyzing the eluted polypeptide by spectroscopic analysis.

21. The method according to any one of claims 1 to 20, wherein the protein comprises a therapeutic protein.

22. The method according to any one of claims 1 to 21, wherein the protein is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a derivative of an antibody or an antibody fragment, and a fusion polypeptide.

23. The method according to any one of claims 1 to 21, wherein the protein is a bispecific T cell engager molecule.

24. The aforementioned proteins include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, avagobomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artitumomab, aselizumab, artinumab, atorizumab, atromimab, tocilizumab, bapineuzumab, basiliximab, bavituximab, and bectumo Mab, belimumab, benralizumab, vertilimumab, besilesomab, bezlotoxumab, bisilomab, vibatuzumab, vibatuzumab meltansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab meltansine, caplacizumab, capromab pendetide, carrumab, katumakisomab, CC49, sedelizumab, certolizumab pegol, sitatuzumab bogatox, sixtumumab, crazakizumab, clenoliximab, cribatuzumab tetraxetan, konatumumab, clenoliximab Nezumab, cr6261, dasetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorulimomab aritox, dorozizumab, duligotuzumab, dupilumab, eclomeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, erucirimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab citucetan, epratuzumab, erenumab, erlizumab, ertzumakisomab, etalacizumab, e Trollizumab, evolocumab, exhibivirumab, fanolesomab, falarimomab, falletuzumab, facinumab, fbta05, felbizumab, fezakinumab, ficratuzumab, figitumumab, frambotumab, fontrizumab, foralumab, folavirumab, fresolimmab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab, gabirimomab, gemtuzumab ozogamicin, gevokizumab, gylenetuximab, glentzumumab vedotin, golimumab, gomiliximab, gs6624, ibarizumabIbritumomab tiuxetan, iclucumab, igobomab, imusilomab, imugatuzumab, incrumab, indatuximab tansine, intetumumab, inorimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itorizumab, ixekizumab, keriximab, rabetsuzumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, livi Bilmab, rigerizumab, lintuzumab, lirirumab, lorbotuzumab meltansine, lucatumumab, lumiliximab, mapatumumab, masurimomab, mapurilimumab, matsuzumab, mepolizumab, meterimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimmab, motabizumab, moxetumomab pasdotox, muromonab-cd3, nacolomabutafe Natox, Namilumab, Naptumomab, Estafenatox, Narunatumab, Natalizumab, Nevacumab, Necitumumab, Nererimomab, Nesbacumab, Nimotuzumab, Nivolumab, Nofetumomab, Merpentan, Okalatuzumab, Oclerizumab, Odurimomab, Ofatumumab, Oraratuzumab, Orokizumab, Omalizumab, Onarutuzumab, Oporutuzumab, Monatox, O Legobomab, Orticumab, Oterixizumab, Oxerumab, Ozanezumab, Ozoralizumab, Padibaximab, Panitumumab, Panobacumab, Pulsatuzumab, Pascolizumab, Pateclizumab, Patrizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexerizumab, Pizilizumab, Pintumomab, Prakurumab, Ponezumab, Priliximab, Pritumumab, PRO 140, quilizumab, lacosumomab, radrezumab, rafibirumab, ramucirumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevilumab, sibro Tuzumab, cifarimumab, siltuximab, simtuzumab, ciprizumab, silumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamlumab, thresomab, suvizumab, tabarmab, tacutuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tapritumomab paptox, tefivazumab, terimomab aritox,Tenatumomab, teneriximab, teprizumab, teprotumumab, tezeperumab, TGN1412, tremelimumab, tisilimmab, tildrakizumab, tigatuzumab, TNX-650, tralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucothuzumab cermoloykin, tubirumab, ubrituximab, urerumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, bell Tuzumab, bepalimomab, besenkumab, vizilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanolimumab, zatuximab, diralimumab, zolimomab aritox, glycoprotein, CD polypeptide, HER receptor polypeptide, cell adhesion polypeptide, growth factor polypeptide, insulin polypeptide, insulin-related polypeptide, coagulation polypeptide, coagulation-related polypeptide, albumin, IgE, blood group antigen The method according to any one of claims 1 to 21, selected from the group consisting of colony-stimulating factor, receptor, neurotrophic factor, interferon, interleukin, viral antigen, lipoprotein, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor α and β, enkephalinase, mouse gonadotropin-related peptide, DNAse, inhibin, activin, integrin, protein A, protein D, rheumatoid factor, immunotoxin, bone morphogenetic protein, superoxide dismutase, surface membrane polypeptide, disintegration promoter, HIV envelope, transport polypeptide, homing receptor, adresin, regulatory polypeptide, immunoadhesin, myostatin, TALL polypeptide, amyloid polypeptide, thymic interstitial lymphocyte necrosis factor, RANK ligand, c-kit polypeptide, TNF receptor, and angiopoietin, and their bioactive fragments, analogs, or variants.

25. Protein polypeptide fragments, Trifluoroacetic acid (TFA), Acetonitrile, and alcohol Elution containing mobile phase B solvent and A chromatography column, including one.

26. The chromatography column according to claim 25, wherein the protein comprises variable region CDRs, for example, HCDR3 of the heavy chain variable region and / or LCDR3 of the light chain variable region.

27. The chromatography column according to claim 25 or 26, wherein the mobile phase B contains 0.05% to 0.09% TFA.

28. The chromatography column according to claim 25 or 26, wherein the eluent contains more of the HCDR3-containing polypeptide than is bound to the column and / or more of the LCDR3-containing polypeptide than is bound to the column.

29. The chromatography column according to any one of claims 25 to 28, wherein the mobile phase B comprises about 35 to 45% acetonitrile, 35 to 45% alcohol, and the TFA in water.

30. The chromatography column according to any one of claims 25 to 29, wherein the mobile phase B comprises about 40% acetonitrile, 40% alcohol, and the TFA in 20% water.

31. The chromatography column according to any one of claims 25 to 30, wherein the alcohol is isopropyl alcohol.

32. A chromatography column according to any one of claims 25 to 31, comprising the elution gradient of the mobile phase B solvent and the polar mobile phase A solvent.

33. The chromatography column according to any one of claims 25 to 32, wherein the mobile phase A comprises TFA and water.

34. The chromatography column according to any one of claims 25 to 33, wherein the mobile phase A contains less than 0.1% TFA.

35. A chromatography column according to any one of claims 25 to 34, comprising porous particles having a particle size of approximately 2 to 5 μm, 2 to 7 μm, 3 to 5 μm, or 3 to 7 μm.

36. The chromatography column according to claim 35, wherein the porous particles have a pore size of about 100 to 500 angstroms, for example, about 300 angstroms.

37. A chromatography column according to any one of claims 25 to 36, comprising fully porous particles having a pore size of approximately 300 angstroms and a particle size of approximately 3 μm.

38. A chromatography column according to any one of claims 25 to 37, comprising a divinylbenzene (DVB) resin.

39. The chromatography column according to any one of claims 25 to 38, wherein the protein comprises a therapeutic protein.

40. The chromatography column according to any one of claims 25 to 39, wherein the protein is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a derivative of an antibody or an antibody fragment, and a fusion polypeptide.

41. The chromatography column according to any one of claims 25 to 39, wherein the protein is a bispecific T cell engager molecule.

42. The aforementioned proteins include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, avagobomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artitumomab, aselizumab, artinumab, atorizumab, atromimab, tocilizumab, bapineuzumab, basiliximab, bavituximab, and bectumo Mab, belimumab, benralizumab, vertilimumab, besilesomab, bezlotoxumab, bisilomab, vibatuzumab, vibatuzumab meltansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab meltansine, caplacizumab, capromab pendetide, carrumab, katumakisomab, CC49, sedelizumab, certolizumab pegol, sitatuzumab bogatox, sixtumumab, crazakizumab, clenoliximab, cribatuzumab tetraxetan, konatumumab, clenoliximab Nezumab, cr6261, dasetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorulimomab aritox, dorozizumab, duligotuzumab, dupilumab, eclomeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, erucirimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab citucetan, epratuzumab, erenumab, erlizumab, ertzumakisomab, etalacizumab, e Trollizumab, evolocumab, exhibivirumab, fanolesomab, falarimomab, falletuzumab, facinumab, fbta05, felbizumab, fezakinumab, ficratuzumab, figitumumab, frambotumab, fontrizumab, foralumab, folavirumab, fresolimmab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab, gabirimomab, gemtuzumab ozogamicin, gevokizumab, gylenetuximab, glentzumumab vedotin, golimumab, gomiliximab, gs6624, ibarizumabIbritumomab tiuxetan, iclucumab, igobomab, imusilomab, imugatuzumab, incrumab, indatuximab tansine, intetumumab, inorimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itorizumab, ixekizumab, keriximab, rabetsuzumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, livi Bilmab, rigerizumab, lintuzumab, lirirumab, lorbotuzumab meltansine, lucatumumab, lumiliximab, mapatumumab, masurimomab, mapurilimumab, matsuzumab, mepolizumab, meterimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimmab, motabizumab, moxetumomab pasdotox, muromonab-cd3, nacolomabutafe Natox, Namilumab, Naptumomab, Estafenatox, Narunatumab, Natalizumab, Nevacumab, Necitumumab, Nererimomab, Nesbacumab, Nimotuzumab, Nivolumab, Nofetumomab, Merpentan, Okalatuzumab, Oclerizumab, Odurimomab, Ofatumumab, Oraratuzumab, Orokizumab, Omalizumab, Onarutuzumab, Oporutuzumab, Monatox, O Legobomab, Orticumab, Oterixizumab, Oxerumab, Ozanezumab, Ozoralizumab, Padibaximab, Panitumumab, Panobacumab, Pulsatuzumab, Pascolizumab, Pateclizumab, Patrizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexerizumab, Pizilizumab, Pintumomab, Prakurumab, Ponezumab, Priliximab, Pritumumab, PRO 140, quilizumab, lacosumomab, radrezumab, rafibirumab, ramucirumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevilumab, sibro Tuzumab, cifarimumab, siltuximab, simtuzumab, ciprizumab, silumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamlumab, thresomab, suvizumab, tabarmab, tacutuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tapritumomab paptox, tefivazumab, terimomab aritox,Tenatumomab, teneriximab, teprizumab, teprotumumab, tezeperumab, TGN1412, tremelimumab, tisilimmab, tildrakizumab, tigatuzumab, TNX-650, tralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucothuzumab cermoloukin, tubilumab, ubrituximab, urerumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, beltuzumab Bepalimomab, besenkumab, vizilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanolimmumab, zatuximab, diralimumab, zolimomab aritox, glycoprotein, CD polypeptide, HER receptor polypeptide, cell adhesion polypeptide, growth factor polypeptide, insulin polypeptide, insulin-related polypeptide, coagulation polypeptide, coagulation-related polypeptide, albumin, IgE, blood group antigen, colony puncture A chromatography column according to any one of claims 25 to 39, selected from the group consisting of excitatory factors, receptors, neurotrophic factors, interferons, interleukins, viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor α and β, enkephalinase, mouse gonadotropin-related peptide, DNAse, inhibin, activin, integrin, protein A, protein D, rheumatoid factor, immunotoxin, bone morphogenetic protein, superoxide dismutase, surface membrane polypeptide, disintegration-promoting factor, HIV envelope, transport polypeptide, homing receptor, adresin, regulatory polypeptide, immunoadhesin, myostatin, TALL polypeptide, amyloid polypeptide, thymic interstitial lymphocyte necrosis factor, RANK ligand, c-kit polypeptide, TNF receptor, and angiopoietin, and their bioactive fragments, analogs, or variants.

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