Method for processing samples for peptide mapping analysis

Abstract

This invention provides an improved method for processing proteins or polypeptides before analysis, which leads to improved recovery of long-chain peptides and subsequent analysis. [Solution] In one embodiment, the digestion sample is incubated at a weakly acidic pH and / or in the presence of chaotrope, wherein the digestion sample is produced by digesting the polypeptide with a protease to produce a digestion sample containing at least two peptides. In one embodiment, the polypeptide is digested with a protease that cleaves the C-terminus of tryptophan to produce a digestion sample containing at least two peptides. In one embodiment, the polypeptide is digested with trypsin in an enzyme:substrate (E:S) weight ratio of about 1:1 to about 1:15 to produce a digestion sample containing at least two peptides. In an exemplary embodiment, the digestion sample contains at least one or two peptides, each containing tyrosine at its C-terminus.

Description

TECHNICAL FIELD

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 080,489 filed on September 18, 2020 and U.S. Provisional Application No. 63 / 236,996 filed on August 25, 2021 is claimed herein, and the entire contents of each of the above are incorporated herein by reference.

[0002] INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS A computer-readable nucleotide / amino acid sequence listing filed concurrently with the present specification and identified as follows is incorporated herein by reference in its entirety: an ASCII (Text) file of 272 kilobytes with the file name "A-2650-WO-PCT_ST25.txt" created on August 25, 2021. BACKGROUND ART

[0003] Mammalian cells are suitable hosts for the production of many protein biopharmaceuticals because they have cellular mechanisms for performing human-like post-translational modifications. However, the production of post-translationally modified proteins by mammalian cells may be affected by high variability due to batch-to-batch variation, process drift, manufacturing changes and other factors. Therefore, there is a need to monitor and control such modifications during the manufacturing process, especially modifications that are important for the in vivo therapeutic function of proteins, such as Critical Quality Attributes (CQA). Product quality analysis performed during protein manufacturing has been limited by the long time requirements for such analysis, but recent efforts have moved the industry toward automated high-throughput technologies combined with multivariate data analysis to provide relevant process knowledge in near real-time. See, for example, Pais et al, Current Opinion in Biotechnology 2014, 30: 161-167.

[0004] Traditionally, protein quality analysis has involved the isolation of proteins from samples obtained from manufacturing processes (e.g., cell culture samples taken from bioreactors), enzymatic digestion of proteins into peptide fragments, and mass spectrometry (MS) analysis of the peptide fragments after chromatographic separation. Depending on the complexity of the three-dimensional structure of the protein analyte, pre-digestion steps, including, for example, denaturation, reduction, and alkylation, may be required to cleave the protein for digestion. Generally, one or more post-digestion steps are included to stop digestion and prepare the yield of peptide fragments for separation and MS analysis. For example, a quench buffer may be added to stop digestion, and / or a buffer change may be performed before adding the fragments to the chromatography column.

[0005] Depending on the sequence of the protein analyte, digestion may produce long-chain peptides with low solubility, thus potentially leading to low recovery rates before analysis. If even the minimum amount of long-chain peptides is not recovered, a significant portion of the protein analyte may not be analyzed. The presence of one or more critical quality agents (CQAs) within the long-chain peptides reduces the usefulness of the overall quality analysis of the protein analyte. The problem becomes even more complex when protein digestion produces several or more long-chain peptides with low solubility and recovery rates, resulting in a lack of attribute information.

[0006] Therefore, there is a need in the art for improved pre-analysis processing methods for proteins or polypeptides that lead to improved recovery and subsequent analysis of long-chain peptides. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Pais et al,Current Opinion in Biotechnology 2014,30:161-167 [Overview of the initiative] [Means for solving the problem]

[0008] This specification describes data showing that a method for processing polypeptides, comprising digesting the polypeptide with a protease to obtain a digested sample, and then incubating the digested sample at a weakly acidic pH and / or in the presence of chaotrope, results in an increased recovery rate of long-chain peptides from the polypeptide. This increased recovery rate of long-chain peptides enables mass spectroscopic (MS) analysis of the polypeptide. The data described herein further demonstrates that the polypeptide processing method disclosed herein also results in an increased recovery rate of modified forms of long-chain peptides, such as deamidation products of long-chain peptides, which in turn enables mass spectroscopic analysis of modified polypeptides. The data described herein further supports the fact that modified forms of long-chain peptides can be separated chromatographically from unmodified forms of long-chain peptides, thereby enabling the detection and monitoring of modified polypeptides. The addition of chaotrope to a sample directly injected into a mass spectrometer for analysis has been counterintuitive, as many chaotropes are known to adversely affect MS performance and are not typically present in the injected sample. Furthermore, data presented herein also support the idea that digesting certain polypeptides with proteases that cleave the C-terminus of Trp residues within long-chain trypsin peptides facilitates polypeptide recovery and analysis.

[0009] Accordingly, a method for processing a polypeptide is provided herein, comprising digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. As used herein, "weakly acidic" pH means an acidic pH of about 4 or more and less than 7. Examples of weakly acidic pH ranges include 4 to 6, 4 to 5, greater than 5 and less than 7, and greater than 6 and less than 7. In an exemplary embodiment, the method comprises digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample in the presence of chaotrope. In an exemplary embodiment, the method comprises digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample at a weakly acidic pH. In an exemplary embodiment, the method comprises digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample at a weakly acidic pH in the presence of chaotrope. In exemplary examples, the method further includes mass spectrometry analysis after incubation of the digested sample. In various embodiments, a method for processing polypeptides includes digesting polypeptides with a protease to produce a digested sample containing at least two peptides, incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH, and analyzing the digested sample by mass spectrometry. In various embodiments, the digested sample is injected directly into a mass spectrometer after incubation. In various examples, no buffer exchange is performed after incubation of the digested sample. In various embodiments, a method for processing polypeptides includes digesting polypeptides with a protease to produce a digested sample containing at least two peptides, incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH, and injecting the digested sample directly into a mass spectrometer after incubation.In exemplary examples, the method results in increased solubility and / or recovery of long-chain peptides, such as long-chain hydrophobic peptides, or long-chain peptides containing one or more Trp residues, compared to the solubility and / or recovery of such peptides when the digested sample is not incubated in the presence of chaotrope and / or at a weakly acidic pH. Therefore, the disclosure provides a method for increasing the solubility of long-chain peptides in a digested sample, comprising incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH, wherein the digested sample is produced by digesting a polypeptide with a protease. In various examples, the solubility of peptides in a digested sample increases compared to the solubility of peptides treated without incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. Furthermore, the disclosure provides a method for increasing the recovery of long-chain peptides in a digested sample, comprising incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH, wherein the digested sample is produced by digesting a polypeptide with a protease. In various embodiments, the recovery rate of peptides from digested samples is increased in the presence of chaotrope and / or without incubation of the digested samples at a weakly acidic pH. As used herein, “long-chain” peptides mean peptides having a length greater than about 50 amino acid residues. In exemplary embodiments, at least one of the peptides in the digested sample is greater than 50 amino acid lengths, greater than 60 amino acid lengths, greater than 70 amino acid lengths, or greater than 80 amino acid lengths. Optionally, at least one of the peptides is greater than 50 amino acid lengths and contains five or more hydrophobic amino acids and / or at least one, at least two, or at least three Trp residues. In various embodiments, polypeptides contain oxidized Trp residues. In some embodiments, the solubility of peptides greater than 50 amino acid lengths in the digested samples is increased in the presence of chaotrope and / or without incubation of the digested samples at a weakly acidic pH.In various embodiments, the recovery rate of peptides longer than 50 amino acids in the digested sample is increased in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH. Optionally, the peptide recovery rate is at least three or four times higher in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH. In various embodiments, the protease is trypsin, chymotrypsin, AccuMAP® Modified Trypsin Solution (available from Promega as catalog number V5285) for use at a weakly acidic pH, pepsin, elastase, pseudotrypsin, or a combination thereof. Optionally, the polypeptide is digested by only one protease. In various embodiments, the one protease cleaves a Trp residue, optionally, the C-terminus of trypsin, or its proteoform or isoform. Alternatively, the polypeptide is digested by at least two proteases. For example, polypeptides are digested by trypsin and elastase, or by trypsin and chymotrypsin. In various embodiments, the method includes incubating the digested sample using mechanical shaking. In exemplary embodiments, the method includes digesting the polypeptide with a protease in the presence of chaotrope. In various embodiments, incubation is performed at a weakly acidic pH, optionally about 4 to about 6. In some embodiments, the weakly acidic pH is less than 5.5 or about 4.8 to about 5.2. In various examples, the method includes incubating the digested sample at a pH of about 5.0 ± 0.1. In exemplary embodiments, the method includes adding a high concentration of chaotrope to the digested sample. In various examples, the chaotrope includes urea, n-butanol, ethanol, guanidine, or salts thereof, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, or thiourea. Optionally, the chaotrope is guanidine or a salt thereof. In the exemplary examples, the chaotrope is guanidine hydrochloride, guanidine nitrate, guanidine thiocyanate, or guanidine carbonate. Optionally, guanidine hydrochloride is added to the digested sample.In various embodiments, the method includes incubating a digested sample in the presence of guanidine at a final guanidine concentration of approximately 2 M, optionally 3 M. In certain examples, the final guanidine concentration is less than 5 M. In various embodiments, the method includes incubating a digested sample in the presence of guanidine hydrochloride to obtain a final guanidine concentration of 4 M. In various embodiments, the method further includes incubating a digested sample in the presence of one or more of organic solvents, alcohols, acetonitrile, urea, surfactants, and / or dimethyl sulfoxide (DMSO). In various embodiments, the surfactant is a nonionic surfactant such as polyoxyethylene or glycoside. In some embodiments, polyoxyethylene is a surfactant of the Tween, Triton, or Brij series, a lipid, or a fatty acid. The method further optionally includes denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide. This method optionally further comprises denaturing, reducing, and / or alkylating the polypeptide before digesting it with a protease. The method optionally further comprises buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide. In various embodiments, the buffer exchange includes using a size exclusion cartridge, optionally a NAP5 cartridge having Sephadex G-25 gel filtration material. Optionally, the buffer exchange includes using a molecular weight cutoff (MWCO) filter. In some examples, the MWCO filter is a flat-bottom MWCO filter. In various embodiments, the method is carried out without using any filter. In various embodiments, the method is carried out using only a gel filter. In various embodiments, the method is carried out with or without using a gel filter. For example, the gel filter may include dextran gel. In various embodiments, the polypeptide is an antigen-binding protein, optionally a bispecific T cell inducer (BiTE®) molecule.In an exemplary embodiment, the BiTE® molecule includes a CD3-binding domain. In an exemplary embodiment, the polypeptide includes a sequence longer than 50 amino acids, longer than 60 amino acids, longer than 70 amino acids, or longer than 80 amino acids, and the sequence includes at least one Trp residue. In an exemplary embodiment, the BiTE® molecule includes a sequence having at least 90% sequence identity with SEQ ID NO: 88 shown in Figure 1. In an exemplary example, the polypeptide includes a sequence having at least 90% sequence identity with SEQ ID NO: 88 (Figure 1), and the sequence includes at least one tryptophan.

[0010] This disclosure further provides a method for processing a polypeptide, comprising digesting the polypeptide with a protease that, for example, cleaves the C-terminus of tryptophan, to produce a digested sample containing at least two peptides. Optionally, the protease cleaves the C-terminus of tryptophan, and at least one of the peptides in the digested sample contains a C-terminal Trp residue. In various embodiments, the recovery rate of the peptide containing the C-terminal Trp is higher than the recovery rate of the peptide processed without digesting the polypeptide with a protease that cleaves the C-terminus of Trp. In various examples, the recovery rate of the peptide containing the C-terminal Trp is 20% or more. In an exemplary embodiment, the method comprises digesting the polypeptide with only one protease. In an exemplary embodiment, the protease is trypsin, or its proteoform or isoform. Optionally, the proteoform is pseudotrypsin. In various embodiments, the polypeptide is digested with a protease (e.g., trypsin) at a weakly acidic pH. In some embodiments, the pH of weak acidity is less than 5.5 or about 4.8 to about 5.2. In various examples, the method includes incubating the digestion sample at a pH of about 5.0 ± 0.1. In exemplary embodiments, the protease is chymotrypsin. In exemplary embodiments, the method includes digesting the polypeptide with two or more proteases, or optionally, only two proteases. Optionally, at least one of the proteases is trypsin. In various embodiments, at least one of the proteases is chymotrypsin. In various examples, the method includes sequentially digesting the polypeptide with trypsin and then chymotrypsin. In specific examples, the method includes digesting the polypeptide with trypsin, and then digesting the polypeptide with chymotrypsin. Optionally, digestion is carried out at a neutral pH, or optionally, at a pH greater than 6.0 and less than 8.5. In various embodiments, the neutral pH is 7.0 or 7.5. In some embodiments, the neutral pH is approximately 7.5 ± 0.1. In various embodiments, the polypeptide is an antigen-binding protein and, optionally, a bispecific T cell-inducing (BiTE®) molecule.In exemplary embodiments, the BiTE® molecule includes a CD3-binding domain. In exemplary embodiments, the polypeptide includes a sequence longer than 50 amino acids, longer than 60 amino acids, longer than 70 amino acids, or longer than 80 amino acids, and the sequence includes at least one Trp residue. In exemplary embodiments, the polypeptide includes a sequence having at least 90% sequence identity with SEQ ID NO: 88 (Figure 1), and the sequence includes at least one tryptophan. In various embodiments, the digested sample includes a peptide containing the amino acid sequence HGNFGNSYISYW (SEQ ID NO: 92). The disclosure further provides a method for processing the BiTE® molecule, comprising digesting the BiTE® molecule with a protease that, for example, cleaves the C-terminus of tryptophan, to produce a digested sample containing at least two peptides, the two peptides being absent in the digested sample obtained by digesting the BiTE® molecule with trypsin at pH 7.5. This disclosure further provides a method for processing a BiTE® molecule, comprising digesting the BiTE® molecule with a protease that cleaves the site in SEQ ID NO: 91, for example, the C-terminus of tryptophan, to produce a digested sample containing at least two peptides. In exemplary examples, the BiTE® molecule is processed for analysis for less than 12 hours, optionally less than about 6 hours, or less than about 4 hours. Optionally, the method further comprises incubating the digested sample at a weakly acidic pH and / or in the presence of a chaotrope to the digested sample, as described herein. Optionally, the method further comprises denaturing, reducing, and / or alkylating the polypeptide. Optionally, the method further comprises denaturing, reducing, and / or alkylating the polypeptide before digesting the polypeptide with a protease. Optionally, the method further comprises buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide.In various embodiments, buffer exchange includes using a size exclusion cartridge and, optionally, a NAP5 cartridge having Sephadex G-25 gel filtration material. Optionally, buffer exchange includes using a molecular weight cutoff (MWCO) filter. In some examples, the MWCO filter is a flat-bottom MWCO filter. In various embodiments, the method is carried out without using any filter. In various embodiments, the method is carried out using only a gel filter. In various embodiments, the method is carried out with or without a gel filter. For example, the gel filter may include dextran gel. In an exemplary embodiment, the MWCO filter is not used in the method. In an exemplary example, the method further includes mass spectrometry analysis of the digested sample. In an exemplary example, the method includes digesting a polypeptide with a protease that cleaves the C-terminus of tryptophan to produce a digested sample containing at least two peptides, and injecting the digested sample into a mass spectrometer. Optionally, the method does not include buffer exchange after digestion.

[0011] Post-translational modifications (PTMs) or attributes of polypeptides processed by the methods disclosed herein are identified by analysis in exemplary embodiments. In exemplary embodiments, the identification of PTMs or attributes of polypeptides processed by the methods disclosed herein is improved compared to the identification of PTMs or attributes when digestion is performed without protease (cleaving the C-terminus of Trp), and / or when the digested sample is not incubated in the presence of chaotrope and / or at a weakly acidic pH. This disclosure further provides methods for monitoring polypeptide attributes. In exemplary embodiments, the method includes (a) processing the polypeptide in a first sample obtained at a first time point according to any one of the methods of the present disclosure; (b) injecting the digested sample obtained in (a) into a mass spectrometer to identify the PTM or attributes of the polypeptide in the first sample; (c) processing the polypeptide in a second sample obtained at a second time point according to any one of the methods of the present disclosure; (d) injecting the digested sample obtained in (c) into a mass spectrometer to identify the PTM or attributes of the polypeptide in the second sample; and (e) comparing the PTM or attributes of the first sample with the PTM or attributes of the second sample. In exemplary embodiments, each of the first and second samples is taken from a cell culture containing cells expressing the polypeptide, and the first time point is different from the second time point.

[0012] In exemplary embodiments of the methods disclosed herein, the method comprises digesting a polypeptide with trypsin in an enzyme-to-substrate (E:S) weight ratio of about 1:1 to about 1:15 to produce a digested sample containing at least two peptides, optionally, the digested sample containing a peptide containing the amino acid sequence HGNFGNSY (SEQ ID NO: 108), a peptide containing the amino acid sequence HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide containing the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 110). In exemplary embodiments, the E:S ratio is about 1:1 to about 1:10. Optionally, the E:S ratio is about 1:2 to about 1:8. In various examples, the E:S ratio is about 1:4 to about 1:6, optionally, about 1:5. In exemplary embodiments, the digestion is carried out at a pH of about 7.0 to about 8.0, optionally, about 7.5. In various embodiments, digestion takes place in less than approximately 12 hours, less than approximately 6 hours, or less than approximately 4 hours. In various examples, digestion takes place in approximately 2 to 4 hours. In various embodiments, digestion takes place in approximately 2 hours or less. In exemplary examples, digestion takes place at a temperature of approximately 35°C to approximately 40°C, or optionally, approximately 37°C. Optionally, digestion takes place in the presence of calcium chloride, or digestion takes place in the absence of calcium chloride. In exemplary examples, the polypeptide is digested by trypsin alone at an E:S ratio of approximately 1:1 to approximately 1:15. In various embodiments, no other proteases are used to digest the polypeptide. In various examples, digestion of the polypeptide with trypsin at this E:S ratio produces a digested sample containing one or more peptides with tyrosine at the C-terminus. In exemplary embodiments, the method includes denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide before digesting the polypeptide with a protease. In an exemplary embodiment, the method includes a buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide, and optionally the buffer exchange includes the use of a size exclusion cartridge. In an exemplary embodiment, the size exclusion cartridge is an NAP5 cartridge having Sephadex G-25 gel filtration material.In exemplary embodiments, the method further comprises incubating the digested sample at a weakly acidic pH in the presence of a chaotrope. Optionally, the chaotrope is guanidine hydrochloride. The weakly acidic pH is about 5 in various embodiments. The method in various examples comprises injecting the peptides of the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis.

[0013] This disclosure further provides a method for processing a polypeptide to produce a digested sample comprising at least two peptides, each containing tyrosine at its C-terminus. In exemplary embodiments, the method comprises digesting the polypeptide with trypsin in an E:S ratio of about 1:1 to about 1:15. In various examples, the digestion is carried out according to the method disclosed herein. Optionally, the digested sample comprises a peptide comprising the amino acid sequence HGNFGNSY (SEQ ID NO: 108), a peptide comprising the amino acid sequence HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 110). In exemplary embodiments, the E:S ratio is about 1:1 to about 1:10. Optionally, the E:S ratio is about 1:2 to about 1:8. In various examples, the E:S ratio is about 1:4 to about 1:6, and optionally, about 1:5. In exemplary embodiments, digestion is carried out at a pH of approximately 7.0 to approximately 8.0, optionally at approximately 7.5. In various embodiments, digestion is carried out for less than approximately 12 hours, less than approximately 6 hours, or less than approximately 4 hours. In various examples, digestion is carried out for approximately 2 hours to approximately 4 hours. In various embodiments, digestion is carried out for approximately 2 hours or less. In exemplary embodiments, digestion is carried out at a temperature of approximately 35°C to approximately 40°C, optionally at approximately 37°C. Optionally, digestion is carried out in the presence of calcium chloride, or in the absence of calcium chloride. In exemplary embodiments, the polypeptide is digested by trypsin alone at an E:S ratio of approximately 1:1 to approximately 1:15. In various embodiments, no other proteases are used to digest the polypeptide. In various embodiments, digestion of the polypeptide with trypsin at this E:S ratio produces a digested sample containing one or more peptides with tyrosine at the C-terminus. In exemplary embodiments, the method includes denaturing, reducing, and / or alkylating the polypeptide before digesting it with a protease. In exemplary embodiments, the method includes buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide, and optionally the buffer exchange includes the use of a size exclusion cartridge.In exemplary embodiments, the size exclusion cartridge is a NAP5 cartridge having Sephadex G-25 gel filtration material. In exemplary embodiments, the method further comprises incubating the digested sample at a weakly acidic pH in the presence of chaotrope. Optionally, the chaotrope is guanidine hydrochloride. The weakly acidic pH is about 5 in various embodiments. In various examples, the method comprises injecting the peptides of the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis. [Brief explanation of the drawing]

[0014] [Figure 1] These molecules exhibit a CD3ε-binding domain or an anti-CD3 (α-CD3) domain, which are common to many BiTE® molecules (canonical BiTE® molecules and half-life extended (HLE) BiTE® molecules). Because multiple amino acids are easily modified by trypsin digestion, a long-chain peptide of approximately 8 kDa containing two complementarity-determining regions (CDRs; highlighted in yellow) that may be CQAs is obtained. Trypsin cleavage sites are shown in light blue. Long-chain peptides containing more than 80 amino acids are underlined. [Figure 2] This is an alignment of the long-chain peptide sequences of the α-CD3 domain common to BiTE(registered trademark) molecules #1 to #3. The amino acid sequence of BiTE molecules #1 to #3 is sequence number 94. [Figure 3A] Figure 3A is a graph of peptide signals (number of ions) plotted as a function of digestion time (minutes) for various trypsin peptides of the BiTE® molecule containing amino acids D104-R154, V155-K178, V198-K239, H350-R436, or A462-K486. The long-chain peptide of the α-CD3 domain is H350-R436. Figure 3B is a photograph of an HPLC vial containing the BiTE® molecule after 48 hours of storage at 5°C. Arrows pointing to fibrous particles are indicated. [Figure 3B]Figure 3A is a graph of peptide signals (number of ions) plotted as a function of digestion time (minutes) for various trypsin peptides of the BiTE® molecule containing amino acids D104-R154, V155-K178, V198-K239, H350-R436, or A462-K486. The long-chain peptide of the α-CD3 domain is H350-R436. Figure 3B is a photograph of an HPLC vial containing the BiTE® molecule after 48 hours of storage at 5°C. Arrows pointing to fibrous particles are indicated. [Figure 4A-4D] These are images of vials containing a solution with BiTE® molecules (Vial 1), a solution with reduced and alkylated BiTE® molecules (Vials 2 and 3), and a solution with BiTE® molecules reduced, alkylated, and digested with trypsin at zero time (Figure 4A) and 10 minutes (Figure 4B) (Vials 4 and 5). Vial 3 was divided into two aliquots and treated with or without guanidine hydrochloride (Figure 4C). Vial 5 was divided into two aliquots and treated with or without guanidine hydrochloride (Figure 4C). Magnified images of vials 3 and 5 at zero time and 10 minutes are shown on the right side of Figures 4A and 4B. A magnified view of the magnified image of vial 5 on the right side of Figure 4B is shown to the right of the magnified image of vial 3. [Figure 5A]The steps for processing polypeptide samples (e.g., mAb or BiTE® molecules in a formulation solution) leading to HPLC-MS analysis are outlined. In Figure 5A, denaturation, reduction, and alkylation of the polypeptide are performed before buffer exchange to change the buffer to a digestion buffer. Buffer exchange is performed, for example, by gel filtration using an NAP5 cartridge. The gel filtration elution profile of the protein separated from guanidine is shown. Next, one or more enzymes are added to the eluted protein to obtain a digestion sample. The digestion sample is incubated at a specific pH as described herein, in or out of the presence of guanidine, e.g., 4M guanidine hydrochloride (GuHCl). The peptide is then injected into an HPLC-MS system for analysis. In Figure 5B, denaturation, reduction, and alkylation have occurred, and the buffer is placed on an MWCO filter. Buffer exchange to change the buffer to a digestion buffer is performed by centrifugation of the buffer using an MWCO filter and adding the digestion buffer to the filtrate. Digestion is performed on an MWCO filter, and the digested sample is incubated at a specific pH as described herein, in or out of the presence of guanidine, e.g., 4M guanidine hydrochloride (GuHCl). After incubation, the digested sample is centrifuged by filtration, and the filtrate containing the digested peptides is injected into an HPLC-MS system for analysis. [Figure 5B]The steps for processing polypeptide samples (e.g., mAb or BiTE® molecules in a formulation solution) leading to HPLC-MS analysis are outlined. In Figure 5A, denaturation, reduction, and alkylation of the polypeptide are performed before buffer exchange to change the buffer to a digestion buffer. Buffer exchange is performed, for example, by gel filtration using an NAP5 cartridge. The gel filtration elution profile of the protein separated from guanidine is shown. Next, one or more enzymes are added to the eluted protein to obtain a digestion sample. The digestion sample is incubated at a specific pH as described herein, in or out of the presence of guanidine, e.g., 4M guanidine hydrochloride (GuHCl). The peptide is then injected into an HPLC-MS system for analysis. In Figure 5B, denaturation, reduction, and alkylation have occurred, and the buffer is placed on an MWCO filter. Buffer exchange to change the buffer to a digestion buffer is performed by centrifugation of the buffer using an MWCO filter and adding the digestion buffer to the filtrate. Digestion is performed on an MWCO filter, and the digested sample is incubated at a specific pH as described herein, in or out of the presence of guanidine, e.g., 4M guanidine hydrochloride (GuHCl). After incubation, the digested sample is centrifuged by filtration, and the filtrate containing the digested peptides is injected into an HPLC-MS system for analysis. [Figure 6] Figure 5A shows graphs of the relative abundances of peptides treated according to the NAP5 buffer exchange method ("Nap5") or Figure 5B (using a "30kDa" or "10kDa" MWCO filter), with or without (+) incubation of digested samples in the presence of guanidine hydrochloride (GuHCl) at various pH values ​​(2, 5, 7.5). "Without GuHCl" means the digested sample was incubated without the addition of guanidine hydrochloride, while "GuHCl" without "Without" means the digested sample was incubated with guanidine hydrochloride. [Figure 7A]Figure 7A is an LC-UV chromatogram showing the digested peptide peaks of four samples, three of which were stressed. The main peak for the long-chain peptide H355-R441 is indicated. Figure 7B is a magnified portion of the chromatogram in Figure 7A, showing the differences in the height of the main peaks of the four samples. Peaks 1, 2, and 3, representing the deamidated versions of the long-chain peptide H355-R441, are shown to the right and left of the main peak. [Figure 7B] Figure 7A is an LC-UV chromatogram showing the digested peptide peaks of four samples, three of which were stressed. The main peak for the long-chain peptide H355-R441 is indicated. Figure 7B is a magnified portion of the chromatogram in Figure 7A, showing the differences in the height of the main peaks of the four samples. Peaks 1, 2, and 3, representing the deamidated versions of the long-chain peptide H355-R441, are shown to the right and left of the main peak. [Figures 8A-8C] Figure 8A shows a section of the LC-MS chromatogram near the long-chain peptide of the unstressed sample. Figure 8B shows a section of the LC-MS chromatogram near the long-chain peptide of the pH7-stressed sample, as well as peaks 1-3 specific to the pH7-stressed sample. Figures 8C-8F are the mass spectra of the main peak in Figure 8A, peak 1 in Figure 8B, peak 2 in Figure 8B, and peak 3 in Figure 8B. [Figures 8D-8F] Figure 8A shows a section of the LC-MS chromatogram near the long-chain peptide of the unstressed sample. Figure 8B shows a section of the LC-MS chromatogram near the long-chain peptide of the pH7-stressed sample, as well as peaks 1-3 specific to the pH7-stressed sample. Figures 8C-8F are the mass spectra of the main peak in Figure 8A, peak 1 in Figure 8B, peak 2 in Figure 8B, and peak 3 in Figure 8B. [Figure 9]FIG. 9A shows the sequence of the long peptide aa355-441 having the upper sequence (SEQ ID NO: 95), and FIG. 9B shows the MS / MS peak of the main peak of the long peptide. The sequence and identity of the long peptide were confirmed by the masses of the b fragment containing the N-terminus and the y fragment containing the C-terminus. [Figure 10] This is a table listing enzymes and their cleavage patterns. The sequences KR, WYFL (SEQ ID NO: 96), FIMYWV (SEQ ID NO: 97), CDEFLMTWY (SEQ ID NO: 98), and VITALS (SEQ ID NO: 99) are shown. [Figure 11] FIG. 1 shows the cleavage of the same sequence (SEQ ID NO: 100) by different enzymes and the peptide recovery rate. [Figure 12] FIG. 2 is a graph of the recovery rate of peptides containing N352 and N355 cleaved by different enzymes. [Figures 13A-13B] containing N352 and N355 from unstressed samples after sequential digestion with trypsin and chymotrypsin depicted at the bottom of FIG. 11

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Chemical Formula

Chemical Formula

[0015] Multi-attribute mapping (MAM) typically involves enzymatic digestion of molecules, where characterization and quantification of key attributes are performed, usually based on mass spectrometry (MS). Modifications in complementarity-determining regions (CDRs) are of particular concern because they can affect the potency and / or safety of the molecule. Trypsin, which cleaves lysine and arginine residues as the C-terminus, is typically used as the optimal enzyme for peptide mapping and MAM for numerous reasons, including the high digestion specificity and frequency of lysine and arginine residues; digestion of trypsin more typically yields peptides with basic residues at the C-terminus that have an optimal length for analysis based on mass spectrometry. However, many bispecific T cell inducers (BiTE®) molecules contain a conserved α-CD3 domain with two CDRs immediately adjacent to the long-chain linker region. These CDRs ( [ka] One of the sequences (SEQ ID NO: 110) contains two asparagine residues (underlined and bold) that are sensitive to deamidation and a tryptophan residue (underlined) that is sensitive to oxidation. This region of the molecule does not contain any residues that can be applied to trypsin digestion (Figure 1). Therefore, the attributes of interest within the CDR domain cannot be monitored by MAM due to the large size of this peptide (approximately 8 kDa), the difficulty in separating modified versions of the peptide by chromatography, poor peptide recovery and / or ionization, and the general challenges associated with interpreting mass spectroscopy for peptides of this size. Furthermore, this linker region lacks residues that are sensitive to commonly used secondary proteases such as Asp-N, Lys-C, and Glu-C. Similarly, many BiTE® molecules also have short-chain linker peptides (approximately 5-7 kDa) immediately adjacent to the target-specific CDR, which are also difficult to monitor by trypsin digestion. For example, the potential CDR aspartate isomerization site is located within the 5.7 kDa linker peptide. Taken together, these attributes can have a significant impact on target and / or CD3 binding that requires MAM-based surveillance.

[0016] This specification describes the development of a method for processing polypeptides that results in increased recovery of long-chain peptides and / or improved cleavage of long-chain peptides, and thus enables enhanced polypeptide analysis. This specification describes the development of a method for processing polypeptides that increases the abundance of a peptide containing two asparagine residues sensitive to deamidation of the CDR domain of a BiTE® molecule and / or a tryptophan residue sensitive to oxidation. Such an increase in the abundance of a peptide allows for monitoring of attributes or PTMs (e.g., deamidation, oxidation) within or near the CDR domain of the BiTE® molecule. In various embodiments, this method provides an increase in the abundance of a peptide containing a C-terminal tryptophan, for example, a peptide containing the sequence of SEQ ID NOs. 92 or 93. In an alternative embodiment, this method provides an increase in the abundance of a peptide containing a C-terminal tyrosine, for example, a peptide containing any one of the sequences of SEQ ID NOs. 108-110. Thus, this specification provides a method for processing polypeptides.

[0017] This disclosure provides a method for processing a polypeptide, comprising digesting the polypeptide with a protease that cleaves the C-terminus of tryptophan to produce a digested sample containing at least two peptides. Optionally, at least one of the peptides in the digested sample contains a C-terminal Trp residue. In various embodiments, the recovery rate of the peptide containing the C-terminal Trp is higher than the recovery rate of the peptide digested in the absence of a protease that cleaves the C-terminus of Trp. In various examples, the recovery rate of the peptide containing the C-terminal Trp is 20% or more. In an exemplary example, the method comprises digesting the polypeptide with only one protease. In an exemplary embodiment, the protease is trypsin, or its proteoform or isoform. Optionally, the proteoform is pseudotrypsin. In various embodiments, the polypeptide is digested with a protease (e.g., trypsin) at a weakly acidic pH, optionally about 4 to about 6, for example, pH 5. In various embodiments, the protease is chymotrypsin. In exemplary examples, the method includes digesting a polypeptide with two or more proteases, or optionally, just two proteases. Optionally, at least one of the proteases is trypsin. Optionally, at least one of the proteases is chymotrypsin. In various embodiments, the method includes sequentially digesting a polypeptide with trypsin and then chymotrypsin. In specific examples, the method includes digesting a polypeptide with trypsin, and then digesting the polypeptide with chymotrypsin. Optionally, the digestion is carried out at a neutral pH, or optionally, at pH 7.5. Optionally, the digestion is carried out for at least 4, 6, 8, 10, or 12 hours.

[0018] In various embodiments, the polypeptide is an antigen-binding protein and, optionally, a bispecific T cell-inducing (BiTE®) molecule. In exemplary embodiments, the BiTE® molecule includes a hypervariable region, which may include a CD3-binding domain. In exemplary embodiments, the polypeptide includes the hypervariable region or a portion thereof, the portion of which is greater than 50 consecutive amino acid lengths, greater than 60 consecutive amino acid lengths, greater than 70 consecutive amino acid lengths, or greater than 80 consecutive amino acid lengths. In exemplary embodiments, the polypeptide includes a sequence greater than 50 consecutive amino acid lengths, greater than 60 consecutive amino acid lengths, greater than 70 consecutive amino acid lengths, or greater than 80 consecutive amino acid lengths, and the sequence includes at least one Trp residue. In exemplary embodiments, the polypeptide includes a binding domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 88 (Figure 1), and includes at least one Trp. In various embodiments, the digested sample includes a peptide of the sequence HGNFGNSYISYW (SEQ ID NO: 92). In various embodiments, the digested sample contains the peptide of sequence AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR (SEQ ID NO: 93). The protease cleaves the C-terminus of tryptophan within the hypervariable region (or a portion thereof), and the digestion of this region by the protease is increased in various embodiments compared to the digestion of this region in the absence of the protease (e.g., compared to digestion by a different protease). In various examples, the polypeptide is processed for peptide mapping analysis for less than 12 hours, optionally less than about 6 hours, e.g., less than about 4 hours. In exemplary examples, the BiTE® molecule is processed for analysis for less than 12 hours, optionally less than 6 hours, or less than about 4 hours. In various configurations, the BiTE® molecule is processed for analysis for approximately 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, 2 to 6 hours, 3 to 6 hours, 4 to 6 hours, or 5 to 6 hours.

[0019] Optionally, the method further comprises incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. Exemplary chaotropes are described herein. In various embodiments, the digested sample is incubated at a pH of about 5.0 in the presence of guanidine at a concentration of about 4 M. Optionally, the method further comprises denaturing, reducing, and / or alkylating the polypeptide before digesting the polypeptide with a protease. Such pre-digestion steps are further described herein. See, for example, “Pre-digestion”. Optionally, the method further comprises a buffer exchange after denaturing, reducing, and / or alkylating the polypeptide and before digesting the polypeptide with a protease. In some examples, the buffer exchange step includes using a size exclusion cartridge, optionally a NAP5 cartridge having Sephadex G-25 gel filter material. Optionally, the buffer exchange includes using a molecular weight cutoff (MWCO) filter. In some examples, the MWCO filter is a flat-bottom MWCO filter. In various embodiments, the method is carried out without using any filters at all. In various embodiments, the method is carried out without using any MWCO filters at all. In various embodiments, the method is carried out using only gel filters, such as dextran gel filters like the NAP5 cartridge.

[0020] Optionally, the method further includes a post-digestion stage. See, for example, “Post-digestion.” For example, the method includes analyzing the peptides of the digested sample. In exemplary examples, the method further includes mass spectrometry analysis of the digested sample. In various embodiments, the method further includes injecting the peptides of the digested sample into a mass spectrometer, optionally, a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis. In exemplary examples, the method includes digesting a polypeptide with a protease that cleaves the C-terminus of Trp to produce a digested sample containing at least two peptides, and injecting the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system. The attributes of the polypeptide are identified, in exemplary embodiments, through peptide mapping analysis, e.g., MAM. In exemplary examples, the identification of the polypeptide PTM or attributes is improved compared to the identification of the PTM when digestion is performed without a protease (cleaving the C-terminus of Trp).

[0021] This disclosure further provides a method for processing a polypeptide, comprising digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. In an exemplary embodiment, the method comprises digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample at a weakly acidic pH in the presence of chaotrope. In an exemplary embodiment, the method comprises digesting the polypeptide with a protease to produce a digested sample containing at least two peptides, and incubating the digested sample at a weakly acidic pH.

[0022] In exemplary examples, the method results in increased solubility and / or recovery of long-chain peptides, such as long-chain peptides containing at least one Trp residue. This increase can be compared to the solubility and / or recovery of the peptide when the digested sample is not incubated in the presence of chaotrope and / or at a weakly acidic pH.

[0023] Accordingly, the present disclosure provides a method for increasing the solubility of long-chain peptides in a digested sample, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH, wherein the digested sample is produced by digesting a polypeptide with a protease. In various examples, the solubility of peptides in the digested sample is increased compared to the solubility of peptides treated without incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH.

[0024] Furthermore, the present disclosure provides a method for increasing the recovery rate of hydrophobic peptides from a digested sample, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH, wherein the digested sample is produced by digesting a polypeptide with a protease. In various embodiments, the recovery rate of peptides from the digested sample is increased compared to the recovery rate of peptides treated without incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH.

[0025] In various embodiments, the increase in solubility and / or recovery is at least or about 1% to about 10% (e.g., at least or about 1%, at least or about 2%, at least or about 3%, at least or about 4%, at least or about 5%, at least or about 6%, at least or about 7%, at least or about 8%, at least or about 9%, at least or about 9.5%, at least or about 9.8%, at least or about 10%) compared to the solubility or recovery of the treated peptide without incubation of the digested sample in the presence of chaotrope and / or at a weakly acidic pH. In exemplary embodiments, the increase in solubility or recovery rate provided by the method of the present disclosure is about 10% to about 100%, optionally about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 10% to about 15%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%. The increase may be relative to a control. In exemplary embodiments, the increase in solubility or recovery rate provided by the method of the present disclosure is greater than 100% compared to a control, for example, an increase of 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%. In exemplary embodiments, the solubility or recovery rate is increased by at least about 1.5 times compared to a control. A preferred control may be the solubility or recovery rate of a peptide treated without incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. Optionally, the peptide recovery rate is at least 3 or 4 times that of the peptide recovered without incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH.

[0026] In an exemplary embodiment, at least one of the peptides in the digested sample is longer than 50 amino acids, longer than 60 amino acids, longer than 70 amino acids, or longer than 80 amino acids. In an exemplary embodiment, at least one of the peptides in the digested sample is longer than 50 amino acids, longer than 60 amino acids, longer than 70 amino acids, or longer than 80 amino acids, and optionally has one trypsin cleavage site, e.g., Arg or Lys, at the N-terminus or C-terminus of the peptide. In an exemplary embodiment, at least one of the peptides in the digested sample is longer than 50 amino acids, longer than 60 amino acids, longer than 70 amino acids, or longer than 80 amino acids, and optionally has one trypsin cleavage site, e.g., Arg or Lys, at the N-terminus or C-terminus of the peptide, and further contains five or more hydrophobic amino acids. Hydrophobicity may be measured or scored according to any one of the hydrophobicity scales known in the art. Generally, the more positive the score, the higher the hydrophobicity of the amino acid. In some cases, hydrophobicity is scored using the Kyte-Doolittle hydrophobicity scale (Kyte J, Doolittle RF (May 1982). "A simple method for displaying the hydropathic character of a protein." J.Mol. Biol. 157(1):105-32). In some embodiments, hydrophobic amino acids have a score greater than approximately 2.5 on the Kyte-Doolittle hydrophobicity scale. In certain embodiments, hydrophobic amino acids are branched or linear C2-C2. 12The polypeptide comprises an alkyl or C4-C8 cycloalkyl, a nitrogen heteroatom, and optionally includes a side chain containing a C4-C8 heterocycle whose heterocycle is imidazole, pyrrole, or indole. For the purposes of this specification, the term "cycloalkyl" encompasses any carbon ring containing two or three carbon rings. In exemplary embodiments, the hydrophobic amino acid comprises a C3-C8 alkyl, and optionally includes a branched C3 alkyl or branched C4 alkyl. In certain embodiments, the hydrophobic amino acid is L-alanine, L-valine, L-leucine, or L-isoleucine. In various embodiments, the polypeptide comprises at least one, at least two, or at least three tryptophan residues that are sensitive to oxidation. In some embodiments, the solubility of peptides longer than 50 amino acids in the digested sample increases in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH compared to the solubility of peptides treated without incubation. In various embodiments, the recovery rate of peptides longer than 50 amino acids in digested samples is increased in the presence of chaotrope and / or without incubation of the digested samples at a weakly acidic pH compared to the recovery rate of peptides treated without incubation.

[0027] This disclosure further provides a method for processing a polypeptide to produce a digested sample comprising at least one or two peptides, each containing tyrosine at its C-terminus. In exemplary embodiments, the method comprises digesting the polypeptide with trypsin at an E:S ratio of about 1:1 to about 1:15. In various examples, the digestion is carried out according to the method disclosed herein. Optionally, the digested sample comprises a peptide comprising the amino acid sequence HGNFGNSY (SEQ ID NO: 108), a peptide comprising the amino acid sequence HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 110). In exemplary embodiments, the E:S ratio is about 1:1 to about 1:10. Optionally, the E:S ratio is about 1:2 to about 1:8. In various examples, the E:S ratio is about 1:4 to about 1:6, and optionally, about 1:5. In exemplary embodiments, digestion is carried out at a pH of approximately 7.0 to approximately 8.0, optionally at approximately 7.5. In various embodiments, digestion is carried out for less than approximately 12 hours, less than approximately 6 hours, or less than approximately 4 hours. In various embodiments, digestion is carried out for approximately 2 hours to approximately 4 hours. In various embodiments, digestion is carried out for approximately 2 hours or less. In exemplary examples, digestion is carried out at a temperature of approximately 35°C to approximately 40°C, optionally at approximately 37°C. Optionally, digestion is carried out in the presence of calcium chloride, or in the absence of calcium chloride. In exemplary examples, the polypeptide is digested by trypsin alone at an E:S ratio of approximately 1:1 to approximately 1:15. In various embodiments, no other proteases are used to digest the polypeptide. In various examples, digestion of the polypeptide with trypsin at this E:S ratio produces a digested sample containing one or more peptides with tyrosine at the C-terminus. In exemplary embodiments, the method includes denaturing, reducing, and / or alkylating the polypeptide before digesting it with a protease. In exemplary embodiments, the method includes buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide, and optionally the buffer exchange includes the use of a size exclusion cartridge.In exemplary embodiments, the size exclusion cartridge is a NAP5 cartridge having Sephadex G-25 gel filtration material. In exemplary embodiments, the method further comprises incubating the digested sample at a weakly acidic pH in the presence of chaotrope. Optionally, the chaotrope is guanidine hydrochloride. The weakly acidic pH is about 5 in various embodiments. In various examples, the method comprises injecting the peptides of the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis.

[0028] digestion In various embodiments, the method includes digesting a polypeptide or cleaving a polypeptide to at least two peptide fragments of the polypeptide. Digestion (or cleavage) can produce at least two fragments of the polypeptide. Furthermore, it is undesirable to completely break down the polypeptide to, for example, a single amino acid.

[0029] Examples of useful proteases (but not all) include trypsin, 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 various embodiments, the protease is trypsin, chymotrypsin, pepsin, elastase, pseudotrypsin, or a combination thereof. In some embodiments, a combination of these proteases is used. In various embodiments, the polypeptide is digested by at least two proteases. For example, the polypeptide is digested by trypsin and elastase, or by trypsin and chymotrypsin. Alternatively, the polypeptide is digested by only one protease. In some embodiments, trypsin alone is used. These and other proteases, including peptide bond selectivity and EC number, are shown in Table A. The suppliers indicated for each protease are illustrative, and many of these proteases are commercially available.

[0030] [Table 1]

[0031] In some embodiments, protein:protease ratios (w / w) of 10:1, 20:1, 25:1, 50:1, or 100:1 can be used. In some embodiments, the ratio is 20:1. In some embodiments, the neutrophil elastase 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 digestion step is 10 minutes to 48 hours, or 30 minutes to 48 hours, or 30 minutes to 24 hours, or 30 minutes to 16 hours, or 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 16 hours, or 1 to 8 hours, or 1 to 6 hours, or 1 to 4 hours. In some embodiments, the digestion step is incubated at a temperature of 20°C to 45°C, or 20°C to 40°C, or 22°C to 40°C, or 25°C to 37°C. In some embodiments, the digestion step is incubated at 37°C. Those skilled in the art can select appropriate conditions (buffer, incubation time, amount of protease, volume, etc.) as in vitro protease digestion is well understood in the art.

[0032] In various embodiments, the method includes digesting a polypeptide with a protease that cleaves the C-terminus of tryptophan, as described herein, to produce a digested sample containing at least two peptides. For example, the method includes digesting the polypeptide with only one protease. In exemplary embodiments, the protease is trypsin, or its proteoform or isoform. Optionally, the proteoform is pseudotrypsin. In various embodiments, the polypeptide is digested with a protease (e.g., trypsin) at a weakly acidic pH, optionally about 4 to about 6, for example, pH 5. In exemplary embodiments, the method includes digesting the polypeptide with two or more proteases, optionally just two proteases. Optionally, at least one of the proteases is trypsin. Optionally, at least one of the proteases is chymotrypsin. In various embodiments, the method includes sequentially digesting the polypeptide with trypsin and chymotrypsin. In certain cases, this method includes digesting the polypeptide with trypsin, followed by digesting the polypeptide with chymotrypsin. Optionally, the digestion is performed at a neutral pH, or optionally at pH 7.5.

[0033] In various embodiments, polypeptides are digested in the presence of chaotropes. Chaotropes are described herein.

[0034] In various cases, this method involves incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. Without being bound by any particular theory, incubating the digested sample in the presence of chaotrope stops or delays the digestion of polypeptides.

[0035] In various embodiments, the method includes incubating the digested sample at a weakly acidic pH. Optionally, a weakly acidic pH is between approximately 4 and less than 7. In exemplary embodiments, a weakly acidic pH is approximately 4 to 6.9, 4 to 6.8, 4 to 6.7, 4 to 6.6, 4 to 6.5, 4 to 6.4, 4 to 6.3, 4 to 6.2, or 4 to 6.1. In various embodiments, a weakly acidic pH is approximately 4 to 6, for example, approximately 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In some embodiments, the pH of a weakly acidic sample is less than 5.5 and / or about 4.8 to about 5.2. In various examples, the pH of a weakly acidic sample is about 5.0 ± 0.1. Optionally, if the pH of the digested sample is neutral to basic, the pH is adjusted to a weakly acidic pH. In various embodiments, formic acid, e.g., 20% (v / v) formic acid, is added to adjust the pH to a weakly acidic pH. Other means of adjusting the pH are known. In various embodiments, if the pH of the digested sample is weakly acidic, no adjustment of the pH of the digested sample is necessary.

[0036] In exemplary embodiments, the method includes incubating the digested sample in the presence of a chaotrope, for example, a high concentration of chaotrope. In various examples, the method includes incubating the digested sample in the presence of urea, n-butanol, ethanol, guanidine, or salts thereof, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, or thiourea. Optionally, the chaotrope includes guanidine or salts thereof. In exemplary embodiments, the method includes incubating the digested sample in the presence of guanidine hydrochloride, guanidine nitrate, guanidine thiocyanate, or guanidine carbonate. Optionally, the method includes adding guanidine hydrochloride to the digested sample. In some embodiments, the method includes adding guanidine to the digested sample to obtain a final guanidine concentration of more than about 2 M, optionally more than 3 M. In specific examples, guanidine is added to the digested sample to obtain a final guanidine concentration of less than 5 M. In various embodiments, the method includes adding guanidine hydrochloride to the digested sample to obtain a final guanidine concentration of approximately 4 M. In various embodiments, the method includes adding guanidine to the digested sample until the final guanidine concentration of the digested sample reaches approximately 4 M.

[0037] In various examples, the method involves incubating a digested sample in the presence of one or more of the following: organic solvents, alcohols, acetonitrile, urea, surfactants, and / or dimethyl sulfoxide (DMSO). In various examples, the surfactant is a nonionic surfactant such as polyoxyethylene or glycoside. In some embodiments, polyoxyethylene is a surfactant of the Tween, Triton, or Brij series, a lipid, or a fatty acid.

[0038] In various embodiments, incubation is carried out using mechanical shaking. In various examples, incubation is performed for at least 30 seconds or at least 60 seconds. In various examples, incubation is performed at a temperature above approximately 4°C.

[0039] In exemplary embodiments of the methods disclosed herein, the method comprises digesting a polypeptide with trypsin in an enzyme:substrate (E:S) weight ratio of about 1:1 to about 1:15 to produce a digested sample containing at least two peptides. In various embodiments, the digested sample contains a peptide containing a C-terminal tyrosine residue. Optionally, the digested sample contains a peptide containing the amino acid sequence HGNFGNSY (SEQ ID NO: 108), a peptide containing the amino acid sequence HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide containing the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 110). In various configurations, the E:S weight ratio is approximately 1:1 to 1:14, 1:1 to 1:13, 1:1 to 1:12, 1:1 to 1:11, 1:1 to 1:10, 1:2 to 1:15, 1:3 to 1:15, 1:4 to 1:15, 1:5 to 1:15, 1:6 to 1:15, 1:7 to 1:15, 1:8 to 1:15, 1:9 to 1:15, 1:10 to 1:15, 1:11 to 1:15, 1:12 to 1:15, 1:13 to 1:15, or 1:14 to 1:15. In an exemplary embodiment, the E:S ratio is approximately 1:1 to 1:10 (e.g., approximately 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:7, 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, 1:1 to 1:2, 1:2 to 1:10, 1:3 to 1:10, 1:4 to 1:10, 1:5 to 1:10, 1:6 to 1:10, 1:7 to 1:10, 1:8 to 1:10, 1:9 to 1:10). Optionally, the E:S ratio is approximately 1:2 to 1:8. In various examples, the E:S ratio is approximately 1:4 to 1:6, and optionally, approximately 1:5. In exemplary properties, digestion occurs at a pH of approximately 7.0 to 8.0, for example, approximately 7.1 to 8.0, 7.2 to 8.0, 7.3 to 8.0, 7.4 to 8.0, 7.5 to 8.0, 7.6 to 8.0, 7.7 to 8.0, 7.8 to 8.0, 7.9 to 8.0, 7.0 to 7.9, 7.0 to 7.8, 7.0 to 7.7, 7.0 to 7.6, 7.0 to 7.5, 7.0 to 7.4, 7.0 to 7.3, 7.0 to 7.2, or 7.0 to 7.1.In various embodiments, digestion is carried out at a pH of approximately 7.3 to 7.7, 7.4 to 7.5, or 7.5. In various embodiments, digestion is carried out for less than approximately 12 hours, less than approximately 6 hours, or less than approximately 4 hours. In various embodiments, digestion is carried out for approximately 2 to 4 hours. In various embodiments, digestion is carried out for approximately 2 hours or less. In exemplary examples, digestion is carried out at a temperature of approximately 35°C to 40°C, optionally, at approximately 37°C. Optionally, digestion is carried out in the presence of calcium chloride, or digestion is carried out in the absence of calcium chloride. In exemplary examples, the polypeptide is digested by trypsin alone at an E:S ratio of approximately 1:1 to 1:15. In various embodiments, no other proteases (other than trypsin) are used to digest the polypeptide. In exemplary embodiments, the method includes denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide before digestion with a protease. In exemplary embodiments, the method includes buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide, and optionally the buffer exchange includes the use of a size exclusion cartridge. In exemplary embodiments, the size exclusion cartridge is a NAP5 cartridge having Sephadex G-25 gel filter material. In exemplary embodiments, the method further includes incubating the digested sample at a weakly acidic pH in the presence of chaotrope. In various embodiments, after trypsin digestion, the digested sample is incubated with chaotrope. Optionally, the chaotrope is guanidine hydrochloride. In exemplary embodiments, the final concentration of guanidine hydrochloride is about 3 M or about 4 M. The weakly acidic pH is about 5 in various embodiments. In various embodiments, the method includes injecting the peptides of the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis.

[0040] Predigestion Methods for processing polypeptides of the present disclosure in various embodiments further include, but are not limited to, one or more pre-digestion treatments, including denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide. The methods further optionally include denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide before digesting the polypeptide with a protease.

[0041] In various embodiments, the method involves denaturing a polypeptide. Polypeptides can be denatured using various techniques and denaturing agents recognized in the art. In some embodiments, multiple denaturing agents are used together, either simultaneously or sequentially. For example, SDS denaturing agents and heat can be combined to denature a polypeptide.

[0042] Protein denaturation can be carried out by any means that disrupts the quaternary, tertiary, or secondary polypeptide structure. For example, the use of chaotropes such as urea and denaturing surfactants (e.g., sodium dodecyl sulfate (SDS)), heat, reducing agents, and agents that inactivate reactive thiol groups to block disulfide reformation. The pH of the polypeptide-containing sample can be manipulated to further accelerate denaturation. These components are often used together to unravel the polypeptide chain.

[0043] Examples of additional chaotropes, in addition to urea, include n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, and thiourea. Urea is preferred in most cases.

[0044] Surfactants are classified into hydrophilic groups: anionic, cationic, nonionic, and amphoteric. Anionic and cationic surfactants are more likely to denature, and examples of these include: SDS, sodium cholate, sodium deoxycholate, sodium glycolate, sodium taurocholate, sodium taurodeoxycholate, N-lauroyl sarcosine, lithium dodecyl sulfate (anionic), and hexadecyltrimethylammonium bromide (CTAB) and trimethyl(tetradecyl)ammonium bromide (TTAB) (cationic). In some cases, amphoteric surfactants may be useful, examples of which include amide sulfobetaine-14 (ASB-14), amide sulfobetaine-16 (ASB-16), C7Bz0, CHAPS, CHAPSO, EMPIGEN® BB, 3-(N,N-dimethyloctylammonio)propanesulfonate intramolecular salt (SB3-8), and d(decyldimethylammonio)propanesulfonate intramolecular salt (SB3-10). Anionic surfactants are preferred, and SDS is particularly preferred.

[0045] The denaturing agent may be heat (for most polypeptides), for example, a high temperature of 30°C or higher. The denaturing agent may include stirring. In some embodiments, essentially or substantially low-salt or salt-free agents may denature the polypeptide.

[0046] The denaturing agent may be a solvent such as ethanol or other alcohols.

[0047] Polypeptide denaturation has been extensively tested and reported; for example, see (Tanford, 1968) for details. Those skilled in the art will understand the methods of denaturing polypeptides, given the properties of the polypeptides and the numerous denaturing agents to be selected.

[0048] In various embodiments, this method involves reducing a polypeptide. A reduced polypeptide is a polypeptide that has been exposed to reducing conditions sufficient to reduce a reducible residue in the polypeptide structure, such as cysteine. If the reduced polypeptide contains a thiol group or a sulfur-containing residue, the thiol group in the reduced polypeptide is reduced. A reduced polypeptide containing a cysteine ​​residue has a sulfur atom of the reduced cysteine ​​residue, which may be denoted as "-SH". A reduced polypeptide may be a disulfide bond-containing polypeptide. A disulfide bond-containing polypeptide can become a reduced polypeptide by exposing it to reducing conditions that induce the rupture of one or more disulfide bonds (disulfide crosslinks) in the disulfide bond-containing polypeptide.

[0049] A reducing agent, reductant, or reducer is an element or compound that loses (or donates) electrons to another chemical species in a redox reaction. Reducing agents allow the disulfide group to become reactive by generating a thiol (-SH) group. Common polypeptide reducing reagents are shown in Table B.

[0050] [Table 2]

[0051] In some embodiments, polypeptide denaturation and reduction are carried out simultaneously. In other embodiments, polypeptide denaturation and reduction are carried out in separate steps.

[0052] Those skilled in the art will understand the method for reducing polypeptides, given the properties of the polypeptide and the reducing reagent to be selected.

[0053] In various embodiments, this method involves alkylating a polypeptide. The "step of inactivating reactive thiol groups" means blocking free thiol groups in the polypeptide to prevent undesirable thiol-disulfide exchange reactions. The alkylating agent is a substance that induces hydrogen substitution by an alkyl group.

[0054] The frequent alkylation of free cysteine ​​following their reduction prevents the formation and reformation of disulfide bonds that could otherwise be formed between the free thiols of cysteine ​​residues. Commonly used alkylating agents include n-ethyl maleimide (NEM), iodoacetamide (IAA), and iodoacetic acid. Other suitable alkylating agents include dithiobis(2-nitro)benzoic acid; acrylamide; 4-vinylpyridine; nitrogen mustards, e.g., chlorambucil and cyclophosphamide; cisplatin; nitrosourea, e.g., carmustine, lomustine, and semustine; alkyl sulfonates, e.g., busulfan; ethyleneimine, e.g., thiotepa; and triazines, e.g., decarbazine. Those skilled in the art are familiar with reagents that can be used to protect sulfhydryl groups and methods for using such reagents.

[0055] This method optionally further includes a buffer exchange before digesting the polypeptide with a protease. In various embodiments, the buffer exchange includes using a size exclusion cartridge, optionally an NAP5 cartridge having Sephadex G-25 gel filter material. Optionally, the buffer exchange step includes using a molecular weight cutoff (MWCO) filter. In some examples, the MWCO filter is a flat-bottom MWCO filter. In various embodiments, this method includes using only a gel filter, for example an NAP5 cartridge having Sephadex G-25 gel filter material.

[0056] In alternative examples, this method does not involve the use of any filters.

[0057] After digestion In various embodiments, the method includes one or more post-digestion treatments or analyses. In various examples, the method includes incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. Incubation may be carried out as described herein. See, for example, the instructions in the “Digestion” section following Table A. In exemplary embodiments, the method includes incubating the digested sample at a pH of about 4–6, optionally at pH 5, in the presence of 4 M guanidine. In various embodiments, incubation is carried out using mechanical shaking. In various examples, incubation is carried out for at least 30 seconds or at least 60 seconds. In various examples, incubation is carried out at a temperature above about 4°C.

[0058] In exemplary embodiments, the method includes analyzing a digested sample. Generally, preferred analytical methods may be chromatography, electrophoresis, and spectroscopy. Some of these analytical methods can be combined. Those skilled in the art can utilize, for example, handbooks that facilitate the selection of suitable analytical methods and appropriate conditions for carrying out those methods, such as (Gunzler and Williams, 2001).

[0059] Chromatography is a method for separating polypeptide fragments in a mobile phase that are processed through a structure in which the phase holds the stationary phase. Because polypeptide fragments differ in size and composition, each fragment has a unique partition coefficient. Due to the different partition coefficients, polypeptides are differentially maintained on the stationary phase. Examples of such methods known in the art include gas chromatography, liquid chromatography, high-performance liquid chromatography, ultrahigh-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, extended adsorption fluidized bed chromatography, reversed-phase chromatography, and hydrophobic interaction chromatography.

[0060] A summary of some known chromatography methods is shown in Table C.

[0061] [Table 3]

[0062] The processed polypeptides can be further analyzed using electrophoresis—gel electrophoresis, free-flow electrophoresis, isoelectric focusing, isokinetic electrophoresis, affinity electrophoresis, immunoelectrophoresis, inverse electrophoresis, capillary electrophoresis, and capillary zone electrophoresis. Overviews and handbooks are available to those skilled in the art, e.g., (Kurien and Scofield, 2012; Lord, 2004).

[0063] Electrophoresis can be used to analyze charged molecules, such as polypeptides, that are transported through a solvent by an electric field and do not exist at their isoelectric point. Polypeptides move at a rate proportional to their charge density. The mobility of polypeptides through an electric field depends on: the strength of the electric field, the net charge on the polypeptide, the size and shape of the polypeptide, the ionic strength, and the properties of the matrix through which the polypeptide moves (e.g., viscosity, pore size). Polyacrylamide and agarose are two common support matrices. These matrices function as porous media and behave like molecular sieves. Polyacrylamide forms a support with a smaller pore size and is particularly useful in the methods disclosed herein, and is ideal for separating the majority of polypeptide fragments.

[0064] Table D shows examples of polypeptide electrophoresis techniques.

[0065] [Table 4]

[0066] The processed polypeptides can be further analyzed using spectroscopic-mass spectroscopy (Rubakhin and Sweedler, 2010), ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy (Norwicka-Janokowska, 1986).

[0067] Table E shows examples of polypeptide electrophoresis techniques.

[0068] [Table 5]

[0069] The principle that enables mass spectrometry (MS) consists of the steps of ionizing a chemical compound to produce charged molecules or molecular fragments, and then measuring the mass-to-charge ratio. In an exemplary MS procedure, the sample is loaded into an MS instrument and vaporized, the components of the sample are ionized by one of various methods that cause the formation of positively charged particles (e.g., by bombarding them with an electron beam), the positive ions are then accelerated by a magnetic field, and as they pass through the electromagnetic field, calculations are performed on the mass-to-charge ratio (m / z) of the particles based on the details of the ion motion, and the ions are detected and classified according to their m / z ratios.

[0070] An example MS instrument has three modules: an ion source that converts gas-phase sample molecules into ions (or, in the case of electrospray ionization, moves ions present in solution into the gas phase); a mass spectrometer that classifies the ions by their mass-to-charge ratio by applying an electromagnetic field; and a detector that measures numerical values ​​of indicator quantities, thus providing data for calculating the abundance of each ion present.

[0071] MS techniques have both qualitative and quantitative uses, including identifying unknown compounds, determining the isotopic composition of elements within a molecule, and determining the structure of a compound by observing its fragmentation. These include gas chromatography-mass spectroscopy (GC / MS or GC-MS), liquid chromatography-mass spectroscopy (LC / MS or LC-MS), and ion mobility spectroscopy / mass spectroscopy (IMS / MS or IMMS).

[0072] Analytical methods (chromatography, electrophoresis, and spectroscopic analysis) can be combined. For example, combinations such as liquid chromatography-mass spectroscopy, capillary zone electrophoresis combined with mass spectroscopy, and ion mobility spectroscopy-mass spectroscopy.

[0073] In exemplary examples, the method further includes injecting the digested sample, after incubation in the presence of a chaotrope and / or at a weakly acidic pH, into a mass spectrometer, such as a liquid chromatography-mass spectroscopy (LC-MS) system.

[0074] Attributes, monitoring method, and polypeptide manufacturing method In exemplary embodiments, a polypeptide comprises a polymer of amino acids, and in various embodiments, a polypeptide comprises one or more post-translationally modified amino acids, e.g., one or more amino acids containing post-translational modifications (PTMs), or one or more other structurally modified amino acids. It will be understood that a polypeptide comprises amino acid residues. For brevity, amino acid residues of a polypeptide may be simply referred to as “amino acids” in this specification.

[0075] In various cases, PTM or modification of one or more amino acids affects the function of a polypeptide. In various cases, the polypeptide is a therapeutic polypeptide. As used herein, the term “therapeutic polypeptide” means any molecule, whether naturally occurring, engineered, or synthetic, comprising at least one polypeptide chain, that is intended to achieve a therapeutic effect when administered to a subject for the treatment of a disease or medical condition. In various cases, PTM or modification of one or more amino acids affects the therapeutic function of a polypeptide.

[0076] In exemplary examples, polypeptides, particularly modified amino acids, are monitored to observe possible changes in the polypeptide structure that may affect the polypeptide's function, such as its therapeutic function. Optionally, the structure of all or some of the amino acids in the polypeptide is monitored. In exemplary embodiments, the structure of an amino acid may be referred to as an “attribute” and characterized in terms of its chemical identity or attribute type and its position in the polypeptide’s amino acid sequence (e.g., the position of the amino acid in which the attribute resides). For example, asparagine and glutamine residues are sensitive to deamidation. Deamidated asparagine at position 10 of the polypeptide amino acid sequence is an example of an attribute. A list of exemplary attribute types for specific amino acids is shown in Table F. Thus, as used herein, “structure” may include, substantially consist of, or be composed of, the attribute types listed in Table F or combinations of two or more attribute types listed in Table F. An attribute is an example of a structure, and it will be understood that whenever “structure” is referred to herein, unless otherwise specified, an attribute is intended as an example of a structure.

[0077] [Table 6]

[0078] Accordingly, this disclosure further provides a method for monitoring the attributes of polypeptides. Without being bound by any particular theory, the methods for monitoring attributes provided herein advantageously require less time and / or achieve superior recovery and analysis of digested peptides, thus enabling advances toward real-time polypeptide monitoring for polypeptide production. In exemplary embodiments, the method comprises (a) processing a polypeptide in a first sample obtained at a first time point according to the method of this disclosure for peptide mapping analysis, and (b) injecting the peptide of the digested sample obtained in (a) into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis in order to identify the attributes of the polypeptide of the first sample. In various embodiments, the method comprises comparing the attributes of the first sample with a standard or control. In alternative or additional embodiments, the method includes (c) processing the polypeptide present in a second sample obtained at a second time point according to the method of the present disclosure for peptide mapping analysis, (d) injecting the peptide of the digested sample obtained in (c) into an LC-MS system for peptide mapping analysis to identify the attributes of the polypeptide of the second sample, and (e) comparing the attributes of the first sample with the attributes of the second sample. In exemplary embodiments, the first sample and / or the second sample are taken from a cell culture containing cells expressing the polypeptide.

[0079] Since therapeutic polypeptides contain two or more amino acids, they may have multiple attributes (e.g., two or more amino acids with altered structures), and their attribute profiles can be described. As used herein, the term “attribute profile” refers to a list of attributes of a therapeutic polypeptide. In various examples, the attribute profile optionally provides chemical identity or attribute type, e.g., deamidation, to the native structure of the therapeutic polypeptide. In various examples, the attribute profile provides the location of the attributes, e.g., the location of the amino acid in which the attribute resides. In some embodiments, the attribute profile provides a description of all attributes present in the therapeutic polypeptide. In other embodiments, the attribute profile provides a description of some of the attributes present in the therapeutic polypeptide. For example, the attribute profile may provide only attributes present in a specific part of the therapeutic polypeptide, e.g., an extracellular domain, a variable region, a hypervariable region, or a CDR. Species of therapeutic polypeptides are characterized by the attributes present in them. Species of therapeutic polypeptides may differ from other species of the same therapeutic polypeptide by having different attribute profiles. If two therapeutic polypeptides have different attribute profiles, these therapeutic polypeptides represent two different species of therapeutic polypeptide. If two therapeutic polypeptides have identical attribute profiles, these therapeutic polypeptides are considered to be the same species of therapeutic polypeptide. In various embodiments, the method of this disclosure includes creating attribute profiles of polypeptides. In exemplary embodiments, the method includes comparing the attribute profiles with a standard attribute profile or a control attribute profile. In alternative or additional embodiments, the method includes creating attribute profiles of polypeptides from a first sample and creating attribute profiles of polypeptides from a second sample. Optionally, the method further includes comparing the attribute profiles of the first sample with the attribute profiles of the second sample.

[0080] In various embodiments, the attribute includes one or more intracellular enzymes that catalyze the attachment of a portion of a polypeptide to one or more amino acid residues, e.g., lipids, sugars, peptides, phosphate groups, ubiquitin tags, methyl groups, or acetyl groups. For example, an intracellular enzyme may act on one or more amino acids of a polypeptide to cause lipidization (e.g., palmitoylation, myristoylation), phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, and / or proteolysis. In exemplary embodiments, the attribute may be due to environmental changes of the polypeptide. In various examples, the attribute may be caused by environmental changes such as changes in pH, salinity, osmotic pressure, pressure, temperature, exposure to light, UV light, etc., exposure to air or oxygen, stirring / shaking, exposure to chemicals or materials (e.g., metals, plastics), or long-term storage, and such environmental changes cause structural changes of one or more amino acids. Environmental changes may include, for example, changes in culture medium, changes in formulation components, or changes in storage conditions. In some embodiments, environmental changes occur during any one or more process steps leading up to administration to the patient (e.g., production of one or more proteins (e.g., recombinant production), collection, purification, formulation, filling, packaging, storage, delivery, and final preparation immediately before administration to the patient). Exemplary modifications that may occur during production include, for example, the removal of residues from the polypeptide of the therapeutic polypeptide and / or protease-mediated cleavage. Exemplary changes that may occur after production of the therapeutic polypeptide but before administration (e.g., during packaging and / or filling, storage and / or transport) include, for example, oxidation, reduction, deamidation, deamination, aggregation, denaturation, precipitation, hydrolysis, aspartate isomerization, and N-terminal and C-terminal modifications. Thus, in various embodiments, changes in attributes may be caused by non-enzymatic modifications (e.g., chemical modifications) that do not involve intracellular enzymes, and therefore, structural changes of the therapeutic polypeptide may be caused by non-enzymatic modifications (e.g., chemical modifications). In various embodiments, modifications occur after administration of the therapeutic polypeptide and occur in vivo (in the subject to which the therapeutic polypeptide is administered).

[0081] Accordingly, this disclosure provides a method for producing polypeptides, comprising: maintaining a cell culture containing polypeptide-producing cells; monitoring the attributes of polypeptides produced by the cells; and modifying one or more conditions to alter one or more attributes of the polypeptides. The modifications may be in accordance with the attributes or attribute profiles (such as the presence, absence, or change of attributes or attribute profiles) detected by the method described herein. In exemplary embodiments, the type of cell culture is a fed-batch culture or a continuous perfusion culture. However, the methods of this disclosure are advantageously not limited to any particular type of cell culture. The cells maintained in the cell culture may be glycosylated competent cells. In exemplary embodiments, glycosylated competent cells are eukaryotic cells, including, but not limited to, yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells are described in the art; see, for example, Frenzel, et al., Front Immunol 4:217 (2013). In exemplary embodiments, eukaryotic cells are mammalian cells. In the exemplary embodiment, the mammalian cells are non-human mammalian cells.In some embodiments, the cells include Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblast cells NIH These include 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or neonatal hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)). Cells that are not competent for glycosylation can also be transformed into glycosylation-competent cells, for example, by introducing genes encoding the relevant enzymes required for glycosylation into them. Exemplary enzymes include, but are not limited to, oligosaccharide transferase, glycosidase, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferase, mannosidase, GlcNAc transferase, galactosyltransferase, and sialyltransferase. In exemplary embodiments, glycosylated competent cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways.In exemplary embodiments, glycosylated competent cells are not genetically modified to alter the activity of one or more of the following: fucosyltransferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinases, GDP-fucosyrphophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase (FX). In exemplary embodiments, glycosylated competent cells are not genetically modified to knock out the gene encoding FX. In exemplary embodiments, glycosylated competent cells are not genetically modified to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexose reductase (RMD). In exemplary embodiments, glycosylated competent cells are not genetically modified to overexpress GNTIII or RMD. In exemplary embodiments, glycosylated competent cells are genetically engineered to alter the activity of enzymes in the de novo or salvage pathway. The cell culture may be maintained according to any set of conditions suitable for the production of recombinant glycosylated proteins. For example, in some embodiments, the cell culture is maintained at a specific pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmotic pressure, etc. In exemplary embodiments, the cell culture is shaken in a CO2 incubator under standard humidified conditions with 5% CO2 before inoculation (e.g., 70 rpm). In exemplary embodiments, about 10 in 1.5 L of medium. 6 The cell culture is seeded at a seeding density of individual cells / mL. As described herein, clones may be selected to produce a chosen unpaired glycan content (e.g., less or more unpaired glycan content than the control). It will be understood that cells derived from the clones may be cultured for the production of the protein or antibody compositions described herein.

[0082] In exemplary embodiments, the methods of the present disclosure include maintaining glycosylated competent cells in cell culture medium at a pH of about 6.85 to about 7.05, for example, in various embodiments, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.90, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.00, about 7.01, about 7.02, about 7.03, about 7.04, or about 7.05.

[0083] In an exemplary embodiment, the method includes maintaining the cell culture at a temperature between 30°C and 40°C. In an exemplary embodiment, the temperature is approximately 32°C to approximately 38°C or approximately 35°C to approximately 38°C.

[0084] In an exemplary embodiment, the method includes maintaining an osmotic pressure of about 200 mOsm / kg to about 500 mOsm / kg. In an exemplary embodiment, the method includes maintaining an osmotic pressure of about 225 mOsm / kg to about 400 mOsm / kg or about 225 mOsm / kg to about 375 mOsm / kg. In an exemplary embodiment, the method includes maintaining an osmotic pressure of about 225 mOsm / kg to about 350 mOsm / kg. In various embodiments, the osmotic pressure (mOsm / kg) is maintained at about 200, 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500.

[0085] In an exemplary embodiment, the method includes maintaining the dissolved oxygen (DO) level of the cell culture at approximately 20% to approximately 60% oxygen saturation during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the DO level of the cell culture at approximately 30% to approximately 50% (e.g., approximately 35% to approximately 45%) oxygen saturation during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the DO level of the cell culture at approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, or approximately 60% oxygen saturation during the initial cell culture period. In an exemplary embodiment, the DO level is approximately 35 mmHg to approximately 85 mmHg or approximately 40 mmHg to approximately 80 mmHg or approximately 45 mmHg to approximately 75 mmHg.

[0086] The cell culture is maintained in any one or more culture media. In an exemplary embodiment, the cell culture is maintained in a medium suitable for cell proliferation and / or supplied with one or more feed media according to any appropriate supply schedule. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing glucose, fucose, lactate, ammonia, glutamine and / or glutamate. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing manganese at a concentration of about 1 μM or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing about 0.25 μM to about 1 μM manganese. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing a negligible amount of manganese. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 50 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 40 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of approximately 30 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of approximately 20 ppb or less during the initial cell culture period. In an exemplary embodiment, the medium contains copper at a concentration of approximately 5 ppb or more, or approximately 10 ppb or more. In an exemplary embodiment, the cell culture medium contains mannose. In an exemplary embodiment, the cell culture medium does not contain mannose.

[0087] Therapeutic polypeptides Polypeptides containing those that bind to one or more of the following can be processed and analyzed by the methods disclosed herein. These include CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34, including those that interfere with receptor binding; HER receptor family proteins, including HER2, HER3, HER4, and EGF receptors; cell adhesion molecules, such as LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and αv / β3 integrins. Growth factors, such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian duct inhibitors, human macrophage inflammatory protein (MIP-I-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), such as fibroblast growth factor including aFGF and bFGF, epidermal growth factor (EGF), in particular transforming growth factors (TGF) including TGF-α and TGF-β, such as TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5, insulin-like growth factor I and II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and bone induction factors. Insulin and insulin-related proteins, including insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor-binding proteins. In particular, coagulation and coagulation-related proteins such as factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin and thrombopoietin; (vii) other blood and serum proteins including but not limited to albumin, IgE and blood group antigens; colony-stimulating factors and their receptors, in particular 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, including 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, including IL-1 to IL-33 and IL-1 to IL-33 receptors (e.g., IL-8 receptor). Examples of viral antigens include AIDS enveloped virus antigens. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactants, tumor necrosis factor alpha and beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptides, DNAse, inhibin, and activin. Integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, degeneration factor (DAF), AIDS envelope, transport proteins, homing receptors, adresin, regulatory proteins, immunoadhesins, and antibodies. Myostatin, TALL-I, amyloid proteins, including, but not limited to, amyloid-beta protein, thymic interstitial lymphocyte necrotizing factor ("TSLP"), RANK ligand ("RANKL" or "OPGL"), TALL proteins containing c-kit, TNF receptors including TNF receptor type 1, TRAIL-R2, angiopoietin, and any of the aforementioned bioactive fragments, analogs, or variants.

[0088] Exemplary polypeptides and antibodies include Activase® (alteplase); alirocumab, Aranesp® (darbepoetin-alpha), Epogen® (epoetin-alpha or erythropoietin); Avonex® (interferon β-1a); Bexxar® (tositumomab); Betaseron® (interferon β); vococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see U.S. Patent No. 8080243); Campath® (aremutuzumab); 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-1); 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 (ezetimabe), 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); mapatuzumab (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; anti-C. difficile toxin A and toxin BC mAbs MDX-066 (CDA-1) and MDX-1388); 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 designated as 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 designated as 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-IGF1R mAb; anti-IGF-1R 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 Examples include mAb (HuMax-ZP3), NVS antibody #1, NVS antibody #2, and amyloid-beta monoclonal antibodies.

[0089] Examples of antibodies suitable for this method and pharmaceutical formulations are shown in Table G. Other examples of suitable antibodies include: infliximab, bevacizumab, cetuximab, palivizumab, avagovomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artitumomab, aselizumab, artinumab, atorizumab, atromimab, tocilizumab, bapinuzumab, basilixima B, bavituximab, vectumomab, belimumab, benralizumab, vertilimumab, besilesomab, bevacizumab, bezlotoxumab, bisilomab, vibatuzumab, vibatuzumab meltansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab meltansine, caplacizumab, capromab pendetide, carlumab, katumakisomab, CC49, sedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, sixtumumab, crazakizumab Clenoliximab, Cribatuzumab Tetraxetan, Conatumumab, Crenezumab, CR6261, Dasetuzumab, Dacrizumab, Darotuzumab, Daratumumab, Demicizumab, Denosumab, Detumomab, Dorlimomab Aritox, Dorizumab, Durigotuzumab, Dupilumab, Eclomeximab, Eculizumab, Edbacomab, Edrecolomab, Ephalizumab, Efungumab, Elotuzumab, Elsirimomab, Enabatuzumab, Enlimomab Pegol, Enokizumab, Enochikumab, Encituximab, Epitumomab Shitux Tan, epratuzumab, erenumab, erlizumab, erzmakisomab, etalacizumab, etrolizumab, evolocumab, exvivirumab, fanolesomab, falilimomab, falletuzumab, facinumab, fbta05, felbizumab, fezakinumab, ficratuzumab, figitumumab, framotuzumab, fontrizumab, foralumab, folavirumab, fresolimmab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab, gabirimomab, gemtuzumab ozogamicin, gevokizumab,Gilentuximab, Grembatumumab Vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab Chiuxetan, Icurcumab, Igobomab, Imsilomab, Imugatuzumab, Incrakumab, Indatuximab Tansine, Infliximab, Intetumumab, Inorimomab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Itorizumab, Ixekizumab, Keriximab Labetuzumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, rivivirumab, rigerizumab, lintuzumab, lirirumab, rorbotuzumab meltansine, lucatumumab, lumiliximab, mapatuzumab, masurimomab, mabrilimumab, matsuzumab, mepolizumab, meterimumab, miratuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motabizumab, moxetumomab Bupasdotox, Muromonab-cd3, Nacolomabutafenatox, Namilumab, Naptumomab estafenatox, Narunatumab, Natalizumab, Nevacumab, Necitumumab, Nererimomab, Nesbacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Okalatuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Oraratuzumab, Orokizumab, Omalizumab, Onartuzumab, Oportuzumab Mabmonatox, olegobomab, orticumab, otelixizumab, oxerumab, ozanezumab, ozoralizumab, padibaximab, palivizumab, panitumumab, panobacumab, pulsatuzumab, pascorizumab, patechlizumab, patrizumab, pemtumomab, perakizumab, pertuzumab, pexerizumab, pizilizumab, pintumomab, pracumubab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, lacosumomab, radrezumab, rafivirumab, ramucirumab, ranibizumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevilumab, cibrotuzumab, cifalimumab, siltuximab, simtuzumab, ciprizumab, silumab, solanezumab, soritomab, sonepcizumab, sontuzumab, stamrumab, thresomab,Suvizumab, Tabalumab, Takatuzumab Tetraxetan, Tadocizumab, Talizumab, Tanezumab, Tapritumomab Paptox, Tefivazumab, Teneriximab, Teprizumab, Teprotumumab, TGN1412, Tremerimumab, Ticilimumab, Childraquizumab, Tigatuzumab, TNX-650, Tocilizumab, Tralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS0 7. Tregalizumab, Tucothozumab, Celmoleukin, Tubilumab, Ubrituximab, Urelumab, Urtoxazumab, Ustekinumab, Bapariximab, Baterizumab, Vedolizumab, Beltuzumab, Bepalimomab, Besenkumab, Vizilizumab, Boroxiximab, Borsetuzumab, Mafodotin, Botumumab, Zaltumumab, Zanolimumab, Zatuximab, Diralimumab, Zolimomab, Aritox.

[0090] Other antibodies include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezeperumab, and trastuzumab, as well as antibodies selected from Table G.

[0091] [Table 7]

[0092] [Table 8]

[0093] In some embodiments, the therapeutic polypeptide is a BiTE® molecule. BiTE® molecules are recombinant bispecific monoclonal antibodies that direct the cytotoxic activity of T cells against cancer cells. They are fusions of two single-chain variable fragments (scFv) of various antibodies or amino acid sequences from four different genes on a single peptide chain of about 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, and the other binds to tumor cells via a tumor-specific molecule. Blinatumomab (BLINCYTO®) is an example of a BiTE® molecule that is specific to CD19. Modified BiTE® molecules (e.g., modified to extend half-life) may also be used in the manner of this disclosure. In various embodiments, the polypeptide is an antigen-binding protein, e.g., a BiTE® molecule. In an exemplary embodiment, the BiTE® molecule includes a domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 88 (Figure 1).

[0094] Exemplary Embodiments Exemplary embodiments of the present invention include, but are not limited to: E1. A method for processing polypeptides, a. Digesting a polypeptide with a protease to produce a digested sample containing at least two types of peptides; b. A method comprising incubating a digested sample in the presence of chaotrope and / or at a weakly acidic pH. E2. The method according to Embodiment 1, optionally further comprising analyzing the digested sample by mass spectrometry, wherein the digested sample is injected directly into the mass spectrometer after incubation. E3. A method for increasing the recovery rate of long-chain peptides from a digested sample, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH, wherein the digested sample comprises at least two peptides and is produced by digesting a polypeptide with a protease. E4. The method according to Embodiment 3, wherein the peptide recovery rate of the digested sample is increased in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH. E5. A method for increasing the solubility of peptides in a digested sample, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH, wherein the digested sample comprises at least two peptides and is produced by digesting a polypeptide with a protease. E6. The method according to Embodiment 5, wherein the solubility of the peptide in the digested sample is increased in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH. E7. The method according to any one of Embodiments 1 to 6, wherein at least one of the peptides in the digested sample is longer than 50 amino acids. E8. The method according to Embodiment 7, wherein at least one of the peptides in the digested sample is longer than 60 amino acids. E9. The method according to Embodiment 8, wherein at least one of the peptides in the digested sample is longer than 70 amino acids. E10. The method according to Embodiment 9, wherein at least one of the peptides in the digested sample is longer than 80 amino acids. E11. The method according to any one of Embodiments 7 to 10, wherein at least one of the peptides in the digested sample is longer than 50 amino acids and contains more than 5 hydrophobic amino acids, optionally more than 10 hydrophobic amino acids. E12. The method according to any one of Embodiments 7 to 11, wherein at least one of the peptides in the digested sample is longer than 50 amino acids and contains one or more tryptophan residues, optionally two or three tryptophan residues. E13. The method according to any one of Embodiments 1 to 12, wherein the solubility of peptides longer than 50 amino acids in the digested sample is increased in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH. E14. The method according to any one of Embodiments 1 to 13, wherein the recovery rate of peptides longer than 50 amino acids in the digested sample is increased in the presence of chaotrope and / or without incubation of the digested sample at a weakly acidic pH. E15. The method according to any one of Embodiments 1 to 14, wherein the peptide recovery rate of the digested sample is increased by at least three or four times compared to the peptide recovery rate when the digested sample is treated in the presence of chaotrope and / or without incubation at a weakly acidic pH. E16. The method according to any one of Embodiments 1 to 15, wherein the protease is trypsin, chymotrypsin, pepsin, elastase, pseudotrypsin, or a combination thereof. E17. The method according to any one of Embodiments 1 to 16, wherein the polypeptide is digested by only one type of protease. E18. The method according to any one of Embodiments 1 to 16, wherein the polypeptide is digested by at least two proteases, and optionally, the polypeptide is digested by only two proteases. E19. The method according to Embodiment 18, wherein the polypeptide is digested by trypsin and elastase, or by trypsin and chymotrypsin. E20. The method according to any one of Embodiments 1 to 19, wherein the polypeptide is digested in the presence of a chaotrope. E21. The method according to any one of Embodiments 1 to 20, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH using mechanical shaking. E22. The method according to any one of Embodiments 1 to 21, comprising incubating the digested sample at a weakly acidic pH, optionally about 4 to about 6. E23. The method of Embodiment 22, in which the pH of weakly acidic is less than 5.5. E24. The method according to Embodiment 23, wherein the pH of a weakly acidic solution is approximately 4.8 to approximately 5.2. E25. The method according to Embodiment 24, wherein the pH of a weakly acidic solution is approximately 5.0. E26. The method according to any one of Embodiments 1 to 25, comprising incubating a digested sample in the presence of a chaotrope. E27. The method according to Embodiment 26, comprising incubating a digested sample in the presence of urea, n-butanol, ethanol, guanidine, or salts thereof, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, and / or thiourea. E28. The method according to Embodiment 26 or 27, comprising incubating a digested sample in the presence of guanidine or a salt thereof. E29. The method according to Embodiment 28, wherein the salt of guanidine is guanidine hydrochloride, guanidine nitrate, guanidine thiocyanate, or guanidine carbonate. E30. The method according to Embodiment 29, wherein the salt is guanidine hydrochloride. E31. The method according to any one of Embodiments 1 to 30, comprising incubating a digested sample in the presence of 2M guanidine. E32. The method according to Embodiment 31, comprising incubating the digested sample in the presence of 3M guanidine. E33. The method according to Embodiment 31 or 32, comprising incubating the digested sample in the presence of less than 5 M guanidine. E34. The method according to any one of Embodiments 1 to 33, comprising incubating a digested sample in the presence of approximately 4 M guanidine. E35. The method according to any one of Embodiments 1 to 34, comprising incubating a digested sample in the presence of one or more of an organic solvent, alcohol, acetonitrile, urea, a surfactant, and / or dimethyl sulfoxide (DMSO). E36. The method according to Embodiment 35, wherein the surfactant is a nonionic surfactant. E37. The method according to Embodiment 36, wherein the nonionic surfactant is polyoxyethylene or glycoside. E38. The method according to Embodiment 37, wherein polyoxyethylene is a surfactant, lipid, or fatty acid of the Tween, Triton, or Brij series. E39. A method for processing a polypeptide, optionally comprising digesting the polypeptide with a protease that cleaves the C-terminus of tryptophan to produce a digested sample containing at least two peptides, wherein at least one peptide contains C-terminal tryptophan. E40. The method according to Embodiment 39, wherein the recovery rate of the peptide containing the C-terminal Trp is increased compared to the recovery rate of the peptide processed without digesting the polypeptide with a protease that cleaves the C-terminus of the Trp. E41. The method according to Embodiment 40, wherein the recovery rate of the peptide containing the C-terminal Trp is greater than 20%. E42. The method according to any one of Embodiments 39 to 41, comprising digesting a polypeptide with a single protease. E43. The method according to any one of Embodiments 39 to 42, wherein the protease is trypsin, or a proteoform or isoform thereof. E44. The method according to Embodiment 43, wherein the proteoform is pseudotrypsin. E45. The method according to Embodiment 43, comprising digesting a polypeptide with trypsin at an acidic pH, optionally about 4 to about 6. E46. The method according to Embodiment 45, wherein the pH is approximately 5. E47. The method according to any one of Embodiments 39 to 46, comprising digesting a polypeptide with at least two proteases, or optionally, only two proteases. E48. The method according to embodiment 47, wherein at least one of the proteases is trypsin. E49. The method according to Embodiment 47 or 48, wherein at least one of the proteases is chymotrypsin. E50. The method according to any one of embodiments 47 to 49, comprising successively digesting a polypeptide with trypsin and chymotrypsin. E51. The method according to Embodiment 50, comprising digesting a polypeptide with trypsin, and then digesting the polypeptide with chymotrypsin. E52. The method according to any one of Embodiments 47 to 51, wherein digestion is carried out at a neutral pH, optionally at pH 7.5. E53. The method according to any one of Embodiments 1 to 52, wherein the polypeptide is an antigen-binding protein and optionally a bispecific T cell-inducing (BiTE®) molecule. E54. The BiTE(registered trademark) molecule is a molecule comprising a CD3-binding domain, as described in Embodiment 53. E55. The method according to any one of Embodiments 39 to 54, wherein the polypeptide comprises a sequence greater than 50 amino acids, and the sequence comprises at least one Trp residue, optionally at least two or three Trp residues. E56. The method according to Embodiment 55, wherein the polypeptide comprises a sequence greater than 60 amino acids, greater than 70 amino acids, or greater than 80 amino acids, and the sequence comprises at least one Trp residue. E57. The method according to any one of Embodiments 1 to 56, wherein the peptide comprises a sequence having at least 90% sequence identity with SEQ ID NO: 88 and comprises at least one Trp. E58. The digested sample comprises a peptide containing the amino acid sequence HGNFGNSYISYW (SEQ ID NO: 92), according to any one of Embodiments 1 to 57. E59. The method according to any one of Embodiments 1 to 58, wherein the polypeptide is processed for peptide mapping analysis for less than 12 hours. E60. The polypeptide is processed for peptide mapping analysis for less than 6 hours, according to the method of Embodiment 59. E61. The method according to Embodiment 60, wherein the polypeptide is processed for peptide mapping analysis for less than 4 hours. E62. The method according to any one of Embodiments 1 to 61, which is carried out using only a gel filter or without using any filter at all. E63. The method according to any one of Embodiments 1 to 62, which is carried out without using any filter. E64. The method according to any one of Embodiments 1 to 61, wherein the method is carried out using only a gel filter. E65. A method according to any one of Embodiments 1 to 61, which includes buffer exchange using a gel filter, or does not include buffer exchange at all. E66. A method for monitoring the attributes of a polypeptide, a) Processing the first polypeptide in the first sample obtained at a first time point according to the method of any one of Embodiments 1 to 65, b) The peptides from the digested sample are injected into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis to identify post-translational modifications (PTMs) of the polypeptides in the first sample, c) Processing the second polypeptide in the second sample, which is the same as or different from the first polypeptide, obtained at a second time point, according to the method of any one of Embodiments 1 to 65. d) The peptides from the digested sample are injected into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis to identify the PTM of the polypeptide in the second sample, e) A method comprising comparing the PTM of a first sample with the PTM of a second sample. E67. The method according to Embodiment 66, wherein the sample is collected from a cell culture containing cells expressing the first polypeptide and / or the second polypeptide. The method according to any one of Embodiments 1 to 67, comprising digesting a polypeptide E68 with trypsin in an enzyme:substrate (E:S) weight ratio of approximately 1:1 to approximately 1:15 to produce a digested sample containing at least two peptides. The method according to Embodiment 68, wherein the weight ratio of E69.E:S is approximately 1:1 to approximately 1:10. The method according to Embodiment 69, wherein the weight ratio of E70.E:S is approximately 1:2 to approximately 1:8. The method according to Embodiment 70, wherein the E:S ratio is approximately 1:4 to approximately 1:6, and optionally approximately 1:5. E72. The method according to any one of embodiments 68 to 71, wherein digestion is carried out at a pH of approximately 7.0 to approximately 8.0. E73. The method according to embodiment 72, wherein digestion is carried out at a pH of approximately 7.5. E74. The method according to any one of embodiments 68 to 73, wherein digestion takes less than approximately 12 hours. E75. The method according to embodiment 74, wherein digestion takes less than approximately 6 hours. E76. The method according to embodiment 75, wherein digestion takes less than approximately 4 hours. E77. The method according to embodiment 76, wherein digestion takes approximately 2 hours to less than 4 hours. E78. The method according to any one of embodiments 68 to 77, wherein digestion is carried out at a temperature of approximately 30°C to approximately 45°C. E79. The method according to Embodiment 78, wherein digestion is carried out at a temperature of approximately 35°C to approximately 40°C, optionally approximately 37°C. E80. The method according to any one of embodiments 68 to 79, wherein digestion is carried out in the presence of calcium chloride. E81. The method according to any one of embodiments 68 to 79, wherein digestion is carried out in the absence of calcium chloride. E82. The method according to any one of embodiments 68 to 81, wherein only trypsin is used to digest polypeptides. E83. The method according to any one of embodiments 68 to 82, wherein the polypeptide is digested solely by trypsin in an E:S weight ratio of approximately 1:1 to approximately 1:15. The method according to any one of Embodiments 68 to 83, wherein when E84 polypeptide is digested with trypsin in the said E:S weight ratio, the digested sample is produced which contains one or more peptides having tyrosine at the C-terminus. E85. The method according to any one of Embodiments 1 to 84, comprising denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide before digesting the polypeptide with a protease. E86. The method according to any one of Embodiments 1 to 85, comprising buffer exchange before digesting the polypeptide with a protease and after denaturing, reducing, and / or alkylating the polypeptide. E87. The method according to any one of embodiments 1 to 86, wherein buffer replacement includes the use of a size exclusion cartridge. E88. The size exclusion cartridge is an NAP5 cartridge having Sephadex G-25 gel filtration material, as described in Embodiment 87. E89. The method according to any one of Embodiments 68 to 88, further comprising incubating the digested sample at a weakly acidic pH in the presence of chaotrope. E90. The method according to Embodiment 89, wherein the chaotrope is guanidine hydrochloride. E91. The method according to Embodiment 89 or 90, wherein the pH is weakly acidic and approximately 5. E92. The method according to any one of Embodiments 1 to 91, comprising injecting peptides from a digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis. E93. A method for processing polypeptides to produce a digested sample containing at least one or two peptides, each having tyrosine at its C-terminus, the method comprising digesting the polypeptides with trypsin in a trypsin:polypeptide ratio of about 1:1 to about 1:15. E94. The method according to embodiment 93, wherein digestion is carried out according to one of the methods of embodiments 69 to 92. E95. The method according to Embodiment 93 or 94, wherein the digested sample comprises a peptide containing the amino acid sequence of HGNFGNSY (SEQ ID NO: 108), a peptide containing the amino acid sequence of HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide containing the amino acid sequence of HGNFGNSYISYWAY (SEQ ID NO: 110). E96. The method according to any one of embodiments 93 to 95, wherein only trypsin is used to digest polypeptides. The method according to any one of embodiments 93 to 96, wherein the weight ratio is approximately 1:1 to approximately 1:10. The method according to any one of embodiments 93 to 96, wherein the weight ratio of E98.E:S is approximately 1:2 to approximately 1:8. The method according to any one of embodiments 93 to 96, wherein the E:S ratio is approximately 1:4 to approximately 1:6, and optionally approximately 1:5. E100. The method according to any one of embodiments 93 to 99, wherein digestion is carried out at a pH of approximately 7.0 to approximately 8.0.

[0095] The following embodiments are provided solely for illustrative purposes and are not intended to limit the scope of the present invention. [Examples]

[0096] In the following examples, we analyzed different bispecific T cell inducers (BiTE®) molecules, each possessing a first binding domain that binds to a first antigen and a second binding domain that binds to the T cell receptor CD3ε. These BiTE® molecules differed in their first binding domains. BiTE® molecule #1 and BiTE® molecule #2 each contained a first binding domain that bound to the B cell maturation antigen (BCMA), while BiTE® molecule #3 contained a first binding domain that bound to CD19. The second binding domain was common to all BiTE® molecules. Figure 1 shows the sequence of the CD3 binding domain common to the BiTE® molecules. As shown in this figure, the CD3 binding domain has two CDRs immediately adjacent to the long linker region. These two CDRs are indicated by the letters highlighted in yellow in Figure 1. Sequence [ka] One CDR containing (SEQ ID NO: 89) contains two asparagine residues sensitive to deamidation and one tryptophan residue sensitive to oxidation. While monitoring these residues during manufacturing is beneficial, the long extension of the CD3-binding domain containing the two CDRs (underlined in Figure 1) lacks a trypsin digestion site, making it difficult to analyze and monitor this region of the molecule. Due to the lack of a trypsin digestion site, this peptide, which is over 80 amino acids, remains intact, has low solubility, and therefore is difficult to recover for analysis. Consequently, attributes of interest within these CDRs cannot be monitored due to the large size of this peptide (approximately 8 kDa), the difficulty of separating modified versions of the peptide by chromatography, poor peptide recovery and / or ionization, and the general challenges associated with interpreting mass spectrometry for peptides of this size.

[0097] The large peptide produced during trypsin digestion of the analyzed BiTE® molecule (having the underlined sequence in Figure 1 and the sequence of Sequence ID No. 91) is referred to herein as the "H350-R436" peptide, because its first amino acid (His) is the 350th amino acid of one of the analyzed BiTE® molecules (hereinafter referred to as BiTE® molecule #1), and Arg is its 436th amino acid. This amino acid sequence is also present in other BiTE® molecules analyzed herein, but this sequence may or may not represent amino acids 350-436 of these other BiTE® molecules. The amino acid numbers in this sequence may differ in these other BiTE® molecules compared to the amino acid numbers of BiTE® molecule #1. For example, in BiTE® molecule #3, amino acids H355-R441 correspond to H350-R436 of BiTE® molecule #1. The alignment of this region of BiTE(registered trademark) molecules #1 to #3 is shown in Figure 2. In this figure, the H350-R436 peptide is labeled "H357-R443". The Cys residues to be reduced are highlighted in yellow. As shown in Figure 2, this region of the BiTE(registered trademark) molecule contains two CDRs of the CD3 binding domain.

[0098] This peptide is not the only region of the BiTE® molecule that is difficult to peptide map. Many BiTE® molecules also have a short-chain linker peptide (approximately 5-7 kDa) immediately adjacent to the CDR of the first binding domain, which can be similarly difficult to monitor by trypsin digestion. For example, of the 5.7 kDa linker peptide, the aspartic acid, which is part of the CDR, is subject to isomerization and therefore requires monitoring, among other things, because this site may affect the binding of the BiTE® molecule. Thus, the studies presented herein aimed to increase the solubility and recovery rate of the long-chain peptide of the BiTE® molecule.

[0099] Example 1 This example demonstrates the insufficient recovery rate of long-chain peptides from BiTE® molecules using conventional peptide mapping methods.

[0100] In the initial study, BiTE® molecule #1 was subjected to trypsin digestion. The trypsin peptide was stored in trypsin digestate (pH 7.5) in a liquid chromatography (LC) vial at 5°C or 37°C for up to 430 minutes. The trypsin digestate was 0.1 M Tris, pH 7.5, and chaotrope-free. To measure the signal of the trypsin peptide, four injections were performed into a liquid chromatography-mass spectroscopy (LC-MS) system at 100-minute intervals. The relative abundance of the trypsin peptide, representing peptide recovery plotted as a function of digestion time, is shown in the chromatogram in Figure 3A. As shown in this figure, the signal for the H350-R436 peptide decreased over time when stored at 37°C, suggesting a low abundance and low recovery rate of this peptide. This peptide is thought to have either aggregated (and therefore could not be separated and analyzed by LC-MS) or adsorbed onto the wall of the LC vial (and was not injected into the LC-MS system).

[0101] Visual inspection was performed on the stored digested samples. Fibrous particles were observed in the digested samples stored in HPLC vials at 5°C for 48 hours (Figure 3B). Without theoretical limitations, this observation suggested that a cluster of trypsin peptides, such as the long-chain peptide H350-R436, was aggregated within the fibrous particles and had low solubility.

[0102] Example 2 This example demonstrates the effect of chaotrope on the solubility of the trypsin peptide in the BiTE(registered trademark) molecule.

[0103] In an initial series of experiments, antibodies were treated with denaturation, alkylation, reduction, digestion, and quenching for peptide mapping analysis. However, there were problems with antibody preparation because peptides spanning one or more CDRs of the antibody were recovered in insufficient amounts for peptide mapping. These poorly recovered peptides contained tryptophan residues. Different chaotropic reagents—(1) NaCl, (2) alcohol, (3) the nonionic surfactant octyl β-D-glucoside (OBG), (4) urea, and (5) guanidine—were tested to see if they could increase the solubility and recovery rate of the peptides. These initial experiments demonstrated that incubating digested samples in the presence of guanidine best recovered CDR-containing trypsin peptides from vials.

[0104] The long-chain H350-R436 peptide of BiTE® molecule #1 contains a Trp residue (Figure 1), and its recovery rate is low, similar to that of Trp-containing peptides with low antibody recovery rates (Example 1). Therefore, a method of processing samples containing BiTE® molecule #1, including incubation of the digested sample in the presence of chaotrope, was evaluated to determine whether it could improve the solubility of the long-chain H350-R436 peptide. If the solubility of the long-chain H350-R436 peptide can be increased, the recovery rate should also increase. In summary, post-digestion solutions containing BiTE® molecule #1 were processed for peptide mapping in the presence or absence of guanidine hydrochloride. Each step of the preparation of BiTE® molecule #1 for peptide mapping analysis was observed in detail in a light chamber. Representative images of the vials are shown in Figures 4A to 4D. As shown in these figures, the solutions were (a) reduced and alkylated (vials 2 and 3), (b) reduced, alkylated, and then digested with trypsin (vials 4 and 5), or (c) untreated (vial 1).

[0105] As shown in Figure 4A, the reduced and alkylated (vials 2 and 3) or reduced, alkylated and digested (vials 4 and 5) solutions showed some turbidity compared to the untreated solution (vial 1). The turbidity in vials 2-5 suggested low peptide solubility. As shown in Figure 4B, after 10 minutes, turbidity remained in vials 2 and 3, but the reduced, alkylated and digested solutions (vials 4 and 5) became clear, although visible particles were observed. Magnified images of the solutions are shown in the right-hand images of Figures 4A and 4B. As shown in the magnified image, the turbidity in vial 5 disappeared due to trypsin digestion, but fibrous particles were formed. A further magnified image of vial 5 containing fibrous particles is shown (bottom of Figure 4C).

[0106] The contents of vials 3 and 5 were each divided into two. Guanidine hydrochloride was added to one aliquot of vial 3 and one aliquot of vial 5 to a final concentration of 4 M, while guanidine hydrochloride was not added to the other aliquot (Figures 4C and 4D). As shown in Figures 4C and 4D, all turbidity and visible particles disappeared only in the vials to which guanidine hydrochloride was added. This effect occurred almost immediately after the addition of chaotrope. In contrast, the vial that was not treated with guanidine HCl (labeled "no GuHCl") remained turbid and / or contained fibrous particles (left-hand vials in Figures 4C and 4D).

[0107] These results suggest that guanidine hydrochloride solubilizes turbid or fibrous particles, and that chaotrope should be included after digestion to enhance the solubility of trypsin peptides before analysis.

[0108] Example 3 This example demonstrates the effect of chaotropes on the recovery rate of trypsin peptides from the BiTE(registered trademark) molecule.

[0109] Solutions containing BiTE® molecules (BiTE® molecule #1 or BiTE® molecule #2) were treated differently for liquid chromatography-mass spectroscopy (LC-MS) peptide mapping, and the relative abundance (representing recovery) of the long-chain H350-R436 peptide of the BiTE® molecule was compared. Each preparation method included denaturation, alkylation, reduction, buffer exchange, digestion, and HPLC-MS analysis. Samples of the digested material after incubation were injected directly into the HPLC-MS system for analysis. Denaturation was performed with 6M guanidine, pH 7.5. Iodoacetic acid (IAA) was added to the denatured material for alkylation, and dithiothreitol (DTT) was added for reduction. Digestion was performed using trypsin at pH 7.5, essentially as described in Example 1. The preparation methods differed as described below.

[0110] In the first method shown in Figure 5A, a solution containing BiTE® molecules was denatured in 6M guanidine (pH 7.5), and IAA and DTT were added for alkylation and reduction, respectively. Buffer exchange of the denatured, alkylated, and reduced polypeptides into a digestion buffer was performed by gel filtration using a NAP5 cartridge, thereby separating the denatured, alkylated, and reduced polypeptides (proteins) from the guanidine. The eluate containing the denatured, alkylated, and reduced polypeptides was digested with trypsin at pH 7.5. To the digested sample, (1) an appropriate amount of 20% (v / v) formic acid was added to obtain a final pH of 2, 5, or 7.5, and a stock solution of guanidine hydrochloride (pH 4) was added to obtain a final guanidine concentration of 4M, or (2) an appropriate amount of 20% (v / v) formic acid was added to obtain a final pH of 2, 5, or 7.5 without adding guanidine hydrochloride. The sample solution containing the digested material was injected directly into the HPLC-MS system for analysis without buffer exchange.

[0111] In the second method shown in Figure 5B, a solution containing BiTE® molecules was denatured in 6 M guanidine (pH 7.5), and IAA and DTT were added for alkylation and reduction, respectively. Denaturation, alkylation, and reduction were performed on a 10 kDa or 30 kDa molecular weight cutoff (MWCO) filter. The 10 kDa MWCO filter was used as a negative control. After denaturation, alkylation, and reduction, the contents above the MWCO filter were filtered and centrifuged. Digestion buffer was added to the filtrate containing the denatured, alkylated, and reduced polypeptides, and this mixture was placed on top of the MWCO filter. Digestion was performed at pH 7.5 using one of two different brands of trypsin. To the digested sample, (1) an appropriate amount of 20% (v / v) formic acid was added to obtain a final pH of 2, 5, or 7.5, and a stock solution of guanidine hydrochloride (pH 4) was added to obtain a final guanidine concentration of 4M, or (2) an appropriate amount of 20% (v / v) formic acid was added to obtain a final pH of 2, 5, or 7.5 without adding guanidine hydrochloride. After incubation, the digested sample was passed through an MWCO filter and centrifuged, and the filtrate was directly injected into the HPLC-MS system for analysis.

[0112] The relative abundance (relative recovery rate) of the long-chain H350-R436 peptide processed by different methods is shown in the graph in Figure 6. As shown in this figure, the methodology in Figure 5A (including gel filtration buffer exchange) resulted in a better overall peptide recovery rate compared to the methodology in Figure 5B (including MWCO filter buffer exchange). As shown in Figure 6, all samples incubated in the presence of guanidine hydrochloride after digestion showed improved recovery rates compared to samples incubated in the absence of guanidine at the same pH. These results also demonstrate that the improvement in recovery rates achieved by incubating digested samples in the presence of guanidine is independent of pH and buffer exchange method (gel filtration vs. MWCO filter). As expected, no detectable peptides were recovered from samples using a 10 kDa MWCO filter. As shown in Figure 6, incubation of digested samples with guanidine hydrochloride at pH 5 resulted in significantly better recovery rates than those obtained using guanidine hydrochloride at other tested pH levels (pH 2 or pH 7.5). For samples treated with a gel filter, incubation of the digested samples at pH 5 yielded a considerable peptide recovery rate, even without guanidine hydrochloride. These results suggest that incubating digested samples at pH 5 in the presence of 4M guanidine significantly increases the recovery rate of long-chain peptides. Although the overall recovery rate of long-chain peptides using MWCO filter buffer exchange was low (Figure 5B), the results obtained using this methodology confirm the observation that incubating digested samples at pH 5 in the presence of 4M guanidine improves the recovery rate of long-chain peptides compared to incubation in the absence of guanidine.

[0113] Given that chaotropes are present in mass spectrometers and many of them are known to negatively affect MS performance, and therefore are not typically included in the solutions injected into the mass spectrometer, the improvement in the recovery rate of long-chain peptides when incubated in the presence of guanidine was surprising.

[0114] Without being limited by theory, at pH 2, the acidic residues Asp and Glu become electrically neutral and hydrophobic, making the long-chain peptides even more hydrophobic (allowing them to adsorb to the vial surface), and at pH 7.5, since this pH (7.5) is close to the pI (pI=9) of the long-chain peptides, the peptides tend to aggregate and / or precipitate, so incubation at a weakly acidic pH yielded the best recovery. In general, the pI values ​​of mAbs and ScFc-BITE proteins are in the range of pI 7.5 to 9, indicating that the average peptide has a pI in the same range. This suggests that the peptides, on average, become less soluble at pH above 7. The higher the pH, the more likely the trypsin peptides are to be subjected to undesirable chymotrypsin-mediated proteolysis and deamidation of asparagine residues.

[0115] These results suggest that incubating digested samples at a weakly acidic pH in the presence of guanidine improves the recovery rate of long-chain peptides during the preparation of protein samples for analysis.

[0116] Example 4 This example demonstrates that the method for processing polypeptides disclosed herein is sufficient for chromatographic separation of long-chain peptides and their deamidation products.

[0117] A first sample of the BiTE® molecular formulation containing 2 mg / ml of BiTE® molecule #3 was processed for peptide mapping according to the methodology in Figure 5A, which involved buffer exchange to digestion buffer using a NAP5 size exclusion cartridge and incubation of the digested sample at pH 5 in the presence of guanidine hydrochloride. Formic acid (20% (v / v)) was added to the digested sample to obtain pH 5.0, and a stock solution of guanidine hydrochloride was added to the digested sample to obtain a final guanidine concentration of 4 M. A second sample containing the same BiTE® molecular formulation as the first sample was stressed by a pH jump before polypeptide processing. In particular, when the formulation was diluted with PBS and incubated at 37°C for 2 weeks, the pH of the formulation shifted from 4.2 to 7. A third sample containing the same BiTE® molecular formulation as the first sample was photostressed by exposure to ultraviolet light. A fourth sample containing the same BiTE® molecular formulation as the first sample was heatstressed by storage at 40°C for 1 month. Such stress can induce polypeptide modifications, such as deamidated polypeptides and oxidized polypeptides. The second, third, and fourth samples were each processed in the same manner as the first sample, with buffer exchange to digestion buffer using a NAP5 size exclusion cartridge, and the digested samples were incubated at pH 5 in the presence of guanidine hydrochloride. The digested samples were then analyzed by peptide mapping.

[0118] The ultraviolet (UV) chromatograms of the samples are shown in Figure 7A. The main peak of the long-chain H355-R441 peptide is shown. The blue line represents the first sample, the red line represents the second sample, the green line represents the third sample, and the magenta line represents the fourth sample. A magnified view of the main peak is shown in Figure 7B. As shown in Figures 7A and 7B, the recovery rate of the long-chain H355-R441 peptide (blue line) in the first sample was good. As shown in Figure 7B, the height of the main peak in the second sample was lower than that of the first sample. Additional peaks were observed to the left and right of the main peak in the second sample, and these peaks were initially assigned as deamidation products of the long-chain H355-R441 peptide, based on the fact that deamidation (the process by which asparagine is converted to aspartic acid and isoaspartic acid residues) results in a mass change of 1 Da. Mass spectroscopy was performed to confirm the initial assignment of deamidation. Figures 8A to 8F are mass spectrometry chromatograms. Figure 8A shows the main peak of the long-chain H355-R441 peptide from the first sample. Figure 8B shows the main peak of the long-chain H355-R441 peptide and the peaks (numbered 1 to 3) assigned to the deamidation product for the second sample. Figures 8C to 8F are the mass spectra of the peaks labeled Main, 1, 2, and 3 in Figures 7B and 8B. The X-axis is on an m / z scale, where m is the mass and z is the charge given to the peptide by the bound protons. Figures 8C to 8F show sections of the mass spectrum around the peptide ion with a charge of 5+ (z=5), given by five bound protons. Due to the abundance of C, H, O, N, and S isotopes, the mass is represented by multiple peaks. The highest isotopic peak represents the average molecular weight. The average molecular mass is calculated as m / z*zz, with the subtraction in the latter part to remove the bound protons. Figure 8C shows the mass spectrum of Figure 8A, Figure 8D shows the mass spectrum of peak 1 in Figure 8B, Figure 8E shows the mass spectrum of peak 2 in Figure 8B, and Figure 8F shows the mass spectrum of peak 3 in Figure 8B. The long-chain H355-R441 peptide was identified by the strict agreement between its measured average mass of 8569 Da (1714.8 * 5 - 5 = 8569 Da, Figure 8C) and its theoretical average mass of 8569 Da.Furthermore, the pre-peak and post-peak of the second sample were identified as deamidation products because their mass was approximately 1 Da greater than that of the main peak (Figures 8D, 8E, and 8F).

[0119] Previous experiments had shown that under specific stress conditions, the long-chain H355-R441 peptide is deamidated at N357 and N360 and oxidized at W431. The pre-peak and post-peak were tentatively identified as the products of deamidation at N357 and N360. During LC-MS / MS peptide mapping, the peptide was also sequenced by fragmentation using a mass spectrometer (Figure 9). The long-chain H355-R441 peptide was identified not only by its precise mass (Figures 8A-8F) but also by numerous fragments that closely matched its sequence and predicted fragmentation pattern (Figure 9).

[0120] These results demonstrate that when polypeptides are processed by the methodology shown in Figure 5A, in which the polypeptides are buffered with digestion buffer using a NAP5-sized exclusion cartridge and the digested samples are incubated in the presence of guanidine hydrochloride at pH up to 5, attributed peptides (deamidation products of the long-chain H355-R441 peptide) can be separated chromatographically from the main peak as pre- and post-peaks. The ability to observe deamidation products at these sites is important, considering that they are part of the molecule's CDR and may affect the binding ability of the BiTE® molecule to CD3. These data suggest that attribute monitoring of the BiTE® molecule is possible by the polypeptide processing methods disclosed herein.

[0121] Example 5 This example demonstrates the evaluation of various enzymes or combinations of enzymes, and the incubation of digested samples in the presence of guanidine at pH 5.

[0122] In the previous example, digestion was performed using trypsin at pH 7.5. Other enzymes were evaluated to improve the recoverability of peptides for peptide mapping and MAM. A summary of the enzymes and their cleavage sites is shown in the table in Figure 10.

[0123] A first sample containing BiTE® molecule #1 was processed according to the methodology shown in Figure 5A, which involves buffer exchange to digestion buffer using a NAP5 size exclusion cartridge and incubation of the digested sample at pH 5 in the presence of guanidine hydrochloride (final guanidine concentration of 4 M). The BiTE® molecule was denatured in guanidine and alkylated and reduced by IAA and DTT. In this study, digestive enzymes were varied to investigate their effect on the recovery rate of long-chain peptides. Digestion with trypsin, chymotrypsin, or pepsin was performed at pH 4. In some cases, sequential digestion was used with a combination of trypsin and elastase, or trypsin and chymotrypsin. In each combination, trypsin was used as the first protease in the two digestions. When chymotrypsin was used as the second protease, chymotrypsin was added directly to the trypsin digestion. However, when elastase was used as a second protease, small trypsin peptides were removed before adding elastase for the second digestion. Each digestion condition was performed three times.

[0124] Previous experiments have shown that the long-chain H355-R441 peptide BiTE® molecule #1 contains Asn residues that are deamidated at N352 and N355 under specific stress conditions. Figure 11 shows the long-chain H350-R436 peptide of BiTE® molecule #1 and how the long-chain peptide is cleaved by each digestion method. The recovery rate of the digested peptides is color-coded. Peptides containing Asn residues that are potentially subject to deamidation are enclosed in red. As shown in Figure 11, sequential digestion using trypsin and chymotrypsin resulted in a recovery rate of over 50% for peptides containing potentially deamidated residues. Figure 12 shows a graph of the recovery rate of peptides containing the target Asn residue for each type of digestion. As shown in Figure 12, the best recovery rate for potentially deamidated peptides was achieved by sequential digestion using trypsin and chymotrypsin.

[0125] The relative abundance of potentially deamidated peptides in the first sample was compared to the relative abundance of the second sample, which contained the same BiTE® molecular formulation as the first sample, but the second sample was stressed by a pH jump as described in Example 4. Each sample was processed for analysis according to the methodology in Figure 5A, in which the digested sample was buffered with digestion buffer using a NAP5 size exclusion cartridge and incubated at pH 5 in the presence of 4 M guanidine hydrochloride. Each sample was digested sequentially with trypsin and chymotrypsin. Figure 13A shows the relative abundance of peptides containing N352 and N355 in the first (unstressed) sample, and Figure 13B shows the relative abundance of peptides containing N352 and N355 in the second (stressed) sample. As shown in Figure 13B, three additional peaks (numbered 1-3) were observed to the left and right of the main peak. These additional peaks were due to stress induced by the pH jump applied to the molecules in the second sample and represented the deamidation products of the peptides containing N352 and N355.

[0126] The assignment of peaks 1 and 2 to the deamidated peptide at N355, and peak 3 to the deamidated peptide at N352, was performed by sequencing after fragmentation of the peptides using mass spectrometry. Exemplary MS peaks obtained by fragmentation and sequencing are shown in Figures 14A to 14D. Figure 14A shows the peaks of the second sample containing the deamidation product, and Figure 14B shows the peaks of the first sample without the deamidation product. Figure 14C is a magnified view of a portion of Figure 14A, and Figure 14D is a magnified view of a portion of Figure 14B. A 1 Da-shifted peak representing deamidated N352 is shown in Figure 14C, and this peak disappears in Figure 14D.

[0127] In summary, we were able to ultimately assign further peaks in the stressed samples to specific amino acids that were deamidated as deamidation products. This was possible because shorter peptides generated during sequential digestion with trypsin and chymotrypsin allowed for better sequence coverage, and fragmentation MS / MS information enabled accurate detection of the deamidation site associated with the 1Da increase. Since chymotrypsin digestion also cleaves the polypeptide at the C-terminus of Trp, it is expected that chymotrypsin digestion would also yield shorter peptides, enabling better sequence coverage and accurate detection of the deamidation site. As recent studies on BiTE® molecule #3 (Bondarenko et al., 2019) and BiTE® molecule #1 (Shi et al., 2020) have shown (data not shown here), N352 has a greater influence on CD3e binding than N355, making accurate evaluation of deamidation at two asparagine residues separated by only three residues important.

[0128] In this example, it was demonstrated that a method of processing polypeptides, which includes buffer exchange of the NAP5 size exclusion cartridge with digestion buffer and incubation of the digested sample in the presence of guanidine hydrochloride at a weakly acidic pH, enables the detection of amino acid attributes separated by only a few residues.

[0129] Example 6 This example describes a method for processing an exemplary sample containing BiTE® molecules, using different enzymes for digestion.

[0130] Data presented herein demonstrates that digesting long-chain peptides common to BiTE® molecules using a protease that cleaves the C-terminus of Trp within the long-chain peptide improves sequence coverage and allows for accurate detection of deamide sites (Example 5). Further proteases that cleave at the same site within the long-chain peptide were evaluated. In one experiment, pseudotrypsin, a proteoform of trypsin, was particularly well-regarded among the proteases.

[0131] Pseudotrypsin is prepared essentially as described in Perutka and Sebela, Molecules 23:2367 (2018). In summary, try to separate the trypsin autodigestion components using a HEMA-BIO1000 SB column (0.75 × 25 cm) in medium-pressure protein liquid chromatography (pH 7.1, flow rate 2 mL / min). Alternatively, an Uno S12 column (15 × 68 mm) may be used instead of the HEMA-BIO column with gradient elution (buffer B containing 1 M NaCl).

[0132] Samples containing BiTE® molecule #1 are processed essentially as described in Example 3. In summary, samples containing BiTE® molecule are denatured, alkylated, reduced, and buffered into digestion buffer by gel filtration using an NAP5 cartridge. In a series of experiments, the digestion buffer contains pseudotrypsin (also known as Ψ-trypsin or psi-trypsin), purified according to the above procedure and Perutka and Sebela, 2018, either alone or in combination with trypsin. Digestion of chymotrypsin alone or trypsin alone is also evaluated. Various trypsin enzymes, including trypsin-1 (Promega V5280), trypsin-2 (Promega, V5111), trypsin-3 (Pierce, 90057), and trypsin-4 (Promega AccuMAP® kit), are also evaluated in this test. Digestion using the above proteases is performed at various pH levels: pH 2, pH 5, and pH 7.5. Digestion samples containing peptides are injected into an LC-MS system for chromatographic separation and peptide mapping.

[0133] The results are expected to support the idea that pseudotrypsin cleaves the long-chain H350-R436 peptide at the C-terminus of Trp, producing two smaller peptides, H350-W361 and A362-R436. This result is also expected to support the idea that trypsin-4 cleaves the long-chain H350-R436 peptide at the C-terminus of Trp at pH 5. Reducing the peptide size allows for better mapping and monitoring of molecular attributes.

[0134] This example supports the use of pseudotrypsin and other enzymes for the digestion of the BiTE® molecule. Since pseudotrypsin is expected to cleave at the same site (the C-terminus of Trp361) as the sequential digestion by trypsin and chymotrypsin, this example confirms that this pseudotrypsin digestion method enables the same peptide analysis and attribute monitoring as described in Example 5.

[0135] Example 7 This example illustrates an exemplary method for digesting a polypeptide sample.

[0136] The BiTE® molecule #1 solution was processed and analyzed as follows: A solution (100 μL) containing BiTE® molecule #1 at a concentration of 1.05 mg / mL was concentrated by filtering the solution through a 10 kDa AMCO filter (14 kx g) pre-wetted with a solution containing 8 M guanidine hydrochloride and 250 mM acetic acid (pH 4.7). This step was performed for 20 minutes. The resulting filtrate (25 μL) contained BiTE® molecule #1 at a concentration of 4.2 mg / mL. Approximately half of the filtrate (12 μL) was denatured and reduced for 30 minutes using AccuMAP® denaturation solution (40 μL), AccuMAP® 10X low pH reaction buffer (7 μL), and 100 mM TCEP (2 μL). Denaturation and reduction were performed for 30 minutes. Next, the solution was alkylated in 300 mM IAM (4 μL) for 30 minutes. Pre-digestion was performed on the alkylated substance in water (12 μL), AccuMAP® 10X low pH reaction buffer (5 μL), and AccuMAP® 10X low pH resistant rLys-C solution (25 μL) for 1 hour. After pre-digestion, water (132 μL), AccuMAP® 10X low pH reaction buffer (20 μL), AccuMAP® 10X low pH resistant rLys-C solution (25 μL), and AccuMAP® denatured trypsin solution (20 μL) were added to the pre-digested substance, and digestion was carried out at pH 5.0 for 4 hours or overnight. 20% (v / v) TFA was added to quench the digestion, and the sample was injected into LC-UV-MS for analysis.

[0137] Figures 15A to 15D show the cleavage of the long-chain peptide H350-R436 of BiTE® molecule #1 and the recovery percentage of each peptide. Figure 15A shows the peptide cleavage and the peptide recovery percentage. As shown in Figure 15A, the long-chain peptide H350-R436 of BiTE® molecule #1 was cleaved after Trp361. Surprisingly, relatively high recovery rates (over 20%) were obtained for the two peptides obtained during cleavage: HGNFGNSYISYW (SEQ ID NO: 92) and AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTGNFGNSYISYW (SEQ ID NO: 93). This cleavage site (the C-terminus of Trp361) is the same as that obtained by sequential digestion with trypsin and chymotrypsin, confirming that this digestion method enables the same peptide analysis and attribute monitoring as described in Example 5.

[0138] Example 8 This example demonstrates trypsin digestion, which yields a trypsin peptide with tyrosine at its terminus.

[0139] The unexpected trypsin peptide containing a C-terminal tryptophan residue, as described in Example 7, prompted further analysis. It was hypothesized that trypsin autodigested during storage, and that damaged trypsin products led to the unexpected observations. To mimic storage conditions that would lead to autodigestion, trypsin (Roche, catalog no. 03 708 969 001) was left at room temperature for an extended period before being added to the digestion buffer for digesting the BiTE® molecule. To compensate for the expected decrease in trypsin activity (caused by trypsin autodigestion), a larger amount of trypsin than recommended by the trypsin manufacturer was added to the digestion buffer.

[0140] Surprisingly and unexpectedly, digestion yielded three trypsin peptides, each containing a C-terminal tyrosine: (1) H350-Y357 containing the sequence HGNFGNSY (SEQ ID NO: 108), (2) H350-Y360 containing the sequence HGNFGNSYISY (SEQ ID NO: 109), and (3) H350-Y363 containing the sequence HGNFGNSYISYWAY (SEQ ID NO: 110). Exemplary extracted ion chromatograms of these three peptides are shown in Figures 16A-16C. As shown in these figures, the three peptides were separated at good abundances with high resolution. Since trypsin is known to cleave the C-terminus of Lys and Arg but not Tyr, these peptides were considered non-canonical. Non-canonical trypsin peptides were particularly interesting because they contain asparagine residues that may be deamidated in or near the antigen-binding domain of the BiTE® molecule. Therefore, monitoring these peptides enables quality control monitoring during the manufacturing of BiTE® molecules.

[0141] Example 9 This example demonstrates the evaluation of trypsin levels for peptide digestion.

[0142] The unexpected trypsin digestion yields of the three peptides containing C-terminal tyrosine residues described in Example 8 prompted further analysis. To investigate the increase in the amount of trypsin used in the study in Example 8, various amounts of trypsin were used in this study. In summary, samples containing BiTE® molecules were prepared in solution. Denaturation, alkylation, and reduction of BiTE® molecules were carried out as described in Example 3 and shown in Figure 5A. In summary, the solution containing BiTE® molecules was denatured in 6M guanidine (pH 7.5), and IAA and DTT were added for alkylation and reduction, respectively. Buffer exchange of the denatured, alkylated, and reduced BiTE® molecules into a digestion buffer was performed by gel filtration using an NAP5 cartridge, thereby separating the denatured, alkylated, and reduced BiTE® molecules from guanidine HCl. A certain amount of trypsin (Roche, catalog no. 03 708 969 001) was added to digestion buffer to obtain a specific enzyme:substrate (E:S) ratio. Table 1 summarizes the amount of trypsin added to the digestion buffer to obtain the indicated E:S ratio. The control ratio is indicated by * in Table 1 and represents the minimum and maximum values ​​of the E:S ratio range recommended by the trypsin manufacturer. Each digestion was performed at pH 7.5 at 37°C for 2 hours.

[0143] [Table 9]

[0144] After digestion, an appropriate amount of 20% (v / v) formic acid was added to each digested sample to obtain a final pH greater than 4, and a stock solution of guanidine hydrochloride (pH 5) was added to obtain a final guanidine concentration of 3M. The solutions containing the digested samples were injected directly into the HPLC-MS system for analysis without buffer exchange.

[0145] Exemplary results are provided in Figures 17A–19B. Figures 17A, 18A, and 19A are graphs of the relative abundances of the H350-Y357 peptide (Figure 17A), H350-Y360 peptide (Figure 18A), and H350-Y363 peptide (Figure 19A) obtained when digested at the indicated E:S ratios. Figures 17B, 18B, and 19B are line graphs of the relative abundances plotted as a function of the E:S ratio. The relative abundance of digestion performed at a 1:1(1) ratio was set to 100%. As shown in Figures 17A, 18A, and 19A, an E:S ratio of 1:5 resulted in a relative abundance of at least 20% for peptides with tyrosine at the end. Surprisingly, the relative abundance of the longest peptide (H350-Y363) was over 40%. As shown in Figures 17B, 18B, and 19B, high relative abundances of non-canonical peptides correlated with high E:S ratios. Linear regression R 2 Since the value was above 0.99, this correlation was strong.

[0146] The relative abundances of canonical peptides (H350-R436) using the same E:S ratio are shown in Figures 20A and 20B. As shown in these figures, unlike the strong correlation observed between the relative abundance of non-canonical peptides with tyrosine at the end and the E:S ratio, the relative abundance of canonical trypsin peptides was inversely correlated with the E:S ratio (Figure 20B).

[0147] Example 10 This example demonstrates the evaluation of different trypsin sources.

[0148] To test whether the results described in Example 9 were specific to the particular trypsin used in the digestion buffer, samples containing denatured, alkylated, and reduced BiTE® molecules were digested using a series of digestion buffers containing trypsins from different suppliers. Denaturation, alkylation, and reduction of the BiTE® molecules were carried out as described in Example 9, and digestion was performed in the digestion buffer at 37°C for 2 hours using an E:S ratio of 1.5. Table 2 lists the catalog numbers of the trypsin suppliers and trypsin products used.

[0149] [Table 10]

[0150] After digestion, formic acid and guanidine hydrochloride (pH 5) were added as described in Example 9. The solution containing the digested sample was injected directly into the HPLC-MS system for analysis without buffer exchange.

[0151] Each trypsin product exhibited unique behavior in terms of relative peptide abundance, but all five products consistently produced the desired non-canonical peptides H350-Y357, H350-Y360, and H350-Y363 (Figure 21). Based on these results, each trypsin product was considered suitable for the production of these non-canonical peptides with an E:S ratio of 1:5. However, Roche's trypsin product was selected for later study because, being a recombinant protein product, it had the lowest possibility of contamination by enzymes other than trypsin, and therefore was expected to provide the most information about the behavior of trypsin itself.

[0152] Example 11 This example demonstrates the evaluation of the effect of calcium on the production of non-canonical trypsin peptides.

[0153] Trypsin is known to have up to two calcium-binding sites, as can be seen in the PDB structure 1SGT. The presence of calcium has been demonstrated to affect the activity, structure, and stability of trypsin (Gilliland, G. and Teplyakov, A. (1970), Structural Calcium (Trypsin, Subtilisin), John Wiley & Sons, Ltd, Hoboken, NJ; and Sipos T, Merkel JR. An effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry. 1970 Jul 7;9(14):2766-75. doi:10.1021 / bi00816a003.PMID:5466615).

[0154] To evaluate the effect of calcium binding on the formation of non-canonical peptides, digestion was performed with or without the addition of calcium chloride and EDTA to the digestion buffer. Samples were prepared as described in Example 9, with or without the addition of 0–125 mM calcium chloride or 10 mM EDTA.

[0155] The inclusion of EDTA reduced the overall level of non-canonical peptides by approximately 60% (Figure 22), suggesting that the level of calcium chloride present in the supplier-provided trypsin is beneficial for the production of non-canonical peptides. Further supplementation with calcium chloride (addition of 0–125 mM) did not significantly increase the production of non-canonical peptides (data not shown).

[0156] Example 12 This example demonstrates the evaluation of digestion pH, digestion time, and digestion temperature.

[0157] The samples were prepared as described in Example 9, with the following exceptions: To evaluate the effect of digestion pH, the digestion buffer was adjusted from pH 7.5 to pH 7.0, 8.0, 8.5, or 9.0. To evaluate the effect of digestion time, digestion was allowed to proceed for 4 hours instead of 2 hours. To evaluate the effect of digestion temperature, digestion was performed at 45°C instead of 37°C.

[0158] Although the relative levels of individual peptides differed, the production of non-canonical peptides was found to be very robust, with an E:S ratio of 1:5 (Figure 22). As shown in Figure 22, non-canonical peptides were produced in consistently high abundances across the entire range of pH values, including 7.0, 7.5, 8.0, 8.5, and 9.0. Increasing the digestion temperature from 37°C to 45°C increased the level of non-canonical peptides by approximately 50% (compared to pH 7.5 at 37°C) (Figure 22). Finally, increasing the digestion time was found to increase the overall level of non-canonical trypsin peptides, with 4 hours of digestion resulting in slightly higher abundances than 2 hours of digestion.

[0159] Example 13 This example illustrates a method for monitoring therapeutic polypeptides for PTM formation during manufacturing.

[0160] A solution containing HLE-BiTE® molecules was denatured in 6M guanidine HCl (pH 7.5), and IAA and DTT were added for alkylation and reduction, respectively. Buffer exchange of the denatured, alkylated, and reduced proteins into a digestion buffer was performed by gel filtration using a NAP5 cartridge, thereby separating the denatured, alkylated, and reduced polypeptides (proteins) from guanidine HCl. The eluate containing the denatured, alkylated, and reduced polypeptides was digested with trypsin in 0.1M Tris at pH 7.5 using an E:S ratio of 1:5. After digestion proceeded at 37°C for 2 hours, the solution was quenched with appropriate amounts of 20% (v / v) formic acid and guanidine hydrochloride (pH 5) to obtain a final guanidine concentration of 3M and a final pH greater than 4. The solution containing the digested sample was injected directly into the HPLC-MS system for analysis without buffer exchange.

[0161] After conventional LC-MS analysis, the observed trypsin peptides account for 97-100% of the HLE-BiTE® molecular sequence. Subsequently, deamidation analysis is performed by creating an extracted ion chromatogram and determining the relative levels of H350-Y357, H350-Y360, and H350-Y363 in both the native and deamidated forms. The relative levels of the deamidated species determine the level of deamidation compared to the native peptide. For example, [ka] (Sequence ID 110) The relative abundance [ka] The level of deamidation in N355 is determined by comparing it with the abundance of (SEQ ID NO: 111).

[0162] To evaluate the performance of the above method with the investigational product, samples containing the HLE-BiTE® molecule were subjected to stress for 4 weeks. Samples were analyzed after stress loading at 0 weeks, 2 weeks, and 4 weeks. Deamidation was attempted to be quantified by evaluating the canonical trypsin peptide H350-R436 and the non-canonical peptide H350-Y363. Figure 23A provides the chromatographic results for the canonical peptide at the indicated time point, and Figure 23B provides the chromatographic results for the non-canonical peptide at the indicated time point. In each of these figures, % indicates the percentage of deamidated species. As expected, the percentage of deamidated species increases with increasing stress duration. As shown in Figure 23A, chromatographic performance was reduced for the canonical peptide, resulting in tailing of the chromatographic peaks and consequently inaccurate and unreliable quantification. In contrast, non-canonical peptides can be accurately identified and quantified by chromatography and mass spectrometry to determine the potential deamidation sites N352 and N355 (Figure 23B).

[0163] These data support the ability to monitor the deamidation of the BiTE® molecule in real time during manufacturing. Such a procedure is important because deamidation is a critical quality attribute that can affect patient safety, immunogenicity, and therapeutic efficacy. The canonical trypsin peptide H350-R436 is unusually large (87 amino acids compared to an average of 12 amino acids in the HLE-BiTE® molecule), resulting in poor chromatographic behavior and interference with the mass spectra of both the native and deamidated species. However, the significantly smaller non-canonical peptide provides clear chromatographic peaks and facilitates analysis of its mass spectrum. In addition, improved LC and MS characterization of the non-canonical trypsin peptide enables accurate quantification of deamidation. This allows trypsin peptide mapping analysis to be used for product stability, lot-to-lot comparison, and evaluation of product and process characteristics.

[0164] All references herein, including publications, patent applications, and patents, are incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated herein by reference, and as if it were described in whole herein.

[0165] The terms “a,” “an,” and “the,” and similar reference subjects used in the descriptions of this disclosure (particularly in the claims below), should be interpreted as encompassing both singular and plural unless otherwise indicated herein or unless explicitly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing,” should be interpreted as unrestricted terms including including, but not excluding, the specified components (i.e., “including, but not limited to”) unless otherwise specified.

[0166] The descriptions of value ranges in this specification are intended merely as shorthand for referring to each individual value and its respective endpoint within that range, unless otherwise specified herein, and each individual value and endpoint is incorporated herein as if it were described individually.

[0167] All methods described herein may be performed in any appropriate order, unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any examples or representative language provided herein (e.g., "such as") 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 that any unclaimed element is essential for the practice of the disclosure.

[0168] Preferred embodiments of the Disclosure are described herein, for example, the best modes known to the inventors for carrying out the Disclosure. Variations of these preferred embodiments will be apparent to those skilled in the art by reading the above description. The inventors expect that those skilled in the art will adopt such variations as needed, and the inventors intend that the Disclosure will be carried out in forms other than those specifically described herein. Accordingly, the Disclosure includes all variations and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements is incorporated herein in all possible variations unless otherwise indicated herein or unless it is clearly inconsistent with the context.

Claims

1. A method for processing a polypeptide to produce a digested sample comprising at least one or two peptides, each containing tyrosine at its C-terminus, the method comprising digesting the polypeptide with trypsin in an enzyme:substrate (E:S) weight ratio of about 1:1 to about 1:

15.

2. The method according to claim 1, wherein the weight ratio of E:S is approximately 1:1 to approximately 1:

10.

3. The method according to claim 2, wherein the weight ratio of E:S is approximately 1:2 to approximately 1:

8.

4. The method according to claim 3, wherein the E:S ratio is approximately 1:4 to approximately 1:6, and optionally approximately 1:

5.

5. The method according to any one of claims 1 to 4, wherein the digestion is carried out at a pH of approximately 7.0 to approximately 8.0, and optionally approximately 7.

5.

6. The method according to any one of claims 1 to 5, wherein the digestion is carried out for less than approximately 12 hours, or optionally less than 6 hours.

7. The method according to claim 6, wherein the digestion is carried out for less than approximately 4 hours, or optionally for about 2 hours to less than 4 hours.

8. The method according to any one of claims 1 to 7, wherein the digestion is carried out at a temperature of approximately 35°C to approximately 40°C, and optionally approximately 37°C.

9. The method according to any one of claims 1 to 8, wherein the digestion is carried out in the presence of calcium chloride.

10. The method according to any one of claims 1 to 8, wherein the digestion is carried out in the absence of calcium chloride.

11. The method according to any one of claims 1 to 10, wherein only trypsin is used to digest the polypeptide.

12. The method according to any one of claims 1 to 11, wherein the polypeptide is digested with trypsin in the E:S weight ratio to produce the digested sample containing one or more peptides having tyrosine at the C-terminus.

13. The method according to claim 12, wherein the digested sample comprises a peptide containing the amino acid sequence of HGNFGNSY (SEQ ID NO: 108), a peptide containing the amino acid sequence of HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide containing the amino acid sequence of HGNFGNSYISYWAY (SEQ ID NO: 110).

14. The method according to any one of claims 1 to 13, comprising denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide before digesting the polypeptide with the protease.

15. The method according to any one of claims 1 to 14, comprising buffer exchange before digesting the polypeptide with the protease and after denaturing, reducing, and / or alkylating the polypeptide.

16. The method according to any one of claims 1 to 15, wherein the buffer replacement comprises the use of a size exclusion cartridge, the size exclusion cartridge being a NAP5 cartridge having Sephadex G-25 gel filtration material.

17. The method according to any one of claims 1 to 16, further comprising incubating the digested sample at a weakly acidic pH in the presence of chaotrope.

18. The method according to claim 17, wherein the chaotrope is guanidine hydrochloride.

19. The method according to claim 17 or 18, wherein the pH of the weakly acidic area is approximately 5.

20. The method according to any one of claims 1 to 19, comprising injecting the peptide of the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis.

21. A method for processing polypeptides, a. Digesting the polypeptide with a protease to produce a digested sample containing at least two types of peptides; b. Incubating the digested sample in the presence of chaotrope and / or at a weakly acidic pH. A method that includes [this].

22. A method for increasing the recovery rate of long-chain peptides from a digested sample, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH, wherein the digested sample comprises at least two peptides and is produced by digesting a polypeptide with the protease.

23. A method for increasing the solubility of peptides in a digested sample, comprising incubating the digested sample in the presence of a chaotrope and / or at a weakly acidic pH, wherein the digested sample comprises at least two peptides and is produced by digesting a polypeptide with a protease.

24. A method for processing a polypeptide, optionally comprising digesting the polypeptide with a protease that cleaves the C-terminus of tryptophan to produce a digested sample containing at least two peptides, wherein at least one peptide contains C-terminal tryptophan.

25. A method for monitoring polypeptide attributes, a. Processing the first polypeptide in the first sample obtained at a first time point according to the method described in any one of claims 1 to 24, b. The peptides from the digested sample are injected into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis, and the PTM of the polypeptide in the first sample is identified. c. Processing a second polypeptide in a second sample obtained at a second time point according to the method of any one of claims 1 to 24, wherein the second polypeptide is the same as or different from the first polypeptide. d. The peptides from the digested sample are injected into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis, and the PTM of the polypeptide in the second sample is identified. e. Comparing the PTM of the first sample with the PTM of the second sample. A method that includes this.

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