Peptide ligands for capture of host cell proteins

Abstract

To provide means for removing one or more host cell proteins from a mixture.SOLUTION: Described are compositions and methods for removing one or more host cell proteins from a mixture. Each composition comprises one or more peptides, where each peptide in the composition has a greater binding affinity for the one or more host cell proteins than for one or more target biomolecules.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 784,104, filed December 21, 2018, and U.S. Provisional Patent Application No. 62 / 771,272, filed November 26, 2018, the entire contents of each of which are hereby incorporated by reference in their entirety. Sequence Listing The sequence listing is submitted with this application in electronic format only and is incorporated herein by reference. The sequence listing text submitted at "030871-9075-WO01_As_Filed_Sequence_Listing.txt" was created on November 22, 2019 and is 10,241 bytes in size. Technical Field The present disclosure relates to the development of peptide ligands for the capture of host cell proteins. In particular, the present disclosure relates to the development of peptide ligands for the capture and removal of host cell proteins when they are present in a mixture with a target biomolecule. [Background technology]

[0002] Removal of host cell proteins (HCPs) is a crucial issue in biomanufacturing given their diversity in composition, structure, and abundance, and their occasional structural homology with the product. Although often referred to as a single impurity, HCPs are composed of various species with diverse abundance, size, function, and composition. Current approaches to HCP clearance in monoclonal antibody (mAb) manufacturing rely on product capture with Protein A followed by removal of residual HCPs in flow-through mode using ion exchange or mixed-mode chromatography. However, recent studies have highlighted the presence of “problematic HCP” species that can degrade mAb products or cause immunogenic responses, co-elute with mAbs from Protein A, and escape capture during polishing steps. These “problematic HCP” species compromise product stability and safety even at trace concentrations. Therefore, effective means to improve HCP clearance are needed. Summary of the Invention

[0003] Disclosed herein are compositions, adsorbents and methods for removing one or more host cell proteins from a mixture comprising one or more host cell proteins and one or more target biomolecules. The compositions comprise one or more peptides each independently comprising a sequence selected from the group consisting of GSRYRY (SEQ ID NO: 1), RYYYAI (SEQ ID NO: 2), AAHIYY (SEQ ID NO: 3), IYRIGR (SEQ ID NO: 4), HSKIYK (SEQ ID NO: 5), ADRYGH (SEQ ID NO: 6), DRIYYY (SEQ ID NO: 7), DKQRII (SEQ ID NO: 8), RYYDYG (SEQ ID NO: 9), YRIDRY (SEQ ID NO: 10), HYAI (SEQ ID NO: 11), FRYY (SEQ ID NO: 12), HRRY (SEQ ID NO: 13), RYFF (SEQ ID NO: 14), DKSI (SEQ ID NO: 15), DRNI (SEQ ID NO: 16), HYFD (SEQ ID NO: 17), and YRFD (SEQ ID NO: 18). Each peptide in the composition has a greater binding affinity for one or more host cell proteins than for one or more target biomolecules. [Brief description of the drawings]

[0004] [Figure 1] Figure 1 is a conceptual diagram of a "polyclonal" synthetic HCP binding resin. Highly specific HCP capture is not only possible but the standard approach for HCP quantification by HCP ELISA via polyclonal α-HCP antibodies, as shown on the left. The method currently used involves the generation of synthetic versions of these polyclonal antibodies with identification of HCP-specific peptides, as shown on the right, allowing broad capture of HCPs without the expense and variability introduced by antibody-based ligands. [Diagram 2] Figure 2 is a graph showing the maximum fluorescence intensity (strongest pixel) distribution of fluorescently screened and manually selected tetrameric combinatorial peptide library beads. For each bead imaged, the maximum fluorescence intensity of the IgG fluorophore (Alexa Fluor 488) is plotted against the maximum fluorescence intensity of the HCP fluorophore (Alexa Fluor 594). Beads identified as potential HCP binding ligands are highlighted in the top figure, as determined by the following criteria: IgG maximum fluorescence <2,500, and HCP maximum fluorescence >10,000. [Figure 3A-3B] Figures 3A and 3B are ClonePix 2 fluorescence images of an unbiased combinatorial linear peptide library on ChemMatrix HMBA resin after incubation with fluorescently tagged IgG and CHO-S HCPs. In Figure 3A, the library is imaged with a ClonePix 2 FITC filter to visualize beads bound to IgG tagged with Alexa Fluor 488. Figure 3B shows the same plate imaged with a ClonePix 2 rhodamine filter to visualize beads bound to CHO HCPs tagged with Alexa Fluor 546. [Figure 4]Figure 4 is a graph showing the ClonPix 2 internal average intensity (average bead intensity) distribution of a hexameric combinatorial peptide library screened by ClonePix 2. For each bead imaged, the internal average intensity of the IgG fluorophore (Alexa Fluor 488) is plotted against that of the HCP fluorophore (Alexa Fluor 546). Beads identified as potential HCP binding ligands are highlighted in the top figure, as determined by the following criteria: IgG maximum fluorescence <2,500, and HCP maximum fluorescence >500. [Diagram 5] FIG. 5 is a chart showing the distribution of amino acid residues of lead tetramer HCP-binding peptide candidates identified by solid-phase fluorescence screening manually selected by combination position. [Figure 6] FIG. 6 is a chart showing the distribution of amino acid residues in lead hexameric HCP-binding peptide candidates identified by solid-phase fluorescence screening sorted with ClonePix 2 by combination position. [Figure 7A-7F]Figures 7A, 7B, 7C, 7D, 7E, and 7F are charts showing protein removal by hexameric hydrophobic cathodic and multipolar (6HP and 6MP, respectively) and tetrameric hydrophobic cathodic and multipolar (4HP and 4MP, respectively) lead HCP binding peptide ligands bound to Toyopearl Amino-650M resin in static binding mode in comparison to commercial Capto Adhere and Capto Q resins (N=3 for each condition). Total protein removal was measured by Bradford assay. CHO-K1 host cell proteins removed were measured by Cygnus CHO HCP ELISA,3G assay kit. Monoclonal antibodies removed were measured by Thermo Fisher EasyTiter kit. Each resin was screened under multiple buffer conditions (Figure 7A = pH 6, 20 mM NaCl; Figure 7B = pH 7, 20 mM NaCl; Figure 7C = pH 8, 20 mM NaCl; Figure 7D = pH 6, 150 mM NaCl; Figure 7E = pH 7, 150 mM NaCl; Figure 7F = pH 8, 150 mM NaCl) and two loading conditions: a loading of approximately 5 mg of HCP per ml of resin and a loading of approximately 10 mg of HCP per ml of resin. [Figure 8A-8B] 8A and 8B are tables showing the data presented in FIGS. 7A-F. [Figure 9A-9B] Figures 9A and 9B are bubble plot distributions of HCPs by abundance, theoretical molecular weight, theoretical isoelectric point, and grand average hydropathic index. Figure 9A shows the host cell protein bubble plot distribution of the null CHO-S clarified harvest material used in this study as the fluorescently tagged HCP population for solid-phase peptide library screening. Figure 9B shows the host cell protein bubble plot distribution of the CHO-K1 IgG-producing clarified harvest material used in this study for secondary screening of lead HCP binding ligands by static binding assessment. [Figure 10]Figure 10 is a chart showing resin HCP target binding ratio (TBR) by resin and buffer condition (N=3). HCP TBR is defined as the percent of HCP removed relative to the feedstream divided by the percent of mAb removed relative to the feedstream in static binding mode. In this analysis, HCP TBR>1 indicates preferential binding to HCP compared to IgG, and HCP TBR<1 indicates preferential binding to IgG. [Figure 11] Figure 11 is a bubble plot distribution of CHO HCP species in the mAb production harvest used as loading material by theoretical molecular weight (MW), isoelectric point (pI), grand average hydropathic index (GRAVY), and calculated percent molar abundance. Each data point represents a unique protein identified with a GRAVY value determined using the GRAVY Calculator. With the exception of the GRAVY values, data was obtained from Thermo Proteome Discoverer. [Figure 12A-12D] Figures 12A, 12B, 12C and 12D are charts showing the distribution of CHO HCPs measured in CHO harvest loading material by protein properties: Figure 12A is the theoretical molecular weight, Figure 12B is the theoretical isoelectric point, Figure 12C is the theoretical grand average hydropathic index (GRAVY), which is a measure of relative hydrophobicity, and Figure 12D is the calculated relative molar abundance. [Figure 13]Figure 13 shows the overlap of HCP binding by peptide-based resins (4HP, 6HP, 4MP, and 6MP) and benchmark resins (Capto Q and Capto Adhere) at 20 mM NaCl and 150 mM NaCl at pH 6, pH 7, and pH 8. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting spectral abundance coefficients were significantly lower than the feed by ANOVA (α=0.05). The "overlap", i.e., the number of unique species of bound proteins at multiple pH conditions in the range tested (pH 6, 7, and 8), is shown in the overlap region of the Venn diagram. [Figure 14] Figure 14 shows overlapping HCPs bound by peptide-based resins (4HP, 6HP, 4MP, and 6MP) and benchmark resins (Capto Q and Capto Adhere) at 20 mM, 150 mM, and pH 6, 7, and 8. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting spectral abundance coefficients were significantly lower than the feed by ANOVA (α=0.05). The "overlap", i.e., the number of unique species of bound proteins at both salt concentrations (20 mM and 150 mM) in the range tested (pH 6, 7, and 8), is shown in the overlap region of the Venn diagram. [Figure 15A-15B]Figures 15A and 15B show overlap of proteins bound by peptide resins at pH 7, 20 mM NaCl. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting dilution-adjusted spectral counts were significantly lower than the spectral counts of the feed by ANOVA (α=0.05). Figure 15A compares the number of unique species bound to the novel peptide resins (4HP, 6HP, 4MP, and 6MP) with the Capto Q benchmark resin, and Figure 15B compares the peptide resins with the Capto Adhere benchmark resin. [Figure 16A-16B] Figures 16A and 16B show the overlap of bound proteins by peptide resins at pH 6, 150 mM NaCl. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting dilution-adjusted spectral counts were significantly lower than the spectral counts of the feed by ANOVA (α=0.05). Figure 16A compares the number of unique species bound to the novel peptide resins (4HP, 6HP, 4MP, and 6MP) with the Capto Q benchmark resin, and Figure 16B compares the peptide resins with the Capto Adhere benchmark resin. [Figure 17] Figure 17 is a table showing in tabular form the spectral abundance coefficients and ANOVA for CHO issue HCP with Capto Q and HCP-bound peptide resins at pH 7, 20 mM sodium chloride. The mean and standard deviation of the spectral abundance coefficients (N=3) are reported for each species. The calculated p-values ​​for the ANOVA comparison of each peptide resin compared to Capto Q are provided. [Figure 18]Figure 18 is a table showing in tabular form the spectral abundance coefficients and ANOVA for CHO problem HCP with Capto Adhere and HCP-bound peptide resins at pH 7, 20 mM sodium chloride. The mean and standard deviation of the spectral abundance coefficients (N=3) are reported for each species. The calculated p-values ​​for the ANOVA comparison of each peptide resin compared to Capto Adhere are provided. [Figure 19] Figure 19 is a table showing in tabular form the spectral abundance coefficients and ANOVA for CHO problem HCP with Capto Q and HCP-bound peptide resins at pH 6, 150 mM sodium chloride. The mean and standard deviation of the spectral abundance coefficients (N=3) are reported for each species. The calculated p-values ​​for the ANOVA comparison of each peptide resin compared to Capto Q are provided. [Figure 20] Figure 20 is a table showing in tabular form the spectral abundance coefficients and ANOVA for CHO problem HCP with Capto Adhere and HCP-bound peptide resins at pH 6, 150 mM sodium chloride. The mean and standard deviation of the spectral abundance coefficients (N=3) are reported for each species. The calculated p-values ​​for the ANOVA comparison of each peptide resin compared to Capto Adhere are provided. [Figure 21] FIG. 21 shows the average chromatograms (N=3) of 4MP, 6HP, and 6HP+4MP resin flow-through binding at 280 nm absorbance as a function of retention time. [Figure 22] Figure 22 shows the retention time and concentration of mAb in the flow-through fraction (N=3) with HCP binding resin. The shaded red area indicates the mean mAb concentration ±1 standard deviation of the titrated cell culture harvest feed. [Diagram 23] FIG. 23 shows the cumulative yield of mAb product (N=3) from flow-through binding with HCP selective resin as a function of resin and residence time. [Figure 24] FIG. 24 is an example of a SEC chromatogram of percent main peak, HMW% main peak, and LMW% main peak analysis. [Diagram 25] Figure 25 shows the high molecular weight percent (HMW%) of the main peak (N=3) from flow-through binding with HCP selective resin as a function of resin and residence time. The slope of the solid blue line shows the HMW% measured in each fraction, while the slope of the green line shows the cumulative HMW% calculated to simulate the HMW% of a pool of all fractions. The shaded area shows the HMW% for the main peak ±1 standard deviation in the titrated cell culture harvest feed. [Figure 26] Figure 26 shows the low molecular weight percent (LMW%) of the main peak (N=3) from flow-through binding with HCP selective resin as a function of resin and residence time. The slope of the solid blue line shows the LMW% measured in each fraction, while the slope of the green line shows the cumulative LMW% calculated to simulate the LMW% of a pool of all fractions. The shaded area shows the LMW% for the main peak ±1 standard deviation in the titrated cell culture harvest feed. [Figure 27] Figure 27 shows a table of the Kruskal-Wallis H test for binding protein isoelectric points as a function of buffer salt concentration. The distribution of isoelectric points for each unique binding protein is plotted by isoelectric point frequency, but is not weighted based on abundance. [Fig. 28A-28B] Figures 28A and 28B show the overlap of bound proteins by peptide resins at pH 6, 20 mM NaCl. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting dilution-adjusted spectral counts were significantly lower than the spectral counts of the feed by ANOVA (α=0.05). Figure 28A compares the number of unique species bound to the novel peptide resins (4HP, 6HP, 4MP, and 6MP) with the Capto Q benchmark resin, and Figure 28B compares the peptide resins with the Capto Adhere benchmark resin. [Figure 29A-29B]Figures 29A and 29B show the overlap of bound proteins by peptide resins at pH 8, 20 mM NaCl. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting dilution-adjusted spectral counts were significantly lower than the spectral counts of the feed by ANOVA (α=0.05). Figure 29A compares the number of unique species bound to the novel peptide resins (4HP, 6HP, 4MP, and 6MP) with the Capto Q benchmark resin, and Figure 29B compares the peptide resins with the Capto Adhere benchmark resin. [Fig. 30A-30B] Figures 30A and 30B show overlap of bound proteins by peptide resins at pH 7, 150 mM NaCl. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting dilution-adjusted spectral counts were significantly lower than the spectral counts of the feed by ANOVA (α=0.05). Figure 30A compares the number of unique species bound to the novel peptide resins (4HP, 6HP, 4MP, and 6MP) with the Capto Q benchmark resin, and Figure 30B compares the peptide resins with the Capto Adhere benchmark resin. [Fig. 31A-31B]Figures 31A and 31B show overlap of bound proteins by peptide resins at pH 8, 150 mM NaCl. Bound proteins were determined as proteins identified by LC / MS / MS in the feed but not in the washed supernatant samples after static binding with each resin, or proteins whose resulting dilution-adjusted spectral counts were significantly lower than the spectral counts of the feed by ANOVA (α=0.05). Panel (A) compares the number of unique species bound to the novel peptide resins (4HP, 6HP, 4MP, and 6MP) with the Capto Q benchmark resin, and panel (B) compares the peptide resins with the Capto Adhere benchmark resin. [Diagram 32] Figure 32 shows cumulative % purity (N=3) values ​​versus injected volume (CV) measured by SEC analysis of flow-through fractions generated by injecting clarified CHO-K1 IgG1 production harvest titrated to pH 6 through 4MP-Toyopearl, 6HP-Toyopearl, and 4MP / 6HP-Toyopearl resins at different residence time values ​​(0.5, 1, 2, and 5 min). Cumulative % purity values ​​were calculated using the following formula:

[0005]

number

[0006] Detailed Description Disclosed herein are methods for predicting the affinity of a candidate molecule for a second molecule.

[0007] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials to those described herein may be used to carry out or test the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0008] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0009] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered as disclosing a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses the range "2 to 4". The term "about" may refer to plus or minus 10% of the indicated number. For example, "about 10%" indicates a range of 9% to 11%, and "about 1" may mean a range of 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding, thus, for example, "about 1" may mean 0.5 to 1.4. For the recitation of numerical ranges herein, each intervening number is expressly contemplated with the same degree of precision, for example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0010] 2. Compositions and methods for removing host cell proteins from a mixture A. Composition Disclosed herein are compositions for use in a method for removing one or more host cell proteins from a mixture comprising one or more host cell proteins and one or more target biomolecules. The mixture can be any suitable mixture comprising one or more host cell proteins and one or more target biomolecules. For example, the mixture can be a cell culture medium. For example, the mixture can be a recombinant cell culture medium. In some embodiments, the cell culture medium can be a Chinese Hamster Ovary (CHO) cell culture medium. Other suitable cell culture mediums may be used in accordance with the compositions and methods described. The composition includes one or more peptides, each of which may bind with greater affinity to one or more host cell proteins than to one or more target biomolecules.

[0011] The one or more target biomolecules can be any suitable target biomolecule.For example, the target biomolecule can be a protein, an oligonucleotide, a polynucleotide, a virus or a virus capsid, a cell or a cell organelle, or a small molecule.The protein can be an antibody, an antibody fragment, an antibody-drug conjugate, a drug-antibody fragment conjugate, an Fc fusion protein, a hormone, an anticoagulant, a blood clotting factor, a growth factor, a morphogenetic protein, a therapeutic enzyme, a modified protein scaffold, an interferon, an interleukin, or a cytokine. The one or more host cell proteins can be any host cell protein that one wishes to remove from the mixture and are independently selected from the proteome of a host cell expressing one or more target biomolecules. Examples of host cell proteins include, but are not limited to, acidic ribosomal protein, biglycan, cathepsin, clusterin, heat shock protein, nidogen, peptidyl-prolyl cis-trans isomerase, protein disulfide isomerase, SPARC, thrombospondin-1, vimentin, histones, endoplasmic reticulum chaperone BiP, legumain, serine protease HTRA1, and putative phospholipase B-like protein.

[0012] The one or more peptides each independently comprise a sequence selected from the group consisting of GSRYRY (SEQ ID NO:1), RYYYAI (SEQ ID NO:2), AAHIYY (SEQ ID NO:3), IYRIGR (SEQ ID NO:4), HSKIYK (SEQ ID NO:5), ADRYGH (SEQ ID NO:6), DRIYYY (SEQ ID NO:7), DKQRII (SEQ ID NO:8), RYYDYG (SEQ ID NO:9), YRIDRY (SEQ ID NO:10), HYAI (SEQ ID NO:11), FRYY (SEQ ID NO:12), HRRY (SEQ ID NO:13), RYFF (SEQ ID NO:14), DKSI (SEQ ID NO:15), DRNI (SEQ ID NO:16), HYFD (SEQ ID NO:17), and YRFD (SEQ ID NO:18). One or more of the peptides may further comprise a linker at the C-terminus of the peptide. C-terminal linkers include linkers according to the following structure: Gly n or [Gly-Ser-Gly] m where 6≧n≧1 and 3≧m≧1. The C-terminal linker can be any suitable linker, including, but not limited to, GSG and GGG.

[0013] In some embodiments, each of the one or more peptides comprises a hexameric hydrophobic / positively charged peptide (6HP) comprising about 25%-35% positively charged residues (R, K, H) and 65%-75% hydrophobic (I, A, F, Y) residues. Examples of these peptides include peptides independently comprising a sequence selected from the group consisting of GSRYRY (SEQ ID NO: 1), RYYYAI (SEQ ID NO: 2), AAHIYY (SEQ ID NO: 3), IYRIGR (SEQ ID NO: 4), HSKIYK (SEQ ID NO: 5), GSRYRYGSG (SEQ ID NO: 19), RYYYAIGSG (SEQ ID NO: 20), AAHIYYGSG (SEQ ID NO: 21), IYRIGRGSG (SEQ ID NO: 22), and HSKIYKGSG (SEQ ID NO: 23).

[0014] In another embodiment, each of the one or more peptides comprises a hexameric multipolar peptide (6MP) comprising one positive (R, K, H) and one negative residue (D); and (iii) a hydrogen-bonding and hydrophobic peptide characterized by hydrogen-bonding (Q, S, Y) and hydrophobic (I, A, F, Y) residues. Examples of these peptides include peptides independently comprising a sequence selected from the group consisting of ADRYGH (SEQ ID NO: 6), DRIYYY (SEQ ID NO: 7), DKQRII (SEQ ID NO: 8), RYYDYG (SEQ ID NO: 9), YRIDRY (SEQ ID NO: 10), ADRYGHGSG (SEQ ID NO: 24), DRIYYYGSG (SEQ ID NO: 25), DKQRIIGSG (SEQ ID NO: 26), RYYDYGGSG (SEQ ID NO: 27), and YRIDRYGSG (SEQ ID NO: 28). In another embodiment, each of the one or more peptides comprises a tetrameric hydrophobic / positively charged peptide (4HP) comprising about 25%-35% positively charged residues (R, K, H) and 65%-75% hydrophobic (I, A, F, Y) residues. Examples of these peptides include peptides independently comprising a sequence selected from the group consisting of HYAI (SEQ ID NO:11), FRYY (SEQ ID NO:12), HRRY (SEQ ID NO:13), RYFF (SEQ ID NO:14), HYAIGSG (SEQ ID NO:29), FRYYGSG (SEQ ID NO:30), HRRYGSG (SEQ ID NO:31), and RYFFGSG (SEQ ID NO:32). In another embodiment, each of the one or more peptides comprises a tetrameric multipolar peptide (4MP) comprising a hydrogen-bonding and hydrophobic peptide characterized by one positive (R, K, H) and one negative residue (D); and (iii) hydrogen-bonding (Q, S, Y) and hydrophobic (I, A, F, Y) residues. Examples of these peptides include peptides independently comprising a sequence selected from the group consisting of DKSI (SEQ ID NO: 15), DRNI (SEQ ID NO: 16), HYFD (SEQ ID NO: 17), YRFD (SEQ ID NO: 18), DKSIGSG (SEQ ID NO: 33), DRNIGSG (SEQ ID NO: 34), HYFDGSG (SEQ ID NO: 35), and YRFDGSG (SEQ ID NO: 36).

[0015] Some embodiments include compositions that include one or more peptides from each of the different groups of tetrameric and hexameric hydrophobic or multipolar peptides (4HP), (4MP), (6HP), (6MP), which may be combined in the composition in any number or combination possible from each group. In one non-limiting embodiment, the composition comprises peptides from the 6HP and 4MP groups, where each peptide independently comprises a sequence selected from the group consisting of GSRYRY (SEQ ID NO:11), RYYYAI (SEQ ID NO:2), AAHIYY (SEQ ID NO:3), IYRIGR (SEQ ID NO:4), HSKIYK (SEQ ID NO:5), DKSI (SEQ ID NO:15), DRNI (SEQ ID NO:16), HYFD (SEQ ID NO:17), YRFD (SEQ ID NO:18), GSRYRYGSG (SEQ ID NO:19), RYYYAIGSG (SEQ ID NO:20), AAHIYYGSG (SEQ ID NO:21), IYRIGRGSG (SEQ ID NO:22), HSKIYKGSG (SEQ ID NO:23), DKSIGSG (SEQ ID NO:33), DRNIGSG (SEQ ID NO:34), HYFDGSG (SEQ ID NO:35), and YRFDGSG (SEQ ID NO:36).

[0016] b. Adsorbent Further described herein is an adsorbent comprising the composition as described above, wherein each peptide of the composition is conjugated to a support. The support may include, but is not limited to, particles, beads, plastic surfaces, resins, fibers, and / or membranes. In some embodiments, the support may include microparticles and / or nanoparticles. Each support may be made of any suitable material, including, but not limited to, synthetic or natural polymers, metals, and metal oxides. Some supports may be magnetic microparticles and / or nanoparticles, such as magnetic beads. Suitable synthetic polymers include, but are not limited to, polymethacrylate, polyethersulfone, and polyethylene glycol. Suitable natural polymers include, but are not limited to, cellulose, agarose, and chitosan. Suitable metal oxides include, but are not limited to, iron oxide, silica, titania, and zirconia. Further described herein is an adsorbent comprising the composition as described above conjugated to a support.

[0017] In some embodiments, the adsorbent comprises a single type of support made from a single type of support material, where all peptides in the composition are conjugated to a support formed from a single type of support material. In these embodiments, the composition may comprise one or more different types of peptides, each conjugated to a single type of support made from a single type of support material. In other embodiments, the adsorbent comprises multiple types of supports. Each type of support may be made of the same type of support material or different types of support materials. In these embodiments, the composition may comprise one or more different types of peptides, each conjugated to a different type of support.

[0018] c. Method The methods of the present invention demonstrate improved removal of host cell proteins from a mixture compared to other methods used in the art. Further described herein is a method for removing one or more host cell proteins from a mixture comprising one or more host cell proteins and one or more target biomolecules. The method includes contacting the mixture with a composition or adsorbent described herein. In one embodiment, contact between the composition or adsorbent and the mixture results in binding of one or more host cell proteins to the composition or adsorbent. In this embodiment, the one or more host cell proteins have a higher binding affinity for the composition compared to the one or more target biomolecules. This results in favorable binding of the composition to the one or more host cell proteins compared to the one or more target molecules. The method of the invention may further comprise washing the composition or adsorbent to transfer one or more unbound target biomolecules into a supernatant or mobile phase, and then recovering the supernatant or mobile phase containing the one or more unbound target biomolecules. In one embodiment, a washing step may also be performed after the contacting step and after recovery of the supernatant or mobile phase. According to the method of the present invention, the method can be carried out under any binding conditions suitable for use with the composition or adsorbent, including both static and dynamic binding conditions. In some embodiments, when the method is carried out under static binding conditions, unbound target biomolecules are collected in the supernatant. In some embodiments, when the method is carried out under dynamic binding conditions, unbound target biomolecules are collected in the mobile phase. The method of the present invention can include flow-through chromatography and weak partitioning chromatography.

[0019] The preferred binding affinity of the composition and / or adsorbent for a host cell protein, relative to one or more target molecules, can be altered by changing: the characteristics and concentration of one or more target proteins; the characteristics and concentration of the host cell protein; the composition, concentration, and pH of the mixture; and / or the loading conditions and residence times of the contact and wash steps. Any of these variables may be altered to result in the increased or decreased binding affinity appropriate in accordance with the methods of the invention. According to the method of the present invention, the contacting step may include a high ionic strength binding buffer or a low ionic strength binding buffer. The low ionic strength binding buffer includes a buffer of 1-50 mM NaCl. In one embodiment, the low ionic strength binding buffer includes 20 mM NaCl. The high ionic strength binding buffer includes a buffer of 100-500 mM NaCl. In one embodiment, the low ionic strength binding buffer includes 150 mM NaCl. According to the methods of the present invention, the contacting step may include a low pH buffer of pH 5 to 6.7. According to the methods of the present invention, the contacting step may include a neutral pH buffer of pH 6.8 to 7.4. According to the methods of the present invention, the contacting step may include a high pH buffer, from pH 7.5 to 9.

[0020] In certain embodiments of the invention, the contacting step comprises a binding buffer of neutral pH and low ionic strength, wherein the buffer comprises 20 mM NaCl and has a pH of pH 7, or the contacting step comprises a binding buffer of low pH and high ionic strength, wherein the buffer comprises 150 mM NaCl and has a pH of pH 6. In this embodiment, each peptide may independently comprise a sequence selected from the group consisting of GSRYRYGSG (SEQ ID NO: 19), RYYYAIGSG (SEQ ID NO: 20), AAHIYYGSG (SEQ ID NO: 21), IYRIGRGSG (SEQ ID NO: 22), HSKIYKGSG (SEQ ID NO: 23), DKSIGSG (SEQ ID NO: 33), DRNIGSG (SEQ ID NO: 34), HYFDGSG (SEQ ID NO: 35), and YRFDGSG (SEQ ID NO: 36). EXAMPLES

[0021] 3. Working Example The accompanying examples, as well as partial scope and specific embodiments of the present disclosure, are provided as illustrative and are not intended to limit the scope of the present disclosure.

[0022] Example 1 Design, construction, and screening of solid-phase combinatorial libraries of linear peptides Targeted capture of difficult-to-remove HR-HCPs is a promising strategy to improve product safety and efficacy. To achieve this goal, this disclosure describes the development of a collection of ligands with specific capture capabilities for HCPs in flow-through mode, utilized as next-generation polishing media in the production of mAbs (Figure 1). A single ligand can either limit overall capture due to lack of promiscuous binding, or provide broad specificity such that it also binds the product. As a result, this disclosure describes the identification of multiple ligands with various specificities for different HCP species to strike a balance between yield and breadth of HCP capture.

[0023] Materials: ChemMatrix HMBA resin used for library synthesis was obtained from PCAS BioMatrix (Saint-Jean-sur-Richelieu, Canada) for synthesis and deprotection. Toyopearl AF-Amino-650M resin, triisopropylsilane (TIPS), and 1,2-ethanedithiol (EDT) for secondary screening synthesis were obtained from MilliporeSigma (St. Louis, MO, USA). N',N'-Dimethylformamide (DMF), dichloromethane (DCM), methanol, and N-methyl-2-pyrrolidone (NMP) were obtained from Fisher Chemical (Hampton, NH, USA). Fluorenylmethoxycarbonyl-(Fmoc-) protected amino acids Fmoc-Gly-OH, Fmoc-Ser(But)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Tyr(But)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Glu(OtBu)-OH were obtained from Chem-Impex International (Wood Dale, IL, USA) in addition to 7-azabenzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate (HATU), diisopropylethylamine (DIPEA), piperidine, and trifluoroacetic acid (TFA). Citric acid, acetonitrile, and formic acid for peptide sequencing were from Fisher Chemical (St. Louis, MO, USA), ReproSil-Pur 120 C18-AQ, 3 μm resin was from Dr. Maisch GmbH (Ammerbuch-Entringen, Germany), and 25 cm × 100 μm PicoTip or IntegraFrit emitter columns were from New Objective (Woburn, MA, USA).

[0024] The CHO-S cell line, CD CHO AGT™ medium, CD CHO Feed A, glutamine, Pluronic F68, and anti-aggregants used to generate HCP-containing harvests for fluorescent tagging were manufactured by Life Technologies (Carlsbad, CA, USA). Antifoam C, sodium phosphate (monobasic), and Tween 20 were obtained from MilliporeSigma (St. Louis, MO, USA). Alexa Fluor 488, 594, and 546 NHS activated esters were obtained from ThermoFisher, and sodium chloride, sodium phosphate (dibasic), sodium hydroxide, hydrochloric acid, BisTris, and Tris were obtained from Fisher Chemical (Hampton, NH, USA). Macrosep Advance 3kDa MWCO centrifugation devices were provided by Pall Corporation (Ann Arbor, MN, USA) and Amicon Ultra-0.5ml 3kDa MWCO filters were manufactured by EMD Millipore (St. Louis, MO, USA). Lyophilized polyclonal human IgG was obtained from Athens Research (Athens, GA, USA). CloneMatrix for ClonePix 2 screening was generously provided by Molecular Devices (Sunnyvale, CA, USA). Model mAb-producing CHO-K1 cell culture harvest used for secondary screening was donated by a local biomanufacturing company. Capto Q and Capto Adhere chromatography resins were generously provided by GE Life Sciences (Marlborough, MA, USA). Pierce Coomassie Plus (Bradford) assay kit for protein quantification and Easy-Titer human IgG (H+L) assay kit were obtained from Thermo Fisher (Rockford, IL, USA). CHO HCP ELISA, 3G kit was obtained from Cygnus Technologies (Southport, NC, USA).

[0025] Solid-phase peptide synthesis and deprotection: Solid-phase peptide synthesis (SPPS) was used for the generation of both the U-CLiP library and the identified ligands screened for this study. One bead one peptide (OBOP) libraries for on-bead fluorescent screening were synthesized on ChemMatrix HMBA resin (loading = 0.6 mmol amine per g resin) for the U-CLiP library, and lead ligand candidates for chromatographic screening were synthesized on Toyopearl Amino-650M resin (loading = 0.6 mmol amine per g resin). Synthesis of all resins was performed on a Syro II automated parallel peptide synthesizer (Biotage). 100 mg quantities of resin were swollen in DMF at 40 °C for 20 min using medium speed vortexing. Coupling was performed with a 3-5-fold molar excess of Fmoc-protected amino acids and HATU relative to the reactive sites on the resin, and a 6-fold molar excess of DIPEA solubilized in NMP. Coupling reactions were carried out at 45 °C for 20 min with medium vortexing. Each coupling reaction was performed 3-4 times per cycle before Fmoc deprotection to maximize reaction completion. For deprotection, the resin was first washed 4 times with DMF and then incubated in 20% piperidine at room temperature for 20 min with medium vortexing, followed by an additional wash step as above. All sequences were synthesized with a C-terminal glycine-serine-glycine (GSG) tail that served as a non-reactive spacer between the peptide sequence and the base matrix. Combinatorial tetrameric (X1-X2-X3-X4-GSG) and hexameric (X1-X2-X3-X4-X5-X6-GSG) U-CLiP libraries were synthesized using a split-couple-recombine method. 26The combinatorial libraries were synthesized as one bead one peptide (OBOP) libraries using 1000 mM NaCl. For the tetramer library, the combinatorial positions consisted of equal ratios of isoleucine (I), alanine (A), glycine (G), phenylalanine (F), tyrosine (Y), aspartic acid (D), histidine (H), arginine (R), lysine (K), serine (S), and asparagine (N). The residues selected for the hexameric library were slightly modified by removing F and N and including glutamine (Q) to facilitate synthesis and sequencing. Deprotection of the side chains for both the combinatorial library and the single ligand resin was performed by washing the resin with approximately 10 mL of DMF five times, then washing the resin with approximately 10 mL of DCM, and drying the resin with compressed nitrogen until the resin was dry to a fine powder (3–5 times). Next, a cocktail of 94% TFA, 1% EDT, 3% TIPS, and 2% deionized water was incubated with the resin for 2 h at room temperature on a rotator (6 ml of deprotection cocktail per 100 mg of resin). The resin was first washed 3-5 times with DMF, then with 20% methanol, and stored in 20% methanol at 2-8 °C.

[0026] CHO-S culture and harvest for host cell protein production: Chinese Hamster Ovary (CHO) cell line was selected as a model system to obtain a typical HCP profile found in biopharmaceutical processing. CHO-S cell culture harvest was donated by the Biomanufacturing Training and Education Center (BTEC) at North Carolina State University and cultured following standard procedures for CHO-S wild type (WT) cell line growth and production. Briefly, CHO cell culture bulk fluid (CCBF) was from a null CHO-S cell line grown in CD CHO AGT™ medium containing 4 mM glutamine and 1 g / L Pluronic F68. Cultures were fed daily with 5% CD CHO Feed A from days 3-10. 0.1% anti-flocculant was also added to the culture to prevent cell clumping. Antifoam C was added at 10 ppm to prevent foaming in the bioreactor. CD CHO AGT™ medium contains no protein or peptide components of animal, plant, or synthetic origin, nor does it contain any undefined lysates or hydrolysates. The cell culture process was operated at a pH set at 7.0±0.30, 37.0°C, and 50.0% dissolved oxygen concentration. After production, the CHO-S harvest was clarified by centrifugation at 8,000×g for 30 min. The supernatant was then filtered through a 0.2 μm PES membrane using a VWR Full Assembly Bottle-Top.

[0027] Fluorescent labeling of IgG and CHO-S HCPs: HCPs and IgG were fluorescently labeled with Alexa Fluor NHS esters according to the manufacturer's recommendations. Briefly, clarified harvest of wild-type CHO-S was concentrated to 2.3 g protein / l (approximately 6x) and diafiltered into 50 mM sodium phosphate, 20 mM sodium chloride, pH 8.3 using a Macrosep Advance 3 kDa MWCO centrifugal device. Lyophilized polyclonal human IgG (Athens Research) was dissolved at a concentration of 5 g / l in 50 mM sodium phosphate, 20 mM NaCl, pH 8.3. 1 mg of Alexa Fluor 596 NHS Ester (AF596) or Alexa Fluor 546 NHS Ester (AF546) for HCP solution (based on the instrument used for fluorescence screening) and 1 mg of Alexa Fluor 488 NHS Ester (AF488) for IgG solution were each dissolved in 100 μl of extra-dry DMF, which was immediately combined with 1 ml of diafiltered harvest (HCP-AF596 or HCP-AF546) or IgG (IgG-AF488) and incubated for 1 h at room temperature on a rotator. After incubation, the samples were diafiltered into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 using Amicon Ultra-0.5 ml 3 kDa MWCO filters to remove unreacted AlexaFluor dye.

[0028] Manual and high-throughput fluorescent screening of solid-phase peptide libraries against IgG and CHO-S HCPs: Hexamer or tetramer deprotected libraries were washed three times with 5x the settled resin volume of 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS) to equilibrate. HCP-AF596 or HCP-AF546 and IgG-AF488 were diluted in 50 mM sodium phosphate, 150 mM sodium chloride, 0.2% Tween, pH 7.4 to a final concentration of approximately 1.3 mg / ml IgG-AF488, approximately 0.58 mg / ml HCP-AF546 or HCP-AF596, 50 mM sodium phosphate, 150 mM sodium chloride, 0.1% Tween 20, and mixed with the washed and equilibrated libraries and incubated overnight at 2-8 °C. After incubation, excess protein solution was removed and the resin beads were washed with 50 mM sodium phosphate, 150 mM sodium chloride, 0.1% Tween 20, pH 7.4 (PBS-T). For manual fluorescence screening, the resin was dispensed into a 96-well plate, one bead per well, in 40 μl of PBS-T, and then imaged under a fluorescence microscope. Lead candidate beads were selected based on the highest intensity observed for mCherry after thresholding based on GFP fluorescence.

[0029] To increase throughput, in collaboration with Molecular Devices, Sunnyvale, CA, a ClonePix 2 colony picker was used for fluorescent imaging and higher throughput sorting of HCP-positive and IgG-negative beads. The colony picker was identified as a possible option for increasing throughput due to (1) its ability to rapidly image and quantify the intensity of large numbers of beads, and (2) the size range of ChemMatrix beads, similar to colonies traditionally selected using the ClonePix instrument. After washing as above following library incubation with fluorescently tagged proteins, they were suspended in a semi-solid matrix to accommodate imaging and picking. The semi-solid matrix was prepared from 2 parts Molecular Devices CloneMatrix and 3 parts 83.3 mM sodium phosphate, 250 mM NaCl, 0.17% Tween 20, producing a matrix in buffer conditions similar to the protein binding conditions used. A fixed volume of approximately 5-10 μL of the incubated library was gently incorporated into the matrix solution and evenly dispensed across a 6-well plate to obtain a target bead density of approximately 100-200 beads per well. The plate was then incubated at 37 °C for 2-18 h to allow the matrix to harden. The plate was imaged using the ClonePix FITC (800 ms exposure, 128 LED intensity) and Rhod (500 ms, 128 LED intensity) laser lines to monitor the presence of Alexa Fluor 488 and Alexa Fluor 546, respectively. Due to slight autofluorescence of the ChemMatrix beads under the FITC filter, the position of the beads (i.e., the "primary configuration" of the ClonePix 2 run) was assigned based on the fluorescence intensity from the FITC filter. Using ClonePix 2, beads were selected for further processing based on the following characteristics: FITC internal mean intensity <2500, Rhod internal mean intensity >100, and radius of 0.05-0.25 mm. Picking was performed in suspension mode with an aspiration volume of 20 μL to pick up the beads and a discharge of 60 μL with the excess volume above the aspirated liquid being water.

[0030] Lead peptide sequencing by LC / MS / MS: Fluorescence-based selected beads were sequenced using an LC / MS / MS approach to determine lead peptide candidates for HCP binding. 24 The procedure was performed as described by. Briefly, beads positive for HCP fluorescence and negative for IgG fluorescence were first treated with 20 μL of 0.2 M acetate, pH 3.7 for 1 h to elute bound proteins. The beads were then washed three times with deionized water and incubated with 10 μL of 38 mM sodium hydroxide, 10% v / v acetonitrile to cleave the peptide from the resin. The cleavage solution was then neutralized with 100 mM citrate buffer, 10% v / v acetonitrile and filtered through a fritted pipette tip to remove particles, after which the resulting solute was dried in a speed vacuum. The powder was then resuspended in 0.1% formic acid for injection into the LC / MS / MS.

[0031] A Waters Q-ToF Premier equipped with a nanoAcquity UPLC system with a nanoflow ESI source was used for manually screened tetramer candidates, while a Thermo Orbitrap Elite equipped with a Thermo EASY-nLC 1000 was used for hexameric peptide sequences from the ClonePix 2 screen. Chromatographic separation of peptide samples was performed using 25 cm × 100 μm PicoTip or IntegraFrit emitter columns packed with ReproSil-Pur 120 C18-AQ, 3 μm resin. Samples were loaded as 10–15 μL injections and separated by a 30 min linear gradient of 300 nL / min mobile phase A (0.1% formic acid) and mobile phase B (0.1% formic acid in acetonitrile) from 5–40% mobile phase B.

[0032] For samples sequenced by Orbitrap Elite, MS / MS sequencing was run as follows: MS / MS by positive ion mode, acquisition-full scan (m / z 350-1250), 60,000 resolution, top 5 data-dependent acquisition mode with two fragmentation events at 27 and 35 normalized collision energy (NCE) higher energy collision dissociation (HCD) acquisitions for each precursor investigated. Raw LC-MS data were processed using Proteome Discoverer 1.4.1.14. Searches were performed using MASCOT with a precursor mass tolerance of 50 ppm and a fragment tolerance of 50 ppm against a FASTA-formatted database of all possible peptide species in the combinatorial library. Specific modifications included dynamic modification of each amino acid residue, including side-chain protecting groups during synthesis, accounting for incomplete side-chain deprotection of the library.

[0033] For samples sequenced by Waters Q-ToF Premier, MS / MS sequencing was run as follows: positive ion mode, acquisition-full scan (m / z 400–1990), MS / MS with top 8 acquisition with data-dependent acquisition disabled. The instrument's default collision energy settings based on charge state recognition were used with fragmentation-based scan collision energy. Raw LC-MS data were processed using ProteinLynx Global Server 2.4. Searches were performed using MASCOT with a precursor tolerance of 50 ppm and a fragment tolerance of 50 ppm against a FASTA-formatted database of all possible peptide species in the combinatorial library. In cases where matches with multiple peptides were found for a particular bead, the peptide was assigned based on the lowest expectation value. Cases where this occurred generally consisted of multiple peptides identified with identical composition but different amino acid residue orders, which is likely a result of the difficulty in distinguishing between inverted combination positions in a degenerate library, especially when the likelihood of fragmentation at a particular position is low.

[0034] Static binding of HCPs to chromatography resins: For secondary screening, cell culture harvests demonstrating mAb production from a CHO-K1 wild-type cell line were obtained for use as feed material. After initial concentration by single-pass tangential flow filtration (SPTFF) using Macrosep Advance 3kDa MWCO centrifuge devices, the clarified cell culture fluid was concentrated approximately four-fold (approximately 1.2 mg per ml of host cell protein) to model the expected HCP profile. The concentrated harvest was then diafiltered into the appropriate Bis-Tris or Tris buffer according to the loading conditions. For pH 6 and 7 conditions, 10 mM Bis-Tris buffer was used, for pH 8 condition, 10 mM Tris was used, and "low" and "high" salt buffers composed of 20 mM NaCl and 150 mM NaCl, respectively were used. Lead candidate Toyopearl resins (6HP, 6MP, 4HP, 4MP) were tested along with Capto Q and Capto Adhere, popular commercial resins in the flow-through polishing step of mammalian IgG production. Resins were dispensed into 1 ml solid phase extraction (SPE) tubes at 25 μL of settled resin volume and washed with 3 × 500 μL of the appropriate loading buffer. Resins were then incubated with diafiltered CHO-S harvest on a rotator for 1 h at HCP loadings of approximately 5 and 10 mg HCP per mL of resin and the resulting supernatants were collected. Resins were then washed with 500 μL of loading buffer and wash and flow-through samples were pooled for analysis.

[0035] Quantification of total protein, host cell protein, and IgG removal: Total protein concentrations of pre- and post-treatment samples were measured by Bradford assay using the Pierce Coomassie Plus (Bradford) assay kit (Thermo Fisher, Rockford, IL). IgG concentrations of monoclonal IgG were determined by Thermo Scientific Easy-Titer Human IgG (H+L) assay kit. Relative CHO HCP abundance was monitored using the Cygnus CHO HCP ELISA, 3G kit. Absolute values ​​of HCP concentrations were not determined using this assay because a generic reference standard was used that did not account for the specific cell line or buffer conditions used. To estimate HCP concentrations, observed concentrations were scaled based on the known HCP content in the feedstream using a correction factor for each buffer condition. Percent removal of HCP, IgG, and total protein was calculated as follows:

[0036]

number

[0037] Table of Identification and Relative Quantification of Host Cell Proteins in CHO-S Null Harvest: Proteomic Identification and Quantification of Null CHO-S Clarified Harvest Material Used for Fluorescence Screening of Solid-Phase Combinatorial Peptide Libraries CHO HCP species are tabulated by abundance calculated by intensity-based absolute quantification (iBAQ) as determined by the null CHO-S clarified harvest material used for fluorescence screening of solid-phase combinatorial peptide libraries (Table 1). Concentrated and diafiltered CHO-S harvest and supernatant samples were prepared for proteomic analysis by filter-assisted sample preparation (FASP) using modified tryptic digests. For LC / MS / MS analysis, an EASY-nLC 1000 UPLC coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, San Jose, CA) was used. Chromatographic separation of FASP-digested samples was performed using a 25 cm × 100 μm PicoTip column (New Objective, Woburn, MA) packed with ReproSil-Pur 120 C18-AQ, 3 μm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Samples were loaded as 15 μL injections and proteins were separated by a 120 min linear gradient of 300 nL / min of mobile phase A (0.1% formic acid in 2% acetonitrile) and mobile phase B (0.1% formic acid in acetonitrile) from 5 to 40% mobile phase B. The Orbitrap was operated as follows: positive ion mode, acquisition - full scan (m / z 400-2000) with 60,000 resolution, MS / MS acquisition using top 5 data-dependent acquisition implementing higher energy collision dissociation (HCD) with normalized collision energy (NCE) settings of 35%. Dynamic exclusion was utilized to maximize depth of proteome coverage by minimizing rematching of previously sampled precursor ions. Real-time locked mass correction using the polydimethylcyclosiloxane ion at m / z 445.120025 was utilized to minimize mass measurement errors of precursor and product ions. Raw LC / MS / MS data were processed using Proteome Discoverer 1.4 (Thermo Fisher, San Jose, CA).The search was performed with a precursor tolerance of 10 ppm and a fragment tolerance of 0.01 Da using the Cricetus griseus subset of the UniProtKB / Swiss-Prot database supplemented with sequence data for bovine serum albumin (accession ID P02769). Database search settings were specific for tryptic digestion with a maximum of one missed cleavage. Specific modifications included dynamic Met oxidation and static Cys carbamidomethylation. Identifications were filtered to a strict protein false discovery rate (FDR) of 1% and a relaxed FDR of 5% using the Percolator node in Proteome Discoverer. Based on the sequence of each identified protein, in addition to calculating the molecular weight (MW), the theoretical isoelectric point (pI) and the grand average of hydropathic index (GRAVY) were calculated as models of empirical isoelectric point and hydrophobicity, respectively. GRAVY is an index of hydrophobicity determined as the sum of the contributions of each amino acid in the protein sequence based on the water vapor transfer free energy and the internal and external distribution of amino acid side chains. Negative GRAVY values ​​indicate hydrophilicity and positive values ​​indicate hydrophobicity. GRAVY values ​​were calculated using the GRAVY Calculator developed by Stephan Fuchs at the University of Greifswald. Theoretical pI and MW were calculated using the ExPASy Bioinformatics Resource Portal Compute pI / Mw tool.

[0038] [Table 1] TIFF2025072372000005.tif213156 TIFF2025072372000006.tif203160 TIFF2025072372000007.tif208160 TIFF2025072372000008.tif208165 TIFF2025072372000009.tif203152 TIFF2025072372000010.tif203157 TIFF2025072372000011.tif203157 TIFF2025072372000012.tif208164 TIFF2025072372000013.tif145158

[0039] mAb harvest to support selection of ligands with high HCP binding activity. Approximately 5 μL of fixed ChemMatrix library resin beads were combined with 10 μL of fluorescent protein and incubated overnight at 2-8 °C to ensure saturation of the resin beads. An aliquot of 288 library beads was sampled from the tetramer X1X2X3X4GSG library and plated individually in a 96-well plate. After imaging each bead by fluorescence microscopy, the distribution of maximum fluorescent intensity or most intense pixels was assessed for emission from Alexa Fluor 488 (IgG) compared to Alexa Fluor 594 (HCP), as shown in Figure 2.

[0040] Beads were selected by applying the following criteria: (i) IgG maximum fluorescence <2,500 based on the fluorescence intensity range observed from negative control beads; (ii) HCP maximum fluorescence Library design and synthesis: The OBOP peptide library used in this study was synthesized using a split-couple-recombine method to discover synthetic ligands that bind to target proteins. The library was synthesized on ChemMatrix resin, which provides high peptide purity and can be used to probe protein binding. Considering that the majority of HCPs present in CHO recovered material are hydrophilic and negatively charged in physiological conditions, the amino acid composition was limited to 12 of the 20 natural amino acids for library construction, namely, histidine, arginine, and lysine (positively charged); isoleucine, alanine, and glycine (aliphatic); phenylalanine and / or tyrosine (aromatic), aspartic acid (negatively charged), serine, and asparagine or glutamine (polar). Notably, narrowing the pool of amino acids reduces library size and screening time, and facilitates sequencing. Two libraries were constructed: tetramer X1X2X3X4GSG and hexamer X1X2X3X4X5X6GSG, where X i(where A represents a combination position that any of the selected amino acids may occupy, and GSG is the C-terminal spacer of Gly-Ser-Gly). Hexamers are small synthetic ligands that are useful for pseudoaffinity and low concentration applications. In addition, we determined whether shorter tetrapeptides could be utilized to obtain comparable capacity and specificity at a lower cost. The GSG spacer included in the library sequences was used as an inert spacer arm to facilitate presentation of the combination segments and as a follow-up sequence for LC / MS / MS peptide sequencing due to the frequent occurrence of both the -GSG and -SG y-ion fragments observed. HMBAChemMatrix resin was chosen for this study, whose hydroxymethylbenzoic acid (HMBA) linker allows for on-resin deprotection of the side chain functional groups of amino acid residues prior to library screening; the linker is also alkaline labile, allowing post-screening cleavage of selected peptides from the ChemMatrix beads for eventual sequencing by LC / MS / MS.

[0041] Detection of CHO HCP specificity by manual tetramer library screening and fluorescence detection: During initial screening of the OBOP combinatorial library, we sought to demonstrate the value of simultaneous positive / negative screening by fluorescent labeling to identify HCP-selective peptide binders. Identification of ligands by binding fluorescently labeled targets is beneficial due to the possibility of high-throughput selection and compatibility with simultaneous positive and negative screening. HCP targets have a very wide range of molecular weights. Alexa Fluor fluorescent dyes were selected for their high fluorescence and photostability. Alexa Fluor 488 was used to label IgG and AlexaFluor 594 or 546 was used to label HCPs to minimize emission overlap and ensure instrument compatibility. Labeled proteins were combined at a HCP:IgG ratio of approximately 1:3, higher than the usual >10,000 protein configurations, to include the top 50% of beads with maximum intensity for HCP (one-sided upper tolerance interval of approximately 13,500, α=0.95). The radial fluorescence intensity at each wavelength was also tracked to establish the typical pattern observed for the selected beads, and manual validation of the selected beads to ensure that the maximum fluorescence signal was not the result of an image artifact or bead defect. This resulted in a population of approximately 20% of beads being selected for sequencing.

[0042] ClonePix 2 Hexamer Library Sorting and Detection of CHO HCP Specificity by Fluorescence Detection: The bead sorting criteria defined by manual sorting were implemented using the ClonePix 2 instrument (Molecular Devices, Sunnyvale, CA) to automate the screening of approximately 7,000 beads randomly sampled from the X1X2X3X4X5X6GSG library. For the ClonePix 2 system, bead selection was based on internal mean intensity parameters developed for the ClonePix system, which were approximately equivalent to the mean fluorescence intensity within the range of beads shown in Figure 3A and Figure 3B. Beads were selected based on the following gates: (i) FITC (green) internal mean intensity <2,500; (ii) Rhodamine (red) internal mean intensity >500, representing a similar ratio (approximately 20%) of selected beads to the total beads screened. The bead selection threshold for HCP fluorescence in this case may appear to be significantly lower than that observed in manual screening, but the difference was expected because this system requires a different Alexa Fluor dye (Alexa Fluor 546, which has a lower reported initial brightness compared to Alexa Fluor 594) in addition to differences in imaging exposure and intensity required to visualize the beads. The internal average intensity profile of selected beads is shown in Figure 4.

[0043] Sequencing of HCP-binding ligand candidates: The selected beads were processed for peptide sequencing. First, the isolated beads were thoroughly rinsed with 0.2 M acetate buffer, pH 3.7 to remove all bound proteins. Special attention was paid to the beads selected with the ClonePix 2 device to remove the CloneMatrix used to immobilize the beads for imaging and picking. Next, the beads were individually treated with 38 mM sodium hydroxide, 10% v / v acetonitrile to cleave the ester bond between the GSG spacer and the HMBA linker; to prevent alkaline degradation of the peptides, exposure to the alkaline solution was limited to 10 min, after which the cleavage solution was neutralized with an equal volume of 100 mM citrate buffer, 10% v / v acetonitrile. The cleaved peptides were then reconstituted in 0.1% formic acid in water and sequenced by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS / MS). Peptide sequences were obtained by searching the acquired MS data against a FASTA database of corresponding tetrameric and hexameric peptides using MASCOT (Matrix Science).

[0044] The resulting sequences, listed in Table 2, were grouped into three classes based on consensus of amino acid composition, namely: (i) hydrophobic / positively charged peptides (HP), containing approximately 25%-35% positively charged residues (R, K, H) and 65-75% hydrophobic (I, A, F, Y) residues; (ii) multipolar peptides (MP), containing one positively charged (R, K, H) and one negatively charged residue (D); and (iii) hydrogen-bonding and hydrophobic peptides, featuring hydrogen-bonding (Q, S, Y) and hydrophobic (I, A, F, Y) residues. Table 1 shows the identification and quantification of CHO HCPs. The majority of HCPs have sequence-based isoelectric points <7 and are likely negatively charged under physiological conditions. Thus, the consistent identification of peptides featuring positive amino acids is consistent with the capture of these species via long-range ionic interactions.

[0045] The sequences identified here were sequenced by comparing LC / MS / MS spectra to a FASTA sequence library of all possible peptide sequences in the combinatorial library from combinatorial library beads identified as HCP-positive and IgG-negative solid-phase fluorescence screening studies.

[0046] [Table 2]

[0047] The distribution of amino acids by combinatorial position shown in Figure 5 (tetramers) and Figure 6 (hexamers) reveals that hydrophobic, and especially aromatic, amino acids are preferentially positioned towards the C-terminus. This phenomenon is especially evident in the hexamer sequences and could be attributed to sequence-based peptide-HCP affinity across multiple HCP species, or unexpected biases in the libraries related to higher synthetic yields of the observed sequences. However, the consensus observed within each library and between the two libraries indicates that there is limited bias in bead selection or sequencing introduced during the two screening methods used in this study (manual sorting and ClonePix 2 sorting).

[0048] Secondary screening of HCP-binding ligands by static binding assessment: A set of 18 peptides selected from the groups listed in Table 1 were individually synthesized on Toyopearl Amino-650M resin and mixed with the following single heterogeneous adsorbents: (i) 6HP containing the sequences GSRYRYGSG (SEQ ID NO: 19), RYYYAIGSG (SEQ ID NO: 20), AAHIYYGSG (SEQ ID NO: 21), IYRIGRGSG (SEQ ID NO: 22), HSKIYKGSG (SEQ ID NO: 23); (ii) 6HP containing the sequences ADRYGHGSG (SEQ ID NO: 24), DRIYYYGSG (SEQ ID NO: 25), DKQRIIGSG (SEQ ID NO: 26); (iii) 4HP containing HYAIGSG (SEQ ID NO:29), FRYYGSG (SEQ ID NO:30), HRRYGSG (SEQ ID NO:31), RYFFGSG (SEQ ID NO:32); and (iv) 4MP containing DKSIGSG (SEQ ID NO:33), DRNIGSG (SEQ ID NO:34), HYFDGSG (SEQ ID NO:35), and YRFDGSG (SEQ ID NO:36). The adsorbents were evaluated using a representative IgG-producing CHO-K1 clarified cell culture harvest to validate binding capacity and selectivity by equilibrium binding studies at various binding buffer pH (6, 7, and 8) and salt concentration (20 mM and 150 mM) values; commercially available resins Capto Adhere (CA) and Capto Q (CQ) were used as controls. Percent protein removal of HCP by HCP ELISA, IgG by Easy-Titer assay, and total protein by Bradford assay is shown in Figures 7A-7F (data tabulated in Figures 8A and 8B).

[0049] During evaluation of protein capture across four peptide-based adsorbents, consistently higher binding of total protein, host cell protein, and mAb was observed in low salt conditions compared to high salt conditions, suggesting that, similar to Capto Q and Capto Adhere, ionic interactions play a central role in the binding mechanism. The relevance of electrostatic interactions in peptide-HCP binding was expected given that the majority of HCPs have theoretical isoelectric points much lower than at neutral pH (pI<6, approx. 46%, pI<7, approx. 66%, pI<8, approx. 71%; see Table 1 and Figures 9A-9B for proteomic composition of feedstreams). Furthermore, all species tested in the secondary screen contained at least one positively charged amino acid residue and were screened in Bis-Tris or Tris buffers where positive buffer ions minimally interfere with ionic interactions from positively charged residues.

[0050] At the same time, the dependence of total protein (HCP+IgG) binding on pH was notably different between Capto Q and peptide ligands, suggesting that binding to peptide resins is intrinsically more multimodal and possibly sequence-based than Capto Q. The differences in mAb binding indeed suggest a distinctive binding selectivity of peptides under the conditions tested compared to the Capto Adhere multimodal adsorbent. For both MP and HP resins, binding conditions were identified where the observed HCP removal was comparable to the values ​​given by Capto Q and Capto Adhere resins, while the percentage of mAb loss was below that of Capto Q. Furthermore, Capto Adhere was found to remove substantially more mAbs compared to all other resins, consistently causing a loss of >70% mAb product in all binding conditions. This indicates that library screening with orthogonal fluorescence methods guided peptide selection to sequences that target HCPs with affinity higher than the mixed-mode level. Interestingly, HCP capture was more robust for the tetrameric ligands compared to the hexameric ligands at higher pH regimes (pH 7 and 8), with 40% more HCPs captured by the tetrameric ligands than by the corresponding hexameric peptides, an effect likely a result of the higher binding selectivity exhibited by peptide ligands with longer sequences, narrowing the range of interaction to fewer HCP species.

[0051] As expected, a decreasing percent removal was observed with increasing protein loading across all adsorbents tested, which helped to identify the range in which HCP binding was observable under static binding conditions. Because both loading conditions were incubated for a sufficient time to allow binding equilibrium, a range of loading conditions was screened to ensure that the percentage of captured HCP was measurable in the static binding supernatant. To summarize the specificity of the peptide ligands, the peptide adsorbents were ranked by their HCP target binding ratio (TBR), defined as the ratio of the amount of host cell protein removed to the amount of mAb lost, where HCP TBR<1 indicates preferential binding to mAb and HCP TBR>1 indicates preferential binding to CHO HCP. HCP TBR values ​​by resin and buffer condition are summarized for low loading conditions (5 mg / ml) in Figure 10. Preferential HCP binding by all four peptide adsorbents was observed in most binding buffers tested, except for the pH 8, 150 mM NaCl condition. Given that the mAb concentration in cell culture harvests, as measured in clarified harvests, is at least two orders of magnitude higher than any single host cell protein species, the identified peptides should have much stronger binding to HCPs compared to mAbs. In addition to the reduced HCP TBR observed at pH 7, 150 mM, the preferential binding to IgG observed at pH 8, 150 mM for peptide resin and Capto Q is likely the result of buffer pH conditions close to or above the isoelectric point of the mAb (measured at approximately 7.6) and higher salt concentrations, which minimized the contribution of ionic interactions to binding.

[0052] Multipolar peptides demonstrated excellent specificity for HCPs and proved to be a valuable alternative to current mixed-mode ligands for mAb polishing. In particular, the tetrameric 4MP resin provided the highest HCP TBR of 4.87 (4.868) at pH 7, 20 mM NaCl, more than double that provided by the commercially available Capto Q (2.226). This result was somewhat unexpected, given the lack of multipolar sorbents used in the context of biopharmaceutical purification in the art. Without wishing to be bound by a particular theory, a binding mechanism for multipolar ligands very similar to the dual ion-pairing mechanism proposed for enantio- and stereoselective multipolar ligands is possible, where strong ionic interactions with positively charged amino acids on the ligand are paired with weaker ionic interactions with negatively charged residues such that the protein target remains bound. This mechanism could be applied to other commercially available multimodal resins such as Capto Adhere as well as hydrophobic / positive ligands, except that the dual ion pair interaction mechanism is replaced by other binding mechanisms (π-π bonds, van der Waals interactions, hydrogen bonds, etc.). If the proposed binding mechanism is confirmed, combining these ligands into a "polyclonal" collection would allow for the capture of a more diverse set of HCPs than each set alone.

[0053] Example 2 Capture of specific HCP species with peptide ligands by proteomic analysis Using the same procedures as illustrated and described in Examples 1 and 2, the role of various binding buffers was further evaluated using different methods for relative quantification of individual HCPs. Relative quantification of individual HCPs using method 2: The relative amount of each protein across samples was calculated based on the spectral count (SpC) of each protein in each individual sample multiplied by the sample volume (Cooper et al., 2010). The spectral abundance factor (SAF) of each protein in the collected supernatant sample (combination of the unbound fraction from static binding and subsequent washes) was calculated as shown in the following formula:

[0054]

number

[0055] The relative abundance of each HCP in the feed samples was calculated based on the normalized spectral abundance factor (NSAF) (Neilson et al., 2013) of each identified protein, as shown in the following formula:

[0056]

number

[0057] Comparison of the relative amounts of individual HCPs in the supernatant and feed samples was performed by analysis of variance (ANOVA) of the SAF of each protein in the corresponding samples using JMP Pro 14. For analysis of bound HCPs, the SAF values ​​were used to compare the residual amount of each HCP in the supernatant obtained by static binding of the corresponding feed sample. A "bound HCP" is defined herein as (i) a protein that was identified in the majority of the feed samples (i.e., proteins with a sum of spectral counts >4 in all replicates, N=3) and (ii) a protein that was not found in the supernatant sample or showed a significantly lower spectral count (p<0.05 by ANOVA) compared to the feed sample. Venn diagrams of bound proteins across peptide-based and benchmark resins were created using the Venn diagram add-in in JMP Pro 14. Non-normal distributions of isoelectric points of depleted proteins were compared with Kruskal-Wallis H tests with 90% confidence intervals using JMP Pro 14.

[0058] Analysis of HCP binding. CHO HCP-targeting peptide ligands discovered in a previous study by screening tetrameric (X1X2X3X4GSG) and hexameric (X1X2X3X4X5X6GSG) peptide libraries included multipolar (MP) and hydrophobic / positive (HP) peptides (Lavoie et al., 2019). MP ligands contain sequences with one positively charged amino acid residue (Arg, His, Lys) and one negatively charged (Asp) amino acid residue, with the remaining combination positions filled with aliphatic or aromatic residues. HP ligands contain sequences with one or two positively charged residues, with the remainder being primarily aromatic residues. Initial characterization of these peptide-based adsorbents led to the identification of buffer conditions that maximized the binding specificity of CHO HCP to IgG product (Example 2). To that end, the peptide-based resin was compared with commercially available resins, Capto Q, a strong anion exchange resin featuring quaternary amine ligands, and Capto Adhere, a mixed-mode resin featuring a combination of strong anion exchange, hydrogen bonding, and hydrophobic functions. Binding studies were performed in static binding mode using a series of different binding buffers (NaCl concentrations of 20 or 150 mM; pH 6, 7, or 8). The salt concentrations and pH of the buffers were chosen to evaluate the performance of the resins in "harvest-like" conditions (150 mM NaCl) and "conventional polishing" conditions (20 mM NaCl). The pH range was restricted to 6-8 to prevent protein instability in the clarified harvest. Feed samples were prepared by diafiltration of cell culture fluid against different buffers and incubated with the equilibrated adsorbent for 1 h, and the supernatants (unbound and wash fractions) were collected and pooled before analysis. The majority of the resins yielded the highest selectivity at 20 mM NaCl, pH 7; based on global quantification of HCPs by ELISA, MP resins were found to have equivalent or increased selectivity for HCPs compared to Capto Q and Capto Adhere ( Lavoie et al., 2019 ).The HP resin, although slightly less selective than Capto Q, showed preferential binding to HCPs and was found to be superior to Capto Adhere under the near-neutral pH conditions tested. The peptide-based resin also proved to be more effective than the commercial resin in HCP binding studies performed under "recovery-like" conditions (150 mM NaCl), suggesting its potential use as an HCP clarification agent prior to Protein A. These conditions were not specifically optimized for flow-through operation of the commercial resin; Capto Q is in practice typically operated under low salt conditions, while Capto Adhere is used at significantly lower pH values ​​to prevent binding of mAb products. However, the purpose of this study is to directly compare the peptide-based resin with the commercial resin under comparable buffer conditions to highlight the ability of the peptide ligand to efficiently and selectively capture HCPs without the need for a level of process optimization.

[0059] In this study, HCPs in supernatant samples from static binding experiments were identified and quantified by bottom-up label-free proteomics, and the resulting values ​​were used to assess the differential binding of various HCP groups by peptide-based resins in comparison to a benchmark commercial resin. In this study, "bound HCPs" were defined as proteins that (i) were detected in the feedstream by LC / MS / MS analysis and (ii) were either not detected in the supernatant (unbound + wash) or had a significantly lower SAF compared to the feed sample (p<0.05 by ANOVA).

[0060] Profile of bound HCPs vs. pH of binding buffer. The number of unique HCPs bound to peptide-based resins and the commercial benchmark resin at different pH conditions is shown in Figure 13. Analysis of the overlapping bound HCPs to the various resins as a function of buffer conditions shows that both 4HP and 6HP resins are characterized by a higher tolerance to pH differences compared to the benchmark and MP resins at both salt concentrations (20 mM and 150 mM). As shown in Figure 13, of all unique bound HCPs across the three values ​​of pH, 4HP and 6HP bound 66.2% (198 of 299 unique proteins) and 69.4% (207 of 298), respectively, at 20 mM NaCl, whereas they bound 58.3% (147 of 199) and 54.1% (151 of 279) at 150 mM NaCl. In comparison, the benchmark anion exchange resin Capto Q yielded 60.7% (179 out of 295) at 20 mM and 33.6% (71 out of 211) at 150 mM. The reduced HCP binding by Capto Q at high salt concentrations was expected given that this resin relies solely on electrostatic binding; furthermore, the significant capture of mAb products (isoelectric point of approximately 7.6) by Capto Q at pH 8 also reduces the number of binding sites available for HCP capture (Lavoie et al., 2019). The mixed-mode resin Capto Adhere showed a high overlap of bound HCPs at low salt concentrations (71.4%, 220 out of 308); however, the promiscuous binding of HCPs was also accompanied by a significant loss of mAb products (>80% at all pH conditions) (Lavoie et al., 2019). Analysis of protein binding at 150 mM NaCl showed a 48.2% (133 out of 276 bound proteins) decrease in the overlap of bound HCPs, indicating a low tolerance to pH variations. The ability of the HP resin to maintain nearly constant binding of HCPs under different pH conditions indicates that peptide ligands feature stronger affinity-like binding activity than commercially available mixed-mode ligands, which often require extensive optimization of process conditions to achieve sufficient product yield and purity.The robustness of HCP capture within the design space of buffer conditions by peptide ligands makes them more suitable for platform processes of mAb purification.

[0061] Turning to the multipolar ligands, the 4MP and 6MP resins showed rather striking differences in HCP binding. The 6MP resin compared well with its HP counterpart in terms of robustness of HCP capture to various pH conditions, with an overlap of bound HCPs of 61.2% (180 out of 294) and 51.9% (122 out of 235) at 20 mM and 150 mM, respectively. On the other hand, the 4MP ligand showed a lower tolerance to pH differences at both 20 mM and 150 mM NaCl, with an overlap of bound HCPs of 40.8% (111 out of 272) and 22.0% (41 out of 186), respectively. A unique feature of the 4MP resin was the inverse relationship between HCP binding and buffer pH. The presence of negatively charged amino acids in the 4MP peptide ligand explains the loss of HCP binding at higher pH, since the net charge of proteins in solution shifts to negative values ​​as the pH of the binding buffer increases.

[0062] A comparison of the distribution of pI values ​​in the bound HCPs under different pH conditions was also performed using the Kruskal-Wallis H test to evaluate the shift in the charge profile of HCPs in the supernatant and feed samples. Considering the non-normal distribution of pI values, as shown in the table in Figure 27, the Kruskal-Wallis H test was employed. If HCP binding by peptide-based resins is dominated by electrostatic interactions, the pI profiles of bound HCPs would be significantly different between different pH conditions; in particular, the median pI is expected to increase with increasing binding pH, since HCPs with higher pI values ​​are negatively charged and would be captured by the positively charged HP ligands. Notably, no significant shift in the isoelectric profile of the bound protein was observed for 4HP resin (p=0.171 and p=0.355 for 20 and 150 mM NaCl, respectively), whereas 6HP resin showed a statistically significant shift only at 150 mM NaCl condition (p=0.392 and p=0.0086 for 20 and 150 mM NaCl, respectively), indicating that the HCP:peptide interactions of 4HP and 6HP are not entirely dependent on electrostatic interactions; for comparison, the conventional anion exchange resin Capto Q shows a significant increase in pI as a function of pH at both salt conditions (p=0.0969 at 20 mM and p=0.0434 at 150 mM). Capto Adhere, whose ligands (2-benzyl, 2-hydroxyethyl, 2-methyl-ammonioethyl) have strong similarity to the HP peptide, showed a non-significant response of the pI distribution of bound HCPs to pH at low salt (p=0.240 at 20 mM) but was significant at high salt (p=0.0130 at 150 mM). For multipolar ligands, a significant correlation between pH of binding and pI profile of bound HCPs was observed only for 4MP resin at high salt conditions (p=0.0028). The presence of both positively and negatively charged residues in the MP ligand makes the interaction with HCPs more complex; the softening of electrostatic repulsions at high ionic strength allows the 4MP ligand to behave in a manner closer to a conventional ion exchanger.Taken together, this indicates a stronger correlation between binding pH and the pI profile of bound HCP at higher ionic strength of the binding buffer (150 mM NaCl versus 20, Figure 27). This result arises not only from a shift in HCP:peptide binding strength at different salt concentrations (Tsumoto et al., 2007), but also from a reduction in nonspecific adsorption of the highly abundant mAb product, which increases the availability of binding sites for HCP capture.

[0063] [Table 3]

[0064] Profile of bound proteins versus ionic strength of binding buffer. The overlap of bound HCPs as a function of ionic strength was further evaluated to compare the tolerance of different ligands to salt concentrations. A comparison of HCP binding at 20 mM and 150 mM NaCl concentrations for all resins and binding pH is reported in Figure 14. In particular, proteomic analysis of supernatant samples obtained with peptide-based resins showed a strong tolerance to 150 mM, a typical salt concentration in clarified cell culture harvests. Indeed, when tested at 150 mM NaCl, especially the 4HP and 6HP ligands maintained a significant portion (60.1-82.7%) of the binding of HCPs exhibited at 20 mM NaCl. As expected for ion-exchange resins, Capto Q showed a significant decrease in the number of bound HCPs as the salt concentration increased, resulting in a decrease in the number of overlapping bound proteins. The percent overlap of bound HCPs by Capto Adhere was close to that obtained with HP resin (69.0%-77.3%), but was also associated with significantly higher binding of the mAb product, as shown in Example 2. The multipolar resins 4MP and 6MP showed substantially different binding behavior as a function of salt concentration. Good salt tolerance comparable to HP resin was observed with 6MP resin, which resulted in an overlap of bound HCPs of 52.9%-66.8%. In contrast, 4MP resin showed a low tolerance to salt concentration, similar to that observed in response to pH conditions.

[0065] Profiles of protein binding by peptide-based and commercial resins. A comparison of HCP species bound to various resins at given binding conditions (pH and salt concentration) was then performed to identify proteins uniquely bound to single or combination resins. Our analysis focused on the optimal binding conditions identified in a previous study (Lavoie et al., 2019), namely pH 7 at 20 mM NaCl and pH 6 at 150 mM NaCl, and the results of overlapping protein binding by various resins are shown as Venn diagrams in Figures 15A and 15B and Figures 16A and 16B. Similar plots for other binding conditions are available in Figures 28-31.

[0066] Proteomic analysis of fractions generated at 20 mM NaCl, pH 7 indicates that there is substantial overlap in bound unique proteins between the peptide and benchmark resins. Capto Q in particular resulted in significant binding of 261 unique proteins, only two of which, EF-HAND2-containing proteins and fatty acid-binding protein (adipocytes), were not bound to any of the peptide resins, although to our knowledge, neither of these have been reported as problematic HCPs. On the other hand, the peptide resin showed significant binding of an additional 20 unique HCP species, including group I problematic HCPs (peptidyl-prolyl cis-trans isomerase, fructose-bisphosphate aldolase, sulfated glycoprotein 1, glyceraldehyde 3-phosphate dehydrogenase, and biglycan). From the perspective of overall product purity, group I Protein A co-eluting HCPs are the most difficult to address, as it has been shown that the majority of these proteins co-elute as a result of association with the product (Aboulaich et al., 2014; Levy et al., 2014) or to histones (Mechetner et al., 2011), which in turn may bind non-specifically to multiple entities. Efficient capture of product-bound species in this group may explain to some extent the loss of IgG observed in previous studies (Lavoie et al., 2019), since some IgG molecules may associate with HCPs retained by the HP ligand. Retention of HCPs by the 6HP peptide was comparable to the performance of Capto Adhere, a commercially available mixed-mode ligand with broad and strong HCP binding capacity under these buffer conditions. 6HP showed significant binding of 15 of 20 additional species, but failed to bind one form of peptidyl-prolyl cis-trans isomerase, plus fructose-bisphosphate aldolase, which was captured only by 4MP.

[0067] Compared to the benchmark mixed-mode resin, the peptide resin bound 280 of the 285 unique species bound by Capto Adhere while also showing significantly lower binding (>2-fold) of mAb products. Four HCP species, including the problematic HCP sulfated glycoprotein 1, in addition to tenascin-X, copper transport protein ATOX1, and procollagen C-endopeptidase enhancer 1, were captured by one or more peptide-based resins but showed no binding to Capto Adhere under these conditions. The majority of species bound by Capto Adhere (270 out of 285) were also captured by the 6HP resin; this was expected given the similarity in potential binding interactions between the two resins, despite the significant differences in mAb product binding.

[0068] Parallel analysis of fractions generated at 150 mM NaCl, pH 6 is summarized in Figure 16 and shows considerable differences in host cell protein capture by the Peptide resin and the benchmark resin. As shown in Figure 16A, the Peptide resin was able to capture 100 of the 106 proteins bound by Capto Q, as well as a number of proteins from group I (heat shock cognate proteins, pyruvate kinase, 60S acidic ribosomal protein P0, elongation factor 2, nidogen-1, elongation factor 1-alpha, cofilin-1, oaf protein-like proteins, aldose reductase-related protein 2, peroxiredoxin-1, biglycan, glutathione s-transferase, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase), glutathione s-transferase, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase. It bound to 128 unique proteins, including problematic HCPs in loop I / II (cathepsin B, matrix metalloproteinase-9, matrix metalloproteinase-19, protein disulfide isomerase, serine protease HTRA1), group I / III (glutathione s-transferase), and group III (phospholipase B-like protein, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1, and peroxidoxin-1). The majority of species (117 of 128) that did not bind Capto Q but bound at least one peptide resin also showed binding to the 6HP resin. Notable exceptions include peptidyl-prolyl cis-trans isomerase, which was bound by 4HP and both MP resins, and biglycan, glutathione s-transferase P, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, which were bound only by 4HP. In comparison, only one of the six HCPs bound exclusively by Capto Q, the 60S acidic ribosomal protein P2, is reported as problematic. The overlap of bound HCPs shown in Figure 16B indicates more extensive binding by Capto Adhere compared to Capto Q, and a larger group of bound proteins shared between the peptide resin and Capto Adhere.Nevertheless, the peptide resin, while binding 40 more unique species than Capto Adhere, showed significantly lower binding to the mAb product.

[0069] Semi-quantitative assessment of binding of "problematic" HCPs by peptide and benchmark resins. To gather a quantitative measure of the differences in HCP binding activity of peptide-based resins, label-free relative quantification based on proteomic analysis of collected fractions was performed by LC / MS / MS. Specifically, a data-dependent acquisition (DDA) method was employed to compare the relative SAF of each HCP species in supernatant samples obtained from static binding studies with peptide-based and benchmark resins Capto Q and Capto Adhere shown in Figure 17, Figure 18, Figure 19, and Figure 20.

[0070] This study was limited to supernatant samples obtained at the conditions that proved to be most effective for HCP binding, i.e., 20 mM NaCl at pH 7 and 150 mM NaCl at pH 6 (Lavoie et al., 2019). The SAF values ​​obtained for problematic HCP species identified in supernatants produced at 20 mM NaCl at pH 7 are listed in the tables in Figure 17 and Figure 18. These values ​​of SAF were compared by ANOVA (N=3) between the peptide-based resin and both benchmark resins (Capto Q comparison in Figure 17 and Capto Adhere comparison in Figure 18) to evaluate the benefits of using peptide ligands for HCP removal. Significantly higher binding of several problematic HCP species was observed with the peptide-based resin compared to Capto Q: cathepsin B, serine protease HTRA1, peptidyl-prolyl cis-trans isomerase, and peroxiredoxin-1. 6HP resin was particularly effective in binding serine protease HTRA1, a group I / II HCP, and peroxiredoxin-1, a group I / III HCP, compared to Capto Q, and was superior to its small molecule cognate, Capto Adhere, in binding serine protease HTRA1. 4HP showed improved binding of cathepsin B, a group I / II HCP, compared to both Capto Adhere and Capto Q. In particular, binding of peptidyl-prolyl cis-trans isomerase by both MP resins was significantly higher compared to Capto Q and comparable to Capto Adhere; however, the capture of this difficult-to-remove species by Capto Adhere comes at a much higher cost in terms of mAb loss compared to MP resin. Fructose-bisphosphate aldolase was also observed to be depleted to levels below the detection limit by only 4MP among the peptide resins, although the difference in mean spectral counts was not statistically significant and was consistent only with more product binding to Capto Adhere.

[0071] The development of salt-tolerant stationary phases for mAb purification is highly sought after as they offer flexibility in process implementation. As a result, the binding of HCP species in 150 mM NaCl at pH 6 was analyzed. Total HCP clearance and HCP-to-IgG binding values ​​determined by ELISA testing showed that all four peptide-based resins performed as well as or better than Capto Q in this condition (Lavoie et al., 2019).

[0072] The SAFs of HCP species at 150 mM NaCl for both peptide-based and benchmark resins were calculated and compared with Capto Q in Figure 19 and Capto Adhere in Figure 20. While there was an overall decrease in HCP binding with increasing salt concentration, a notable improvement in capture with the peptide ligand was also observed compared to Capto Q. The HP resin was the most versatile in HCP capture, showing significantly higher binding for the majority of species in this subset compared to the other resins. In particular, 4HP showed significantly lower spectral abundance (higher binding) compared to Capto Q for 21 of the 37 problematic HCPs (group I HCPs heat shock cognate protein; pyruvate kinase; actin, cytoplasmic 1; phosphoglycerate mutase 1; vimentin; clusterin; elongation factor 2; nidogen-1; sulfated glycoprotein 1; glutathione S-transferase P; alpha-enolase; cofilin-1; aldose reductase-related protein; elongation factor I-alpha; group I / II proteins cathepsin B; matrix metalloproteinase-9; matrix metalloproteinase-19; serine protease HTRA1; group II proteins sialidase I; endoplasmic reticulum BiP; and group III proteins phospholipase B-like protein and procollagen-lysine, 2-oxogluarate 5-dioxygenase 1). Furthermore, five of the 37 tracked species bound more effectively to 4HP compared to Capto Adhere (pyruvate kinase, vimentin, clusterin, sulfated glycoprotein 1, and the serine protease HTRA1). The remaining species in both cases showed no significant differences in spectral abundance, and as a consequence, no problematic HCPs were found to be captured more effectively by Capto Q than by 4HP.6HP resin also successfully bound these HCPs compared to Capto Q and showed significantly lower spectral abundances for 22 of the 37 investigated species, including group I HCPs heat shock cognate protein; pyruvate kinase; actin, cytoplasmic 1; phosphoglycerate mutase 1; vimentin; clusterin; elongation factor 2; nidogen-1; sulfated glycoprotein 1; cofilin-1; aldose reductase-related protein; elongation factor I-alpha; group I / II proteins lipoprotein lipase; cathepsin B; matrix metalloproteinase-9; matrix metalloproteinase-19; serine protease HTRA1; group II proteins sialidase I; endoplasmic reticulum BiP; group I / III proteins peroxiredoxin-1; and group III proteins phospholipase B-like protein and procollagen-lysine, 2-oxogluarate 5-dioxygenase 1.

[0073] Compared to Capto Adhere, seven of the 37 species were bound more effectively by 6HP, including heat shock cognate proteins, pyruvate kinase, vimentin, clusterin, phospholipase B-like protein, cofilin-1, and the serine protease HTRA1. Only one HCP, the group I HCP peptidyl-prolyl cis-trans isomerase, showed statistically higher binding to Capto Adhere. Species captured more effectively by 4HP and 6HP compared to the benchmark resins showed good agreement, as expected given the similarity of the peptide functional groups.

[0074] Among peptide-based resins, 4MP showed the lowest improvement in HCP binding compared to Capto Q and Capto Adhere; nevertheless, improved capture of problematic HCPs was observed and was noted to be associated with the lowest mAb product binding, as detailed in a previous study (Lavoie et al., 2019). Thirteen of the 37 species examined showed significantly lower spectral abundance (higher binding) compared to Capto Q, including the group I HCPs pyruvate kinase, vimentin, clusterin, elongation factor 2, nidogen-1, sulfated glycoprotein 1, and elongation factor 1-alpha; the group I / II HCPs cathepsin B and serine protease HTRA1; the group II HCPs sialidase 1 and endoplasmic reticulum BiP; and the group III HCPs phospholipase B-like protein and procollagen-lysine, 2-oxogluarate 5-dioxygenase 1. One HCP, cathepsin D, a group I / II HCP, was bound more effectively by Capto Q than 4MP, but overall, significantly improved binding performance was observed. Capto Adhere binding of problematic HCPs outperformed 4MP for only five, namely heat shock cognate protein, cathepsin B, sulfated glycoprotein 1, phospholipase B-like protein, and endoplasmic reticulum BiP; however, the high mAb product binding observed with this resin would reduce its feasibility of implementation. 4MP outperformed Capto Adhere for a single protein, the serine protease HTRA1, a group I / II HCP. It is noteworthy that although the 4MP resin returned the lowest HCP binding performance, it outperformed the quaternary amine ligand (Capto Q) currently used in depth filter media for removing HCPs in recovery solutions characterized by salt concentrations comparable to those considered here, both in quantitative and qualitative measurements ( Gilgunn et al., 2019 ; Singh et al., 2017 ).

[0075] Finally, 6MP, with the sole exception of pyruvate kinase and lipoprotein lipase, behaved similarly to 6HP in improving the clearance of HCP species compared to Capto Q. No statistically significant differences were observed in the binding of 37 problematic HCPs compared to Capto Adhere; however, significantly lower binding of mAb products was reported, confirming previous findings of improved selectivity compared to Capto Adhere (Lavoie et al., 2019).

[0076] Example 3 Trapping of HCP species by peptide ligands under dynamic binding conditions In this example, the performance of selected peptide resins (4MP, 6HP, and a mixture of peptides from both resins, 6HP+4MP) was evaluated under dynamic binding conditions to further characterize the ability of these resins to remove HCPs from direct application of mAb-produced harvest. In Examples 1-3, pH 6.0, the lowest pH condition tested, demonstrated the most selective clearance of HCPs at salt conditions that most closely simulated those of the harvest. As a result, clarified cell culture harvest titrated to pH 6.0 was used to test these resins under dynamic binding conditions. 4MP and 6HP were selected due to their versatility in capturing HCPs from previous studies (Examples 1-3). Although 6HP was observed to exhibit the highest affinity for mAb products of the peptide resins tested (K p,mAb= 0.96, pH 6, 150 mM conditions), also showed the highest number of unique protein binding. 4MP was included as a candidate for the highest observed HCP selectivity among the resins tested. The resulting impurity profiles, determined by size exclusion chromatography, indicate that in dynamic binding mode, 6HP and 4MP ligands are useful for high yield impurity capture. 4MP was shown to bind more selectively to high molecular weight impurities, while 6HP was more effective at binding low molecular weight impurities. Furthermore, mixing these resins to create 6HP+4MP resin was shown to be as effective at removing both high and low molecular weight impurities as the individual resins.

[0077] Materials. Toyopearl AF-Amino-650M resin for the preparation of peptide resins was obtained from Tosoh Corporation (Tokyo, Japan). Fluorenylmethoxycarbonyl-(Fmoc-) protected amino acids Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Glu(OtBu)-OH, azabenzotriazole tetramethyluronium hexafluorophosphate (HATU), diisopropylethylamine (DIPEA), piperidine, and trifluoroacetic acid (TFA) were obtained from CheMImpex International (Wood Dale, IL, USA). Kaiser test kit, triisopropylsilane (TIPS), and 1,2-ethanedithiol (EDT) were obtained from Millipore Sigma (St. Louis, MO, USA). N,N'-Dimethylformamide (DMF), dichloromethane (DCM), methanol, and N-methyl-2-pyrrolidone (NMP) were obtained from Fisher Chemical (Hampton, NH, USA).

[0078] CHO-K1 mAb-producing clarified cell culture harvest for dynamic binding studies was generously provided by Fujifilm Diosynth Biotechnologies (Durham, NC, USA). Sodium phosphate (monobasic), sodium phosphate (dibasic), hydrochloric acid, sodium hydroxide, BisTris, ethanol, and sodium chloride were obtained from Fisher Scientific (Hampton, NH, USA). Vici Jour PEEK 2.1 mm ID, 30 mm empty chromatography columns, and 10 μm polyethylene frits were obtained from VWR International (Radnor, PA, USA). Yarra 3 μm SEC-2000 300 × 7.8 mm size-exclusion chromatography columns were obtained from Phenomenex Inc. (Torrance, CA, USA). Repligen CaptivA Protein A chromatography resin was generously provided by LigaTrap Technologies (Raleigh, NC, USA).

[0079] Solid-phase peptide synthesis and side-chain deprotection. 6HP peptides RYYYAI-GSG (SEQ ID NO:2), HSKIYK-GSG (SEQ ID NO:5), GSRYRY-GSG (SEQ ID NO:1), IYRIGR-GSG (SEQ ID NO:4), AAHIYY-GSG (SEQ ID NO:3), and 4MP peptides DKSI-GSG (SEQ ID NO:15), DRNI-GSG (SEQ ID NO:16), HYFD-GSG (SEQ ID NO:17), and YRFD-GSG (SEQ ID NO:18) were synthesized via conventional Fmoc / tBu chemistry on a Toyopearl AF-Amino-650M (approximately 0.1 mmol of amine per mL of resin loading, 0.6 mL of loading volume per reaction vial) using a Biotage SyroII automated parallel synthesizer as described in Examples 1-3. Prior to synthesis, Toyopearl resin was swollen in DMF at 40 °C for 20 min. All amino acid couplings were performed by incubating the resin with Fmoc-protected amino acids (3 equivalents relative to the amine functional group density of the resin), HATU (3 eq.), and DIPEA (6 eq.) at 65 °C for 20 min. To ensure complete conjugation, multiple amino acid couplings were repeated at each position; reaction completion was monitored by Kaiser test. Following amino acid conjugation, Fmoc deprotection was performed using 20% ​​v / v piperidine in DMF for 10 min at room temperature, followed by extensive DMF washing; for the 6HP sequence, a second deprotection step using 40% v / v piperidine in DMF for 3 min at room temperature was included for the last two positions. After chain elongation, the peptides were washed with DMF, DCM, and deprotected by acidolysis using a cocktail containing 95% TFA, 3% TIPS, 2% EDT, and 1% water (10 mL per mL of resin) for 2 h at room temperature with gentle agitation. The resin was drained, washed successively with DCM, DMF, and methanol, and stored in 20% v / v aqueous methanol. An aliquot of the peptide-Toyopearl resin was analyzed by Edman degradation to verify the peptide sequence.4MP-Toyopearl resin was prepared by mixing equal amounts of DKSI-GSG-Toyopearl (SEQ ID NO:15), DRNI-GSG-Toyopearl (SEQ ID NO:16), HYFD-GSG-Toyopearl (SEQ ID NO:17), and YRFD-GSG-Toyopearl resin (SEQ ID NO:18); similarly, 6HP-Toyopearl resin was prepared by mixing equal amounts of RYYYAI-GSG-Toyopearl (SEQ ID NO:2), HSKIYK-GSG-Toyopearl (SEQ ID NO:5), GSRYRY-GSG-Toyopearl (SEQ ID NO:1), IYRIGR-GSG-Toyopearl (SEQ ID NO:4), and AAHIYY-GSG-Toyopearl (SEQ ID NO:3); finally, 4MP / 6HP-Toyopearl resin was prepared by mixing equal amounts of all peptide-Toyopearl resins.

[0080] Capture of CHO HCPs in dynamic mode using 4MP-Toyopearl, 6HP-Toyopearl, and 4MP / 6HP-Toyopearl resins. Dynamic binding experiments were performed using an AKTA Pure 25 L FPLC (GE Healthcare Life Sciences, Chicago, IL, USA). A volume of 0.1 mL of 6HP-Toyopearl, 4MP-Toyopearl, and 6HP / 4MP-Toyopearl resins were wet-packed into a Vici Jour PEEK 2.1 mm ID, 30 mm column, washed with 20% v / v ethanol (approximately 10 CV), deionized water (3 CV), and finally equilibrated with 10 mM Bis-Tris buffer supplemented with 150 mM sodium chloride, pH 6.0 (10 CV) at 1.0 mL / min. A volume of 10 mL of clarified CHO-K1 mAb product harvest titrated to pH 6.0 was loaded onto the column at a flow rate of either 0.2 mL / min (residence time, RT: 0.5 min), 0.1 mL / min (RT: 1 min), 0.05 mL / min (RT: 2 min), or 0.02 mL / min (RT: 5 min). Flow-through fractions were collected in 1 mL increments, resulting in 17 fractions per injection. After loading, the column was washed with 20 CV of equilibration buffer at the corresponding flow rate, and pooled wash fractions were collected until the absorbance at 280 nm decreased below 50 mAU. All flow-through runs were performed in triplicate, and the resin was discarded after use (no elution or regeneration was performed).

[0081] Quantification of mAbs in flow-through samples by analytical protein A chromatography (PrAC). mAb concentrations in the titrated harvest and flow-through fractions were determined by analytical protein A chromatography using a Waters Alliance 2690 Separation Module System equipped with a Waters 2487 Dual Absorbance Detector (Waters Corporation, Milford, MA, USA). Repligen CaptivA Protein A resin was packed into a Vici Jour PEEK 2.1 mm ID × 30 mm column (0.1 mL) and equilibrated with PBS pH 7.4. A volume of 10 μL was injected for each sample or standard, and the analytical method proceeded as outlined in Table 4. The eluate was monitored by absorbance at 280 nm (A280) and concentrations were determined based on the peak area of ​​the A280 elution peak. A standard curve was generated using 0.1, 0.5, 1.0, 2.5, and 5.0 mg / mL of pure mAb.

[0082] [Table 4] To assess the recovery of the mAb product, the pooled yield as a function of CV was calculated using the following formula:

[0083]

number

[0084] Quantification of low molecular weight (LMW) and high molecular weight (HMW) HCPs in the flow-through fraction by size-exclusion chromatography (SEC). Next, the flow-through fraction was analyzed by analytical SEC using a Yarra 3μm SEC-2000 300mm×7.8mm column operated in an isocratic mode for 40 minutes using PBS at pH 7.4 as the mobile phase. A 50 μL volume of sample was injected and the eluate was continuously monitored by UV spectroscopy at an absorbance of 280 nm (A280). The values of the relative abundance of HWM and LMW HCPs in the flow-through fraction were calculated as % of the main peak. First, the total integrated area of all peaks was calculated; next, based on the retention time relative to the main product peak of approximately 150 kDa determined using a standard molecular weight ladder, the integrated peak area was divided into three sections (Figure 24); the HMW and LMW peak areas were defined as the integrated area of all peaks at retention times shorter and longer than the retention time of the main peak, respectively; peaks associated with ultra-low molecular weight impurities (MW < 10 kDa) were excluded from the LMW region; finally, the values of "HMW% of main peak" and "LMW% of main peak" were calculated using the following equations.

[0085] [Number] TIFF2025072372000021.tif11150where A メイン , A HMW , and A HMW are the integrated main area of 150 kDa (corresponding to mAb), the high molecular weight peak area (MW > 150 kDa), and the low molecular weight peak area (10 kDa < MW < 150 kDa), respectively. The cumulative HMW% and LMW% of the main peak were calculated using the following equations.

[0086] [Number] TIFF2025072372000023.tif13150where HMW% 累積,fis the cumulative HMW% in fraction f, A HMW,i is the HMW peak area in the i-th fraction, A LMW,i is the LMW peak area in the i-th fraction, and A mAb,i is the main peak area of ​​the i-th fraction. Finally, the cumulative mAb purity was calculated using the following formula:

[0087]

number

[0088] Proteomic analysis of flow-through fractions by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Feed and flow-through samples were first processed by filter-assisted sample preparation (FASP) using a modified trypsin digestion method adapted from the work of Wisniewski et al. (Wisniewski et al., 2009). Briefly, 30 μL of the flow-through sample was denatured in 5 mM dithiothreitol at 56 °C for 30 min, washed twice with 8 M urea and once with 0.1 M Tris-HCl buffer in a 3 kDa MWCO Amicon Ultra 0.5 mL spin filter (EMD Millipore, Darmstadt, Germany), and alkylated with 0.05 M iodoacetamide for 20 min at room temperature. Samples were washed again with 8 M urea, 0.1 M Tris-HCl, 50 mM ammonium bicarbonate, and finally trypsinized overnight at 37 °C using 15 μg / mL sequencing-grade modified trypsin at a trypsin:protein ratio of approximately 1:100. After trypsinization, samples were washed again with 50 mM ammonium bicarbonate, evaporated to dryness in a SpeedVac, reconstituted in 1 mL of 2% aqueous acetonitrile, 0.1% formic acid (mobile phase A), and then further diluted 1:5 with mobile phase A before injection. Proteomic analysis using nanoLC-MS / MS was performed at the Molecular Education, Technology, Research and Innovation Center (METRIC) at North Carolina State University. Samples were loaded as 2 μL injections, and proteins were separated using a 60 min linear gradient of 300 nL / min of mobile phase A and mobile phase B (0.1% formic acid in acetonitrile) from 0 to 40% mobile phase B. The Orbitrap operational parameters were: (i) positive ion mode, (ii) full scan (m / z 400–1400) at a resolution of 120,000 in acquire-MS mode, (iii) MS / MS acquisition using the top 20 data-dependent acquisition implementing higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) setting of 27%; dynamic exclusion was employed to minimize rematching of previously sampled precursor ions.The resulting nanoLC-MS / MS data were processed using Proteome Discoverer 2.2 (Thermo Fisher, San Jose, CA) by performing a search against the Cricetulus griseus (Chinese hamster) CHOgenome / EMBL database with a precursor tolerance of 5 ppm and a fragment tolerance of 0.02 Da. Database search settings were specific for trypsin digestion and included modifications such as dynamic Met oxidation and static Cys carbamidomethylation. Identifications were filtered to a strict protein false discovery rate (FDR) of 1% and a relaxed FDR of 5% using the Percolator node in Proteome Discoverer.

[0089] Relative quantification of individual HCPs and bound protein analysis. Relative quantification of HCPs in the flow-through samples was obtained from MS-derived spectral counts (SpC) of each HCP (Cooper et al., 2010). The percent removal of individual proteins in the collected supernatant samples (combination of the unbound fraction from static binding and subsequent washes) was calculated as shown in the following formula:

[0090]

number

[0091] For analysis of bound HCPs, protein spectral counts were used to compare flow-through fractions obtained with 4MP-Toyopearl, 6HP-Toyopearl, and 4MP / 6HP-Toyopearl resins. "Bound HCPs" are defined herein as (i) proteins that were identified in the majority of feed samples (i.e., proteins with sum of spectral counts >4 in all experiments, N=3) and (ii) proteins that were not found in the supernatant samples or showed significantly lower spectral counts (p<0.05 by ANOVA) compared to the feed samples. Venn diagrams of bound proteins across peptide-based and benchmark resins were created using the Venn diagram add-in in JMP Pro 14 (Figures 28-31). Non-normal distributions of isoelectric points of depleted proteins were compared with Kruskal-Wallis H-tests with 90% confidence intervals using JMP Pro 14.

[0092] HCP-selective peptide resin in dynamic binding mode. HCP target peptides 6HP (GSRYRYGSG (SEQ ID NO: 19), HSKIYKGSG (SEQ ID NO: 23), IYRIGRGSG (SEQ ID NO: 22), AAHIYYGSG (SEQ ID NO: 21), and RYYYAIGSG (SEQ ID NO: 20)) and 4MP (YRFDGSG (SEQ ID NO: 36), DKSIGSG (SEQ ID NO: 33), DRNIGSG (SEQ ID NO: 34), and HYFDGSG (SEQ ID NO: 35)) were synthesized individually on Toyopearl AF-Amino-650M resin as described in Examples 2-3. The resulting resins were mixed in equal amounts to generate adsorbents (i) 6HP-Toyopearl resin containing five 6HP peptides, (ii) 4MP-Toyopearl resin containing four 4MP peptides, and (iii) 6HP+4MP-Toyopearl resin containing all nine peptides. The three sorbents were packed into 0.1 mL columns and equilibrated with 10 mM Bis-Tris supplemented with 150 mM sodium chloride, pH 6.0. A volume of 10 mL of clarified CHO-K1 IgG1 production harvest (approximately 1.7 g total protein per L and approximately 1.4 mg / mL mAb) was loaded onto the column at various residence times (0.5, 1, 2, and 5 min), resulting in a total protein loading of approximately 170 mg protein per mL of resin. The eluate was continuously monitored by UV spectroscopy at 280 nm and collected in incremental fractions of 1 mL. The resulting chromatograms (Figure 21) show no noticeable differences; when HCP species are present in low abundance compared to the mAb product (approximately 1:5 HCP:IgG), the A280 signal of the eluate is mainly determined by the mAb.

[0093] mAb Binding and mAb Product Yield. Binding of mAb products to peptide resins was monitored for this study to assess possible product loss. The mAb concentrations in each fraction and feed, as determined by analytical Protein A chromatography, are reported in Figure 22. When examining the mAb concentration for each resin, higher concentrations of mAb were observed compared to the feed concentration, which corresponds to the stabilization of the A280 dynamic binding chromatograms shown in Figure 21. This effect was particularly pronounced for the 6HP and 6HP+4MP resins, where the increase in maximum concentration for each resin correlates with the increase in residence time. In Examples 1-3, higher mAb product binding was observed in static binding mode for 6HP resin compared to 4MP resin, and a larger fraction of the feed HCP bound to the peptide resin compared to the mAb product was further noted. Given the stronger binding of mAb by 6HP resin, the observed increase in concentration is likely a result of partitioning. This is supported by previous studies in static binding mode (Examples 1-3), where the K of mAb products for 6HP was significantly higher than that of 6HP. p 4MP 69 (At pH 6 and 150 mM sodium chloride, K p,mAb = 0.96 compared to 0.75 for 4MP). The higher observed affinity of 6HP for the mAb product likely corresponds to a larger fraction of mAb bound at low loading in the dynamic binding mode. This increase in binding is an order of magnitude higher than the HCP K p (K for 4MP and 6HP at pH 6 and 150 mM NaCl under static binding conditions, respectively) p,HCP = 7.3 and 6.1), which may explain this trend. Upon harvest loading, highly abundant mAb molecules weakly bind and saturate the ligand, so that upon introduction of more harvest, the higher affinity HCP displaces the weakly bound mAb, resulting in the observed increased mAb concentration. This indicates that these ligands are optimally worked with WPC for direct application of titrated harvest.

[0094] To assess recovery of mAb product, the pooled yield as a function of loading was calculated as shown in the following formula for comparison with the residence times and resins shown in Figure 23. Note that the calculated pooled yield does not take into account the column wash.

[0095]

number

[0096] For the conditions tested, all resins exceeded 80% mAb product yield with a loading of 120 mg total protein / mL, an approximate loading where the mAb fraction concentration falls to the feed concentration. This observation, coupled with the improvement in yield observed with increasing residence time, further supports a weak partitioning of the loaded protein. For residence times of 1, 2, and 5 min, the pooled yield was greater than 90% for all resins with 200 mg / mL, the highest loading tested.

[0097] Clearance of high - and low - molecular weight impurities by HCP - selective peptide resins. Also, the titrated feed and flow - through fractions were analyzed by size - exclusion chromatography (SEC) to derive a qualitative correlation between the clearance of high - molecular weight (MW>150 kDa) and low - molecular weight (10 kDa<MW<150 kDa) HCPs and the type of ligand, protein loading, and residence time. Next, as summarized in Figure 24, the total area under all the signals observed in the relevant range of the protein was determined, and subsequently, the absorbance chromatogram of the results monitored at 280 nm was interpreted by separating the integration region into three distinct regions: (i) high - molecular weight (HMW), (ii) main peak (IgG), and (iii) low - molecular weight (LMW). This allowed the inventors to obtain a preliminary understanding of the conditions for optimizing the clearance of high - and low - molecular weight HCP impurities. For this purpose, the chromatogram was divided into three regions: (i) high - molecular weight (HMW, SEC retention time<12.8 minutes), (ii) main peak (mAb product and potential HCPs having a similar hydrodynamic radius), and (iii) low - molecular weight (LMW, SEC retention time 13.6 - 20 minutes). Using the integrated chromatogram areas corresponding to these regions, the fraction ratios and cumulative ratios of HMW:main peak area, i.e., "HMW%", and LMW:main peak area, i.e., "LMW%", were calculated using the equations outlined above and compared between different resins, loading amounts, and residence times. Figures 25 and 26 report the values of the HMW% and LMW% results of the fractions (solid lines) and cumulative (dashed lines) versus loading CV obtained at different residence times using 4MP - Toyopearl, 6HP - Toyopearl, and 4MP / 6HP - Toyopearl resins, respectively. The plots of the cumulative HMW% and LMW% of the main peak represent the simulated HMW% and LMW% obtained by pooling the flow - through fractions.

[0098] A relatively slow increase in flow-through HMW% as the loading of harvest onto the resin proceeded was consistently observed across all residence times, indicating that the peptide-based resin has high binding strength and capacity for HMW HCPs. Notably, when operated at a residence time of 5 min, the 4MP-Toyopearl resin provided highly effective capture of HMW HCPs, reaching a cumulative HMW% of 5.8% with 84% mAb yield at the loading cut-off value (60 CV, corresponding to a loading of approximately 102 mg of protein per mL of resin); this corresponds to a capture of 70% of the fed HMW HCPs. At maximum loading (10 CV or 170 mg / mL loading) with 91% mAb yield, a 9.6% HMW% was observed, corresponding to a removal of 51% of the fed HMW HCPs. In contrast, the 6HP-Toyopearl resin operated at a 5 min residence time provided only 8.0% HMW% at 60 CV cut-off loading, corresponding to 59% removal of HMW HCPs, and 11.8% HMW% at maximum loading, corresponding to 11.8% HMW HCP removal.

[0099] Most notably, the combined 4MP / 6HP-Toyopearl resins provided a remarkable 2-4 fold reduction of HMW species during the early stages of loading (10-30 CV), while at cut-off loading, an HMW% of 6.5% was obtained, corresponding to removal of 65% of the HMW HCPs in the feed, and at maximum loading, 10.9%, corresponding to removal of 44%. This indicates that the 4MP- and 6HP-Toyopearl resins target different HMW HCPs and should be operated together to perform mAb purification in flow-through mode. At a residence time of 1 min, technically relevant operating conditions, the HMW% at cut-off loading was approximately 10% for 4MP-Toyopearl and 6HP / 4MP-Toyopearl resins, corresponding to a capture of 49% of the HMW HCPs fed, and 12.4% for 6HP-Toyopearl, corresponding to a capture of 36.4%; at maximum loading, instead, the HMW% increased to 12.5% ​​and 13.2% for 4MP-Toyopearl and 6HP / 4MP-Toyopearl resins, corresponding to a removal of 36% and 32% of the HMW HCPs fed, compared to 14.7% (25% removed) for 6HP alone. Taken together, these results indicate a cooperative effect in HCP binding by 4MP and 6HP peptides. This confirms previous studies on HCP capture by peptide ligands (Examples 1-3) that showed that the populations of HCPs bound by the two groups of peptides overlap to some extent, but also contained many species that were uniquely captured by 4MP and 6HP.

[0100] The corresponding analysis of LMW HCPs showed the opposite trend compared to that of HMW HCPs, with 6HP and the combined 6HP / 4MP ligands showing higher binding strength and capacity compared to the 4MP ligand. The 4MP-Toyopearl resin indeed showed low clearance of LMW HCPs, with capture of <25% of the supplied protein at loadings above 60 CV, where values ​​of mAb yield would be industrially realistic (>80%) over all residence times. On the other hand, the 6HP-Toyopearl and 6HP / 4MP-Toyopearl resins captured about 37% of the supplied LMW HCPs at the loading cut-off value (60 CV, corresponding to mAb yield >80%) and 25% at maximum loading (100 CV, mAb yield >90%) when operated with a residence time of 5 min; instead, 29% and 34% capture, respectively, was obtained at the loading cut-off value and about 18% capture at maximum loading when operated with a residence time of 5 min. Improved clearance of LMW species was consistently observed when operating at higher residence times, especially for 6HP-Toyopearl and 6HP / 4MP-Toyopearl resins. As noted above (Examples 1-3), previous studies in static binding mode showed substantial differences in binding of individual HCPs by different resins, which supports the observed differences in both %HMW and %LMW to main peak trends between the two ligand sets. Proteomic analysis of cell culture harvests indicates that species with MW<100 kDa make up the majority of the HCP population, suggesting that clearance of total HCPs may depend on resins with high binding strength and capacity for LMW species. Under this premise, the above results are consistent with previous data generated in static binding mode in Examples 2-3, where statistically significant clearance of a larger number of unique HCPs was observed with 6HP resin compared to 4MP.

[0101] To easily compare the purification performance of peptide-based resins, mAb purity values ​​of the flow-through fractions were calculated using the following formula:

[0102]

number

[0103] Proteomic analysis of flow-through fractions. Global HCP removal values ​​represent only one aspect of the purification activity enabled by the 4MP and 6HP ligands. Indeed, previous studies in static binding mode have demonstrated the ability of these ligands to remove "problematic" HCPs, i.e. species that co-elute with mAb products from Protein A columns (group I), species that cause mAb degradation (group II), and species that have been reported to be highly immunogenic (group III). Targeting and removing these species as early as possible in the purification process holds great promise for enhancing product safety and improving downstream bioprocessing performance.

[0104] To assess the binding of individual HCPs by peptide-based resins, the relative abundance of each species was measured by LC / MS / MS-based proteomic analysis and compared to that of the feedstream by analysis of variance (ANOVA). The method of qualitative bound protein analysis utilized in this study is described in detail in Examples 1-3. Briefly, an HCP is considered bound if (i) it is identified in the feed but not in the flow-through, or (ii) the spectral abundance coefficient (a measure of relative concentration calculated using the formula below) measured in the flow-through sample is statistically lower (α≦0.05 by ANOVA) compared to the spectral abundance in the feed.

[0105]

number

[0106] Figure 33 compares the total number of HCPs captured by the 6HP / 4MP-Toyopearl resin at various loading values ​​(CV) at 1 min RT out of 661 species identified in the feedstream. As expected, the highest number of bound proteins was observed at the lowest loading condition tested (40 CV), with a total of 292 bound proteins, representing approximately 44% of all species identified in the feedstream. A cut-off loading of 60 CV showed that 169 HCP species (approximately 26%) were captured by the 6HP / 4MP ligand. A total of 114 HCP species (approximately 17% of species identified in the feed) were observed to bind at all loading conditions, indicating strong binding to the peptide ligand. Most notably, a notable number of known "problematic" HCP species identified in Examples 2-3 were included in this set of 114 high-binding species, as summarized in Table 5.

[0107] Analysis of bound HCP was repeated for fractions generated at 2 min RT, as shown in Figure 34. A slight decrease in the number of bound proteins was observed at 40 CV loading, 283 bound species at 2 min RT compared to 292 bound species at 1 min RT, which may be attributed to slight variation in results. On the other hand, a significant increase in the number of bound species was observed at 60 CV cut-off loading, 215 bound species at 2 min RT (33%) compared to 169 bound at 1 min RT. This increase in bound HCP is consistent with the increase in mAb purity at higher retention times as shown by both SEC and ELISA analysis. At 2 min RT, 117 HCP species were observed to be bound in all four loading conditions, as well as 114 bound at 1 min RT.

[0108] The ability of the 6HP / 4MP peptides to capture a significant portion of the HCPs present in the feedstream is highly remarkable from a thermodynamic standpoint. These proteins are present individually at concentrations ranging from 0.1 to 1 μg / mL, so the molar concentrations are likely 1 to 10 nM. At the same time, the antibodies are present at a concentration of about 1.4 mg / mL, which corresponds to a concentration of about 10 μM. The ability of the peptides to selectively capture HCPs without adjusting the protein concentration or the salt composition, concentration, and pH in the feed is therefore remarkable.

[0109] [Table 5]

[0110] The "problematic" HCP species captured under all four loading conditions are summarized in Table 5. Proteomic analysis showed that 23 HCPs known to be "problematic" due to their ability to evade Protein A purification, or to degrade mAbs by direct proteolytic activity or by degrading stabilizers during storage, or due to their demonstrated high immunogenicity, were effectively captured by the 4MP / 6HP-Toyopearl resin across all values ​​of loading (CV) and residence time. Of particular note is the capture of cathepsins B and D, which have been implicated in mAb degradation via heavy chain C-terminal fragmentation leading to the formation of mAb aggregates, the serine proteases HTRA1 and protein disulfide isomerase A6, both degradative HCPs found in Protein A eluates, putative phospholipase B-like 2, a potent immunogen, and legumain, a potent protease that forms acidic charge variants by deamidating asparagine residues in mAbs.

[0111] The results of this example demonstrate that the peptide-based resin of the present invention enables antibody purification in flow-through mode by combining selective capture of high and low molecular weight HCP impurities with high product yield. When used individually, the 6HP and 4MP ligands function to preferentially capture HCP species in the LMW and HMW regions, respectively. When combined, the peptide ligand ensemble results in a significant reduction in HCP levels in the cell culture harvest while still providing good product yields. Notably, at a 60 CV cut-off loading (~102 mg / mL), a ~36% reduction in LMW% and ~50% reduction in HMW% was obtained in combination with ~85% mAb yield when run at a 1 min residence time.

[0112] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof.

Claims

1. 1. A composition for use in a method of removing one or more host cell proteins from a mixture comprising one or more host cell proteins and one or more target biomolecules, the composition comprising one or more peptides each independently comprising a sequence selected from the group consisting of GSRYRY (SEQ ID NO:1), RYYYAI (SEQ ID NO:2), AAHIYY (SEQ ID NO:3), IYRIGR (SEQ ID NO:4), HSKIYK (SEQ ID NO:5), ADRYGH (SEQ ID NO:6), DRIYYY (SEQ ID NO:7), DKQRII (SEQ ID NO:8), RYYDYG (SEQ ID NO:9), YRIDRY (SEQ ID NO:10), HYAI (SEQ ID NO:11), FRYY (SEQ ID NO:12), HRRY (SEQ ID NO:13), RYFF (SEQ ID NO:14), DKSI (SEQ ID NO:15), DRNI (SEQ ID NO:16), HYFD (SEQ ID NO:17), and YRFD (SEQ ID NO:18); A composition, wherein each peptide in the composition has greater binding affinity for one or more host cell proteins than for one or more target biomolecules.

2. 2. The composition of claim 1, wherein the one or more target biomolecules is a protein, an oligonucleotide, a polynucleotide, a virus or viral capsid, a cell or organelle, or a small molecule.

3. 3. The composition of claim 2, wherein the protein is an antibody, an antibody fragment, an antibody-drug conjugate, a drug-antibody fragment conjugate, an Fc fusion protein, a hormone, an anticoagulant, a blood clotting factor, a growth factor, a morphogenic protein, a therapeutic enzyme, an engineered protein scaffold, an interferon, an interleukin, or a cytokine.

4. The composition of claim 1 , wherein the one or more host cell proteins are independently selected from the proteome of a host cell expressing the one or more target biomolecules.

5. 5. The composition of claim 4, wherein the one or more host cell proteins are independently selected from the group including acidic ribosomal protein, biglycan, cathepsin, clusterin, heat shock protein, nidogen-1, peptidyl-prolyl cis-trans isomerase B, protein disulfide isomerase, SPARC, thrombospondin-1, vimentin, histones, endoplasmic reticulum chaperone BiP, legumain, serine protease HTRA1, and putative phospholipase B-like protein.

6. The composition of any one of claims 1 to 5, wherein one or more of the peptides further comprises a linker at the C-terminus of the peptide.

7. The linker is Gly n or [Gly-Ser-Gly] m The composition of any one of claims 1 to 6, comprising: (wherein 6 > n > 1 and 3 > m > 1).

8. 8. The composition of any one of claims 1 to 7, wherein each peptide independently comprises a sequence selected from the group consisting of GSRYRY (SEQ ID NO:1), RYYYAI (SEQ ID NO:2), AAHIYY (SEQ ID NO:3), IYRIGR (SEQ ID NO:4), and HSKIYK (SEQ ID NO:5).

9. 8. The composition of any one of claims 1 to 7, wherein each peptide independently comprises a sequence selected from the group consisting of ADRYGH (SEQ ID NO:6), DRIYYY (SEQ ID NO:7), DKQRII (SEQ ID NO:8), RYYDYG (SEQ ID NO:9), and YRIDRY (SEQ ID NO:10).

10. 8. The composition of any one of claims 1 to 7, wherein each peptide independently comprises a sequence selected from the group consisting of HYAI (SEQ ID NO:11), FRYY (SEQ ID NO:12), HRRY (SEQ ID NO:13), and RYFF (SEQ ID NO:14).

11. 8. The composition of any one of claims 1 to 7, wherein each peptide independently comprises a sequence selected from the group consisting of DKSI (SEQ ID NO: 15), DRNI (SEQ ID NO: 16), HYFD (SEQ ID NO: 17), and YRFD (SEQ ID NO: 18).

12. 8. The composition of any one of claims 1-7, wherein each peptide independently comprises a sequence selected from the group consisting of GSRYRY (SEQ ID NO:11), RYYYAI (SEQ ID NO:2), AAHIYY (SEQ ID NO:3), IYRIGR (SEQ ID NO:4), HSKIYK (SEQ ID NO:5), DKSI (SEQ ID NO:15), DRNI (SEQ ID NO:16), HYFD (SEQ ID NO:17), and YRFD (SEQ ID NO:18).

13. An adsorbent comprising a composition according to any one of claims 1 to 12 conjugated to a support.

14. The adsorbent of claim 13, wherein all of the peptides in the composition are conjugated to a single support.

15. 15. The adsorbent of claim 14, wherein the adsorbent comprises multiple supports and one or more peptides are conjugated to a single support.

16. 17. The adsorbent of claim 16, wherein the one or more peptides conjugated to a single support are all the same peptide or are different peptides.

17. The adsorbent of any one of claims 13 to 16, wherein the support comprises a non-porous or porous particle, a non-porous or porous membrane, a plastic surface, or a fiber.

18. 18. The adsorbent of claim 17, wherein the support comprises polymethacrylate, polyethersulfone cellulose, agarose, chitosan, iron oxide, silica, titania, or zirconia.

19. 1. A method for removing one or more host cell proteins from a mixture comprising one or more host cell proteins and one or more target biomolecules, comprising: a. contacting the mixture with a composition according to any one of claims 1 to 12 or an adsorbent according to any one of claims 13 to 18. The method includes:

20. b. washing the composition or adsorbent to remove one or more unbound target biomolecules into the supernatant or mobile phase; and c. Recovering the supernatant containing the one or more unbound target biomolecules.

20. The method of claim 19, further comprising:

21. The method of any one of claims 19 to 20, wherein the contacting step comprises a high ionic strength binding buffer or a low ionic strength binding buffer.

22. 22. The method of claim 21, wherein the low ionic strength binding buffer comprises 1 to 50 mM NaCl.

23. 22. The method of claim 21, wherein the high ionic strength binding buffer comprises 100-500 mM NaCl.

24. 24. The method of any one of claims 19 to 23, wherein the contacting step comprises a low pH buffer of pH 5 to 6.

7.

25. 24. The method of any one of claims 19 to 23, wherein the contacting step comprises a neutral pH buffer of pH 6.8 to 7.

4.

26. 24. The method of any one of claims 19 to 23, wherein the contacting step comprises a high pH buffer of pH 7.5 to 9.

27. 22. The method of any one of claims 19 to 21, wherein the contacting step comprises a neutral pH and low ionic strength binding buffer, the buffer comprising 20 mM NaCl and having a pH of 7.

28. 22. The method of any one of claims 19 to 21, wherein the contacting step comprises a low pH and high ionic strength binding buffer, the buffer comprising 150 mM NaCl and having a pH of 6.

29. 29. The method of any one of claims 19-28, wherein each peptide independently comprises a sequence selected from the group consisting of GSRYRYGSG (SEQ ID NO: 19), RYYYAIGSG (SEQ ID NO: 20), AAHIYYGSG (SEQ ID NO: 21), IYRIGRGSG (SEQ ID NO: 22), HSKIYKGSG (SEQ ID NO: 23), DKSIGSG (SEQ ID NO: 33), DRNIGSG (SEQ ID NO: 34), HYFDGSG (SEQ ID NO: 35), and YRFDGSG (SEQ ID NO: 36).

30. The method according to any one of claims 19 to 29, wherein the method is carried out under static binding conditions.

31. The method according to any one of claims 19 to 29, wherein the method is carried out under dynamic binding conditions.

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