Method for analyzing recombinant polyclonal proteins

Abstract

Provided herein is a method for analyzing and characterizing a composition comprising a recombinant polyclonal protein. Uses of methods related to the production, manufacture, distribution, storage and therapeutic use of the ABPs are also provided.

Description

1. Technical Field This article provides a method for analyzing a library containing at least 100 antigen-binding proteins (ABPs). Various uses and applications of the method related to the production, manufacture, distribution, storage, and therapeutic use of ABPs are also provided. 2. Background Technology Passive immunization (McDonagh, 1966) provides a protective strategy for immunocompromised individuals who do not respond to active vaccines. For example, intravenous immunoglobulin (IVIg) is a broad-spectrum polyclonal antibody therapy derived from plasma from thousands of human donors. IVIg is used as an antibody replacement therapy for patients with humoral immunodeficiency (Lucas et al., 2010; Resnick et al., 2012). However, IVIg has low titers of antibodies against many common pathogens, leading to significant morbidity and mortality in immunocompromised patients (Orange et al., 2010). To increase antipathogen titers, some teams have developed high-titer plasma-derived antibodies, often referred to as hyperimmune agents (Bozzo & Jorquera, 2017). Hyperimmune agents are typically derived from donor plasma shortly after administration of an active vaccine, such as HyperHEP B (Grifols), which has high titers against hepatitis B virus.

[0003] Hyperimmune preparations derived from donors recently administered live vaccines are an excellent option for passive immunization, but scaling up such products commercially is a challenge (Kreil et al., 2012). Importantly, it can be difficult to identify strong responders willing to be vaccinated and repeatedly donate plasma. Therefore, manufacturing batches of hyperimmune preparations inevitably originate from different groups of donors, leading to batch-to-batch variability. Antipathogenic titers vary considerably among hyperimmune preparations, ranging from as low as 2 to 3 times (Schampera et al., 2017) to as high as 50 times (Kreil et al., 2012). Therefore, in some cases, physicians can simply administer larger doses of IVIg (Polilli et al., 2012). Physicians and patients will benefit from more consistent, higher-titer hyperimmune preparations that are easier to manufacture on a large scale.

[0004] Many of these problems can be addressed by using recombinant DNA technology combined with microfluidic and molecular genomics strategies to generate multivalent hyperimmune globulins. General strategies previously used to generate recombinant multivalent hyperimmune globulins against SARS-CoV-2 or Zika virus, as described in PCT / US2020 / 030878 (submitted April 30, 2020); PCT / US2021 / 037232 (submitted June 14, 2021); and PCT / US2021 / 044523 (submitted August 4, 2021), are incorporated herein by reference in their entirety. Multivalent hyperimmune globulins contain thousands of antigen-binding proteins (ABPs) with binding specificity to therapeutic target antigens. Compared to IVIg, RPPs have shown greater therapeutic efficacy against viral infections, demonstrating their potential as novel therapeutic agents.

[0005] To facilitate the broad therapeutic use of antigen-binding protein (ABP) libraries, it is crucial to generate high-quality compositions containing thousands of ABPs in substantial yields while maintaining their properties and quality. Therefore, there is a need to develop a method for testing the quality and identity of mixtures containing hundreds or thousands of unique ABPs. 3. Summary of the Invention ABP libraries are complex products representing novel therapeutic approaches. Controlling this mixture is complex. Some batch-to-batch variability is expected, and slight variations in population distribution have been noted without affecting critical quality attributes (CQA). It is important to manufacture this product consistently, for example, by using the same seed culture regardless of batch size to maintain consistent cell age across batches. Additionally, the manufactured product needs to be analyzed to ensure the safety, consistency, strength, quality, and potency of the mixture.

[0007] This paper presents a novel method for testing ABP libraries to assess and ensure the quality and identity of ABP. This method can be used in the production, distribution, storage, and therapeutic applications of the libraries.

[0008] Therefore, one aspect of this disclosure provides a method for analyzing a test library containing antigen-binding proteins (ABPs), comprising: measuring the charge state distribution of the test library, wherein the test library contains at least 100 ABPs; comparing the charge state distribution with a reference distribution, wherein the reference distribution is the charge state distribution of a reference library; and determining the quality of the test library based on the comparison.

[0009] In some embodiments, the charge state distribution is measured in step (a) by cation exchange-performance liquid chromatography (CEX-HPLC). In some embodiments, CEX-HPLC is performed with a pH gradient from mobile phase A to mobile phase B, wherein mobile phase A has a low pH from pH 5 to pH 7, and mobile phase B has a high pH from pH 9 to pH 11. In some embodiments, mobile phase A has a low pH from pH 5 to pH 6 or from pH 5.5 to pH 6. In some embodiments, mobile phase B has a high pH from pH 10 to pH 11 or from pH 10 to pH 10.5.

[0010] In some embodiments, CEX-HPLC is performed using a column containing a strong cation exchanger. In other embodiments, CEX-HPLC is performed using a column containing a weak cation exchanger.

[0011] In some embodiments, CEX-HPLC is performed at a flow rate between 0.25 mL / min and 2 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate between 0.5 mL / min and 1.5 mL / min, between 0.75 mL / min and 1 mL / min, or between 0.75 mL / min and 0.85 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate of 0.5 mL / min, 0.75 mL / min, or 1.0 mL / min.

[0012] In some implementations, CEX-HPLC is performed at 20-40°C, 25-35°C, 25°C, 30°C, or 35°C.

[0013] In some implementations, CEX-HPLC is performed using 5-100% B gradient, 10-100% B gradient, 15-100% B gradient, or 20-100% B gradient.

[0014] In some implementations, CEX-HPLC is performed with gradient times of 30–60 min. In some implementations, CEX-HPLC is performed with gradient times of 30–60 min, 30–45 min, 30–40 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0015] In some embodiments, CEX-HPLC is performed using a column packed with resin having a particle size of less than 3 µm, less than 2.9 µm, less than 2.8 µm, less than 2.7 µm, less than 2.6 µm, or less than 2.5 µm. In some embodiments, CEX-HPLC is performed using a column packed with resin, which may be a porous or non-porous resin.

[0016] In some embodiments, the reference distribution is measured by cation exchange-high performance liquid chromatography (CEX-HPLC). In some embodiments, the reference distribution is measured by cation exchange-high performance liquid chromatography (CEX-HPLC) under the same conditions as those used to measure the charge state distribution in (a).

[0017] In some implementations, the reference library contains the same ABP as the test library. In some implementations, the test library and the reference library are produced from the same production cell line or its progeny. In some implementations, the reference library has already been analyzed by sequencing. In some implementations, the reference library is from a different batch than the test library.

[0018] In some implementations, the reference library contains one antibody. In some implementations, the reference library contains multiple antibodies. In some implementations, the reference library has been generated by mixing multiple monoclonal antibodies.

[0019] In some implementations, the reference library contains a subset of at least 100 ABPs from the test library. In some implementations, the test library and the reference library are generated separately.

[0020] In some implementations, the test library contains at least 500 ABPs, at least 1,000 ABPs, at least 2,000 ABPs, at least 3,000 ABPs, at least 4,000 ABPs, at least 5,000 ABPs, at least 6,000 ABPs, at least 7,000 ABPs, at least 8,000 ABPs, at least 9,000 ABPs, or at least 10,000 ABPs.

[0021] In some embodiments, in step (b), the charge state distribution is compared with a reference distribution based on peak retention time. In some embodiments, in step (b), the charge state distribution is compared with a reference distribution based on peak area, peak height, or number of peaks.

[0022] In some embodiments, in step (c), the test library is determined to have better quality when its charge state distribution is closer to the reference distribution. In some embodiments, peak size, peak retention time, or peak number is compared between the test library and the reference library. In some embodiments, peak size, peak retention time, and peak number are compared between the test library and the reference library. In some embodiments, in step (c), the test library is determined to have good quality when its charge state distribution is at least 50%, 60%, 70%, 80%, 90%, 96%, 97%, 98%, or 99% identical to the reference distribution in terms of peak size, peak retention time, or peak number.

[0023] In some embodiments, step (c) further determines the quality of the test library based on the potency of the test library measured by ELISA. In some embodiments, step (c) further determines the quality of the test library based on analysis of the test library by size exclusion chromatography (SEC)-HPLC.

[0024] In some embodiments, SEC-HPLC is performed using an SEC column containing small-particle resin with a particle size less than 2.9 µm, 2.8 µm, 2.7 µm, 2.6 µm, or 2.5 µm. In some embodiments, SEC-HPLC is performed using a BEH stationary phase. In some embodiments, SEC-HPLC is performed using a mobile phase with a pH between 6.7 and 7.3, 6.8 and 7.2, 6.9 and 7.1, or about 7.0. In some embodiments, SEC-HPLC is performed using a mobile phase containing a NaCl concentration between 450 mM and 550 mM, 480 mM and 520 mM, or about 500 mM.

[0025] In some embodiments, in step (c), the quality of the test library is further determined based on at least one of the following: (i) the amino acid sequences of the IgG1 and IgK frameworks, optionally verified by LC-MS reduced peptide mapping; (ii) the disulfide bond linkages between the IgG1 and IgK constant regions, optionally verified by LC-MS non-reduced peptide mapping; (iii) the size heterogeneity of the test library, optionally characterized by multi-angle light scattering (MALS); (iv) the melting temperature of the test library, optionally measured by differential scanning calorimetry (DSC); (v) the glass transition temperature of the test library, optionally measured by differential scanning calorimetry (DSC); (vi) analysis of released N-glycans; (vii) quantification of total sialic acid; and (viii) hemagglutination assay.

[0026] In some embodiments, the test library is a pharmaceutical composition comprising ABP and pharmaceutically acceptable excipients. In some embodiments, the test library is a protein isolated from a host cell culture. In some embodiments, the test library has been prepared by a process comprising: generating at least 100 ABPs by culturing a production cell line; and purifying at least 100 ABPs.

[0027] In some implementations, purification is performed by at least one of the following steps: (i) affinity chromatography, (ii) low pH virus inactivation, (iii) hydrophobic interaction chromatography or membrane filtration, (iv) multimode anion exchange chromatography or membrane filtration, (v) multimode cation exchange chromatography, (vi) anion exchange chromatography or membrane filtration, (vii) cation exchange chromatography, (viii) virus filtration, and (ix) ultrafiltration and / or percolation.

[0028] In some embodiments, purification is performed by two, three, four, five, or all six of the following steps: (i) affinity chromatography, (ii) low-pH virus inactivation, (iii) hydrophobic interaction chromatography or membrane filtration, (iv) multimode anion exchange chromatography or membrane filtration, (v) multimode cation exchange chromatography, (vi) anion exchange chromatography or membrane filtration, (vii) cation exchange chromatography, (viii) virus filtration, and (ix) ultrafiltration and / or percolation. In some embodiments, purification is performed by two, three, four, five, six, seven, eight, or all nine of the following steps: (i) affinity chromatography, (ii) low-pH virus inactivation, (iii) hydrophobic interaction chromatography or membrane filtration, (iv) multimode anion exchange chromatography or membrane filtration, (v) multimode cation exchange chromatography, (vi) anion exchange chromatography or membrane filtration, (vii) cation exchange chromatography, (viii) virus filtration, and (ix) ultrafiltration and / or percolation.

[0029] In some embodiments, ABP is an antibody. In some embodiments, ABP is an antibody specific to an antigen. In some embodiments, the antigen is a viral or bacterial antigen.

[0030] In some embodiments, the method further includes preparing a pharmaceutical drug comprising a test library. In some embodiments, the method further includes selecting the test library for preparing a pharmaceutical composition if the test library meets acceptance criteria.

[0031] In some implementations, a test library meets acceptance criteria when it has a charge distribution that is at least 50%, 60%, 70%, 80%, 85%, 90%, 96%, 97%, 98%, or 99% identical to a reference distribution in terms of peak size, peak retention time, and / or number of peaks. In some implementations, acceptance criteria include one or more factors selected from the following: a. SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to the polyclonal antibodies accounted for 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more of the entire peak. b. SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to high molecular weight (HMW) accounted for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak; and c. SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to low molecular weight (LMW) accounted for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak.

[0032] In some implementation schemes, acceptance criteria include one, two, or three factors selected from a to c.

[0033] In some embodiments, the method further includes preparing a pharmaceutical composition containing a test library. In some embodiments, the pharmaceutical composition is prepared after a test library that meets one or more of the acceptance criteria has been selected.

[0034] In another respect, this disclosure provides a pharmaceutical composition comprising a test library and prepared by any of these methods.

[0035] Another aspect of this disclosure relates to a method for analyzing a test library containing antigen-binding proteins (ABPs), comprising: (a) measuring the size heterogeneity of the test library, wherein the test library contains at least 100 ABPs; (b) comparing peaks corresponding to high molecular weight substances (HMW), polyclonal antibody monomers (pAb peaks), and low molecular weight substances (LMW); and (c) determining the quality of the test library based on the comparison.

[0036] In some implementations, the size heterogeneity of the test library is measured by size exclusion chromatography-HPLC.

[0037] In some embodiments, SEC-HPLC is performed using an SEC column containing small-particle resin with a particle size less than 2.9 µm, 2.8 µm, 2.7 µm, 2.6 µm, or 2.5 µm. In some embodiments, SEC-HPLC is performed using a BEH stationary phase. In some embodiments, SEC-HPLC is performed using a mobile phase with a pH between 6.7 and 7.3, 6.8 and 7.2, 6.9 and 7.1, or about 7.0. In some embodiments, SEC-HPLC is performed using a mobile phase with a NaCl concentration between 450 mM and 550 mM, 480 mM and 520 mM, or about 500 mM. In some embodiments, SEC-HPLC is performed at a temperature between 28°C and 32°C, 29°C and 31°C, or about 30°C.

[0038] In some implementations, the test library meets the acceptance criteria if: a. the SEC (size exclusion chromatography)-HPLC of the test library shows that the peak corresponding to the polyclonal antibody accounts for 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more of the total peak; b. the SEC (size exclusion chromatography)-HPLC of the test library shows that the peak corresponding to the high molecular weight (HMW) accounts for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak; and / or the SEC (size exclusion chromatography)-HPLC of the test library shows that the peak corresponding to the low molecular weight (LMW) accounts for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak.

[0039] In some embodiments, the method further includes preparing a pharmaceutical composition containing a test library.

[0040] In another aspect, this disclosure provides a pharmaceutical composition comprising a test library prepared by the methods disclosed herein. 4. Description of the attached drawings Figure 1 It is the charge state of an ABP library (rHBIG) that has binding specificity to hepatitis B virus antigen, obtained through bioinformatics analysis.

[0041] Figure 2 The charge state of IgG is shown, depending on the pH of the buffer solution.

[0042] Figure 3 The HPLC chromatograms of the same rHBIG sample from three independent runs are shown.

[0043] Figure 4HPLC chromatograms of rHBIG obtained using three different column batches of Proteomix SCX-NP1.7 (4.6 x 100 mm) columns are provided: S / N 2A54701 (LN DW054), S / N 0A60382 (LNDW166), and S / N 9A60383 (LN 430794).

[0044] Figure 5 HPLC chromatograms of rHBIG obtained at three different flow rates (0.5 mL / min, 0.75 mL / min and 1.0 mL / min) are provided.

[0045] Figure 6 HPLC chromatograms of rHBIG obtained using three different gradients (15-100% B, 5-100% B, and 10-100% B) are provided.

[0046] Figure 7 HPLC chromatograms of rHBIG obtained at three different gradient times (35 min, 45 min, and 60 min) are provided.

[0047] Figure 8 HPLC chromatograms of anti-HBV plasma hyperimmune preparation (HyperHEP), ABP library with binding specificity to hepatitis B virus antigen (rHBIG), ABP library with binding specificity to CoV antigen (rCIG), or recombinant monoclonal antibody (anti-CTLA-4) are provided.

[0048] Figure 9 HPLC chromatograms of an ABP library (rHBIG) with binding specificity to hepatitis B virus antigen and six individual antibodies (PN-6103.02, 6104.02, 6105.02, 6115.02, 6116.02, 6117.02) in the library are provided.

[0049] Figure 10 HPLC chromatograms of rHBIG obtained using two different columns (Proteomix SCX NP1.7 4.6x100 mm and MabPac SCX-105 µm, 4.6x250 mm) are provided.

[0050] Figure 11 SEC-HPLC chromatograms of rHBIG from two different batches (Tox DS and GMP DS) are provided.

[0051] Figure 12 SEC-HPLC chromatograms of rHBIG in the forced degradation study are provided.

[0052] Figure 13 SEC-MALS results for rHBIG from two different batches (Tox DS and GMP DS) are provided.

[0053] Figure 14 HPLC chromatograms of rHBIG Tox DS and GMP DS are provided. 5. Detailed Implementation 5.1. Definition Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of this invention are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edition , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al. Current Protocols in Molecular Biology Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), these references are incorporated herein by reference. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The terminology, laboratory procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well-known and commonly used in the art. Standard techniques are applicable to chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0055] Unless otherwise stated, the following terms shall be understood to have the following meanings: The term "antibody" is used in its broadest sense herein and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to antigens or epitopes. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is formed by V... H -V L The antigen-binding domain formed by the dimer.

[0056] The terms "recombinant polyclonal antibody," "recombinant polyclonal protein," or "RPP" refer to more than one recombinant antibody that collectively contains more than one antigen-binding domain that specifically binds to an antigen or epitope, or multiple antigens and epitopes. A recombinant polyclonal antibody can be a complete antibody or a variant or derivative thereof. In some embodiments, the antigen-binding domain binds to the antigen or epitope with similar specificity and affinity to naturally occurring antibodies. In some embodiments, the recombinant polyclonal antibody is a mixture of antibodies. In some embodiments, the recombinant polyclonal antibody contains scFv. In some embodiments, the recombinant polyclonal antibody contains a substitute scaffold. In some embodiments, the recombinant polyclonal antibody consists of a substitute scaffold. In some embodiments, the recombinant polyclonal antibody consists essentially of a substitute scaffold. In some embodiments, the recombinant polyclonal antibody contains antibody fragments. In some embodiments, the recombinant polyclonal antibody consists essentially of antibody fragments.

[0057] The term "antigen-binding domain" refers to the part of an antibody that can specifically bind to an antigen or epitope.

[0058] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to an antibody that has a structure substantially similar to that of naturally occurring antibodies and has a heavy chain containing an Fc region.

[0059] The term "immunoglobulin" refers to a class of structurally related proteins, such as antibodies, that typically consist of two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In a "complete immunoglobulin," all four chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Paul... Fundamental Immunology 7th Edition, Chapter 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. In short, each heavy chain typically contains a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy-chain constant region typically contains three structural domains, abbreviated as C. H1 C H2 and CH3 Each light chain typically contains a light chain variable region (V). L ( ) and the light chain constant region. The light chain constant region typically contains a structural domain, abbreviated as C. L .

[0060] An "antibody fragment" contains a portion of a complete antibody, such as the antigen-binding region or variable region of the complete antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0061] The term "antigen-binding protein" (ABP) refers to a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domains bind to the antigen or epitope with similar specificity and affinity to naturally occurring antibodies. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP is composed of antibodies. In some embodiments, the ABP is substantially composed of antibodies. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP is composed of an alternative scaffold. In some embodiments, the ABP is substantially composed of an alternative scaffold. In some embodiments, the ABP comprises antibody fragments. In some embodiments, the ABP is composed of antibody fragments. In some embodiments, the ABP is substantially composed of antibody fragments.

[0062] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. A substantially homogeneous group of antibodies comprises antibodies that are substantially similar and bind to the same epitopes, except for variants that are typically present during monoclonal antibody production. Such variants are usually present only in small quantities. Monoclonal antibodies are typically obtained through a process involving the selection of a single antibody from a plurality of antibodies. For example, the selection process may involve choosing a unique clone from a library of multiple clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody may be further modified, for example, to improve affinity for the target ("affinity maturation"), humanize the antibody, improve its production in cell cultures, and / or reduce its immunogenicity in subjects.

[0063] The term "polyclonal antibody" refers to a mixture of at least two monoclonal antibodies. Polyclonal antibodies can be monospecific or multispecific.

[0064] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0065] "Isolated antibody" or "isolated nucleic acid" is an antibody or nucleic acid that has been isolated and / or recovered from components of its native environment. Components of the native environment may include enzymes, hormones, and other protein or non-protein substances. In some embodiments, the isolated antibody is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, for example, by using a rotary cup sequencer. In some embodiments, the isolated antibody is purified to homogenization by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and detected by Coomassie blue or silver staining. The isolated antibody includes in situ antibodies from recombinant cells, since at least one component of the antibody's native environment is absent. In some aspects, the isolated antibody or isolated nucleic acid is prepared by at least one purification step. In some embodiments, the isolated antibody or isolated nucleic acid is purified to at least 80 wt%, 85 wt%, 90 wt%, 95 wt%, or 99 wt%. In some embodiments, the isolated antibody or isolated nucleic acid is purified to at least 80 vol%, 85 vol%, 90 vol%, 95 vol%, or 99 vol%. In some embodiments, the isolated antibody or isolated nucleic acid is provided as a solution containing at least 85 wt%, 90 wt%, 95 wt%, 98 wt%, 99 wt% to 100 wt% antibody or nucleic acid. In some embodiments, the isolated antibody or isolated nucleic acid is provided as a solution containing at least 85 vol%, 90 vol%, 95 vol%, 98 vol%, 99 vol% to 100 vol% RPP or nucleic acid.

[0066] Regarding antibody binding to target molecules, the terms "binding," "specific binding," "specifically bound to," "specifically bound to," "selectively bound," and "selectively bound to" refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., binding to non-target molecules) to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen. Specific binding can be measured, for example, by measuring the binding to a target molecule and comparing it to the binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics an epitope recognized on a target molecule. In this case, if the binding of the antibody to the target molecule is competitively inhibited by the control molecule, specific binding is indicated.

[0067] 5.2. Other Interpretive Conventions The ranges listed in this document should be understood as abbreviations of all values ​​within that range, including the listed endpoints. For example, the range 1 to 50 should be understood as including any number, combination of numbers, or subrange that comes from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0068] 5.3. Methods for analyzing ABP This disclosure provides a method for analyzing a library containing ABP (“test library”).

[0069] In some implementations, the method includes measuring the charge state (e.g., pI (isoelectric point)) distribution of the test library. The method may also include comparing the charge state distribution with a reference distribution, wherein the reference distribution is the charge state distribution of a reference library. The method may further include determining the quality of the test library based on the comparison.

[0070] In some embodiments, the method includes measuring the size heterogeneity of the test library. In some embodiments, the method includes characterizing high molecular weight substances (HMW), polyclonal antibody monomers (pAb peaks), and low molecular weight substances (LMW) in the ABP library.

[0071] In some implementations, the methods disclosed herein are used alone or in combination with other methods. In some implementations, the methods disclosed herein are performed simultaneously or sequentially with other analytical methods.

[0072] 5.3.1. Measurement of charge state The methods disclosed herein include measuring the charge state (e.g., pI) of a test library. Various methods known in the art for measuring charge state can be used. In some embodiments, cation exchange-performance liquid chromatography (CEX-HPLC) is used to measure charge state. In some embodiments, HPLC data or spectra display the charge state distribution. Other methods for separating antibodies using pH gradients based on the differential charge state (pI values) of the component antibodies are available in the various embodiments disclosed herein.

[0073] CEX-HPLC can be performed with a pH gradient from mobile phase A to mobile phase B. In some embodiments, mobile phase A has a low pH and mobile phase B has a high pH. In some embodiments, mobile phase A has a pH from pH 5 to pH 7. In some embodiments, mobile phase A has a pH from pH 5 to pH 6. In some embodiments, mobile phase A has a pH from pH 5 to pH 5.5. In some embodiments, mobile phase A has a pH from pH 5.5 to pH 6. In some embodiments, mobile phase B has a pH from pH 9 to pH 11. In some embodiments, mobile phase B has a pH from pH 10 to pH 11. In some embodiments, mobile phase B has a pH from pH 10 to pH 10.5. In some embodiments, mobile phase B has a pH from pH 10.5 to pH 11.

[0074] In some embodiments, CEX-HPLC is performed at a flow rate between 0.25 mL / min and 2 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate between 0.5 mL / min and 2 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate between 0.5 mL / min and 1.5 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate between 0.75 mL / min and 1.5 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate between 0.75 mL / min and 1 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate between 0.75 mL / min and 0.85 mL / min. In some embodiments, CEX-HPLC is performed at a flow rate of 0.5 mL / min, 0.75 mL / min, or 1.0 mL / min.

[0075] In some embodiments, CEX-HPLC is performed at a temperature between 20 and 40°C. In some embodiments, CEX-HPLC is performed at a temperature between 25 and 35°C. In some embodiments, CEX-HPLC is performed at a temperature between 27.5 and 32.5°C. In some embodiments, CEX-HPLC is performed at 25°C, 30°C, or 35°C.

[0076] In some embodiments, CEX-HPLC is performed at a temperature of at least 25°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 26°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 27°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 28°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 29°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 30°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 35°C. In some embodiments, CEX-HPLC is performed at a temperature of at least 40°C.

[0077] In some implementations, CEX-HPLC is performed using gradients of 1-100% B, 5-100% B, 10-100% B, 15-100% B, or 20-100% B.

[0078] In some embodiments, CEX-HPLC is performed with gradient times of 30–60 min. In some embodiments, CEX-HPLC is performed with gradient times of 30–60 min, 30–45 min, or 30–40 min. In some embodiments, CEX-HPLC is performed with gradient times of 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0079] In some embodiments, CEX-HPLC is performed using a CEX column containing a weak cation exchanger. In other embodiments, CEX-HPLC is performed using a CEX column with a strong cation exchanger. The strong cation exchanger may contain acidic functional groups, such as sulfonic acids, which are ionized across the entire pH range. The weak cation exchange adsorbent has surface functional groups, such as carboxylic acids, which are negatively charged at high pH but neutral at low pH.

[0080] In some embodiments, the method includes using a CEX column filled with resin having a particle size less than 3 µm. In some embodiments, the particle size is less than 2.9 µm, less than 2.8 µm, less than 2.7 µm, less than 2.6 µm, less than 2.5 µm, less than 2.4 µm, less than 2.3 µm, less than 2.2 µm, less than 2.1 µm, less than 2.0 µm, less than 1.9 µm, less than 1.8 µm, less than 1.7 µm, less than 1.6 µm, or less than 1.5 µm. In some embodiments, the particle size is between 2.3 µm and 2.9 µm, between 2.5 µm and 2.8 µm, between 2.5 µm and 2.8 µm, between 1.5 µm and 2.5 µm, between 1.5 µm and 2.0 µm, between 1.0 µm and 2.0 µm, or between 1.0 µm and 1.5 µm.

[0081] In some embodiments, the method includes using a CEX column filled with a porous resin. In some embodiments, the method includes using a CEX column filled with a non-porous resin.

[0082] In some implementations, the CEX column is a Proteomix SCX or a MabPac SCX-10.

[0083] 5.3.2. Measurement of dimensional heterogeneity In some embodiments, the methods disclosed herein include measuring the size heterogeneity of the test library. Various methods for measuring size heterogeneity can be used. In some embodiments, the SEC-HPLC method, which has been improved for high-resolution analysis, is used. The high-resolution SEC-HPLC method can characterize high molecular weight substances (HMW), polyclonal antibody monomers (pAb peaks), and low molecular weight substances (LMW) in the ABP library.

[0084] Size exclusion chromatography (SEC) separates proteins based on their hydrodynamic radius (size). The stationary phase of the column contains porous particles through which molecules diffuse using the mobile phase. Smaller particles are trapped in the pores and elute for longer periods, while larger particles do not enter the pores and elute more quickly than smaller particles. SEC is used to separate protein monomers from their dimers and high molecular weight (HMW) aggregates, fragments, and other low molecular weight (LMW) impurities. In some embodiments, the eluted protein is monitored by UV absorbance at 280 nm. The area under the curve (AUC) quantitatively corresponds to the amount of protein. In some embodiments, the peak area percentages of monomers, HMW, and LMW are reported.

[0085] In some embodiments, the method includes the use of an SEC column filled with resin having a particle size less than 3 µm. In some embodiments, the particle size is less than 2.9 µm, less than 2.8 µm, less than 2.7 µm, less than 2.6 µm, less than 2.5 µm, less than 2.4 µm, or less than 2.3 µm. In some embodiments, the particle size is between 2.3 µm and 2.9 µm, between 2.5 µm and 2.8 µm, or between 2.5 µm and 2.8 µm.

[0086] In some embodiments, the SEC method uses a stationary phase to reduce residual silanol groups. In some embodiments, the method uses a BEH stationary phase. In some embodiments, the material has the empirical formula SiO2(O) 1.5 SiCH2CH2SiO 1.5 ) 0.25 They can be synthesized by co-condensation of 1,2-bis(triethoxysilyl)ethane (1 equivalent) with TEOS (4 equivalent).

[0087] In some embodiments, the SEC method uses a mobile phase with a pH between pH 6.5 and pH 7.5. In some embodiments, the mobile phase has a pH between pH 6.75 and pH 7.25. In some embodiments, the mobile phase has a pH between pH 6.8 and pH 7.2. In some embodiments, the mobile phase has a pH between pH 6.9 and pH 7.1. In some embodiments, the mobile phase buffer has a pH of 7.0.

[0088] In some embodiments, the mobile phase contains a NaCl concentration between 250 mM and 750 mM. In some embodiments, the mobile phase contains a NaCl concentration between 300 mM and 700 mM. In some embodiments, the mobile phase contains a NaCl concentration between 400 mM and 600 mM. In some embodiments, the mobile phase contains NaCl concentrations of 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, or 550 mM.

[0089] In some embodiments, the SEC method is performed at a column temperature between 20°C and 40°C. In some embodiments, the SEC method is performed at a column temperature between 22°C and 38°C. In some embodiments, the SEC method is performed at a column temperature between 25°C and 35°C. In some embodiments, the SEC method is performed at a column temperature between 28°C and 32°C. In some embodiments, the SEC method is performed at a column temperature between 29°C and 31°C. In some embodiments, the SEC method is performed at a column temperature of 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0090] 5.3.3. ABP and ABP Library Each member of the ABP library (test library or reference library) described herein is a polypeptide that specifically binds to an antigen, such as an antibody or antibody fragment. In some embodiments, the antigen is a viral antigen or a bacterial antigen. In some embodiments, the ABP comprises homologous pairs of heavy and light chain CDR sequences. In some embodiments, the ABP is an scFv. In some embodiments, the ABP is a full-length antibody. The ABP can also be any synthetic or genetically engineered protein.

[0091] An ABP library may contain at least 100, at least 1000, or at least 2000 ABPs. In some embodiments, the ABP library contains 100-5000 ABPs. In some embodiments, the ABP library contains 100-2000 ABPs. In some embodiments, the ABP library contains 100-1000 ABPs. In some embodiments, the ABP library contains 1000-3000 ABPs. In some embodiments, the ABP library contains 2000-3000 ABPs.

[0092] In some embodiments, the library comprises one or more components other than ABP. In some embodiments, the library comprises one or more therapeutic agents other than ABP. In some embodiments, the library is a pharmaceutical composition comprising ABP and excipients.

[0093] In some implementations, the test library contains at least 500 ABPs, at least 1,000 ABPs, at least 2,000 ABPs, at least 3,000 ABPs, at least 4,000 ABPs, at least 5,000 ABPs, at least 6,000 ABPs, at least 7,000 ABPs, at least 8,000 ABPs, at least 9,000 ABPs, or at least 10,000 ABPs.

[0094] In some implementations, the test library contains fewer than 50 ABPs, fewer than 100 ABPs, fewer than 200 ABPs, fewer than 300 ABPs, fewer than 400 ABPs, fewer than 500 ABPs, fewer than 1000 ABPs, fewer than 2000 ABPs, fewer than 3000 ABPs, fewer than 4000 ABPs, and fewer than 5000 ABPs.

[0095] In some implementations, the reference library contains at least 500 ABPs, at least 1,000 ABPs, at least 2,000 ABPs, at least 3,000 ABPs, at least 4,000 ABPs, at least 5,000 ABPs, at least 6,000 ABPs, at least 7,000 ABPs, at least 8,000 ABPs, at least 9,000 ABPs, or at least 10,000 ABPs.

[0096] In some implementations, the reference library contains fewer than 50 ABPs, fewer than 100 ABPs, fewer than 200 ABPs, fewer than 300 ABPs, fewer than 400 ABPs, fewer than 500 ABPs, fewer than 1000 ABPs, fewer than 2000 ABPs, fewer than 3000 ABPs, fewer than 4000 ABPs, and fewer than 5000 ABPs.

[0097] In some embodiments, multiple ABPs in the library have binding affinity for the same antigen. In some embodiments, multiple ABPs in the library have binding affinity for two or more antigens. In some embodiments, the library contains ABPs from a single donor. In some embodiments, the library contains ABPs from multiple donors.

[0098] The library can be a test library or a reference library. In some embodiments, the test library and the reference library contain the same ABPs. In some embodiments, the test library and the reference library contain different groups of ABPs. In some embodiments, the test library contains a subset of the ABPs in the reference library. In some embodiments, the reference library contains a subset of the ABPs in the test library. In some embodiments, the reference library contains a subset of at least 100 ABPs in the test library.

[0099] In some implementations, the reference library contains one antibody. In some implementations, the reference library contains multiple antibodies. In some implementations, the reference library has been generated by mixing multiple monoclonal antibodies.

[0100] In some implementations, the library contains ABP that has already been analyzed by sequencing. In some implementations, the test library and the reference library are generated from the same production cell line or its progeny. In some implementations, the reference library has already been analyzed by sequencing. In some implementations, the reference library is from a different batch than the test library. In some implementations, the test library and the reference library have been generated separately.

[0101] In some embodiments, the test library has been prepared by a process including: generating at least 100 ABPs by culturing production cell lines; and purifying at least 100 ABPs. In some embodiments, purification is performed by at least one of the following steps: affinity chromatography, low-pH virus interaction chromatography, hydrophobic interaction chromatography or membrane filtration, multimode anion exchange chromatography or membrane filtration, anion exchange chromatography or membrane filtration, multimode cation exchange chromatography, cation exchange chromatography, virus filtration, and ultrafiltration and / or percolation. In some embodiments, purification is performed by two, three, four, five, six, or all of the following steps: affinity chromatography, low-pH virus interaction chromatography, hydrophobic interaction chromatography or membrane filtration, multimode anion exchange chromatography or membrane filtration, anion exchange chromatography or membrane filtration, multimode cation exchange chromatography, cation exchange chromatography, virus filtration, and ultrafiltration and / or percolation.

[0102] 5.3.4. Analysis of test libraries The methods disclosed herein may include comparing the charge state distribution (i.e., the test distribution) of a test library with the charge state distribution (i.e., the reference distribution) of a reference library. In some embodiments, the comparison is performed by comparing the HPLC spectra of the test library and the reference library.

[0103] This comparison can be used to assess the safety, identity, strength, quality, and potency of the library. In some implementations, this analysis is used to assess the physiological and chemical properties of the test library. A set of qualified release criteria and additional characterization methods can be used to characterize and control the test library. Analytical methods and acceptance criteria can be developed and tailored based on information obtained from characterization of process development batches.

[0104] The reference distribution can be measured using methods used to measure the charge state of the test library. In some embodiments, CEX-HPLC is used to measure the reference distribution. In some embodiments, the reference distribution is measured using methods and conditions used to measure the charge state of the test library. In some embodiments, the reference distribution is measured by cation exchange-high performance liquid chromatography (CEX-HPLC) under the same conditions as those used to measure the charge state distribution of the test library.

[0105] In some embodiments, the reference distribution is obtained through bioinformatics analysis. In some embodiments, the reference distribution is obtained by analyzing the amino acid sequence of the ABP. In some embodiments, the reference distribution and the average molecular weight, charge state, and extinction coefficient of the library components are calculated using the component amino acid sequences.

[0106] In some implementations, the reference distribution is obtained based on sequence analysis of ABP. In some implementations, the reference distribution is obtained based on sequence analysis of ABP expected to be present in the test library. In some implementations, the reference distribution is obtained based on sequence analysis of ABP in a reference library.

[0107] In some embodiments, the analytical method includes comparing the charge state of the test library and the reference library. In some embodiments, the comparison includes comparing peak retention time, peak area, peak height, and / or peak number. In some embodiments, the comparison is performed by identifying overlapping peaks. In some embodiments, the comparison is performed by comparing the area under the HPLC spectrum (AUC) of the test library and the reference library. In some embodiments, the comparison indicates the safety, identity, intensity, quality, or potency of the libraries.

[0108] In some implementations, a test library is determined to have better quality when its charge state distribution is closer to that of a reference distribution. In some implementations, a test library is determined to have good quality when its charge state distribution is at least 90%, 96%, 97%, 98%, or 99% identical to the reference distribution in terms of peak retention time, peak area, peak height, and / or number of peaks. In some implementations, a test library is determined to have good quality when its charge state distribution is at least 90%, 96%, 97%, 98%, or 99% identical to the reference distribution in terms of area under the curve (AUC).

[0109] In some implementations, the overlap of the main peaks between the test library and the reference library is compared.

[0110] In some implementations, the overlap of charge states between the test library and the reference library is related to the library’s security, identity, strength, quality, or effectiveness.

[0111] In some implementations, one or more additional factors are considered to determine the quality or characterization of the test library.

[0112] In some implementations, one or more physical properties of the test library are measured to determine whether they are within acceptance limits. Physical properties are selected from one or more of the following: color, clarity, visible particles, pH, osmotic pressure, PS20, protein concentration, antigen binding, size variants, denatured size distribution, bacterial endotoxins, sterility, and sub-visible particles.

[0113] In some embodiments, a test library is acceptable when it is not darker than the reference material. In some embodiments, a test library is acceptable when it is not more milky white than the reference material. In some embodiments, a test library is acceptable when it is substantially free of visible particles. In some embodiments, a test library is acceptable when its pH is between pH 3 and pH 6, between pH 4 and pH 5, between pH 4.5 and pH 5, or between pH 4.8 and pH 4.9. In some embodiments, a test library is acceptable when its osmolarity is between 300 and 500 mOsm / kg, between 300 and 400 mOsm / kg, between 325 and 375 mOsm / kg, or between 340 and 360 mOsm / kg. In some embodiments, a test library is acceptable when it contains polysorbate 20 (PS20) at concentrations of 0.001% to 0.1% w / v, 0.005% to 0.05% w / v, 0.075% to 0.03% w / v, or 0.01% to 0.03% w / v. In some embodiments, a test library is acceptable when the protein concentration is 20 to 40 mg / mL, 25 to 35 mg / mL, or about 30 mg / mL.

[0114] In some embodiments, a test library is acceptable when its binding to the target antigen is comparable to that of the reference material. In some embodiments, a test library is acceptable when its binding to the target antigen is 25%-300%, 50%-200%, or 50%-150% of that of the reference material. In some embodiments, binding is measured by ELISA. In some embodiments, a test library is acceptable when its neutralizing activity against the target (e.g., a virus) is comparable to that of the reference material. In some embodiments, a test library is acceptable when its neutralizing activity against the target is 25%-300%, 50%-200%, or 50%-150% of that of the reference material.

[0115] In some embodiments, the test library is acceptable when HPLC SEC (size exclusion chromatography) indicates that the peak corresponding to the polyclonal antibody accounts for more than 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the total signal. In some embodiments, the test library is acceptable when HPLC SEC indicates that the peak corresponding to high molecular weight (HMW) molecules is less than 10%, less than 7.5%, less than 5%, or less than 2.5%. In some embodiments, the test library is acceptable when HPLC SEC indicates that the peak corresponding to low molecular weight (LMW) molecules is less than 10%, less than 7.5%, less than 5%, or less than 2.5%.

[0116] In some implementations, the test library is acceptable when the denaturation size distribution shows that 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the signal corresponds to the intact antibody. In some implementations, the test library is acceptable when the denaturation size distribution shows that 70%, 75%, 80%, 85%, 90%, or 95% of the signal corresponds to the heavy or light chain of the antibody.

[0117] In some implementations, the quality of the test library is further determined based on antigen binding measured by ELISA. The antigen binding of the test library can be compared with reference materials.

[0118] In some embodiments, the quality of the test library is further determined based on analysis of the test library by size exclusion chromatography (SEC)-HPLC. In some embodiments, the quality of the test library is further determined based on the amino acid sequences of the IgG1 and IgK frameworks. The amino acid sequences of the IgG1 and IgK frameworks can be determined by LC-MS reduced peptide mapping. In some embodiments, the quality of the test library is further determined based on disulfide bond linkages between the IgG1 and IgK constant regions. In some embodiments, the disulfide bond linkages between the IgG1 and IgK constant regions are determined by LC-MS non-reduced peptide mapping. In some embodiments, the quality of the test library is further determined based on its size heterogeneity. In some embodiments, the size heterogeneity of the test library is characterized by multi-angle light scattering (MALS) technology. In some embodiments, the quality of the test library is further determined based on its melting temperature. In some embodiments, the melting temperature is measured by differential scanning calorimetry (DSC). In some embodiments, the quality of the test library is further determined based on its glass transition temperature. In some embodiments, the glass transition temperature of the test library is measured by differential scanning calorimetry (DSC). In some embodiments, the quality of the test library is further determined based on the analysis of released N-glycans. The N-linked glycans of the library can be released by enzymatic cleavage (PNGase F) under reducing and denaturing conditions. The N-glycans can be fluorescently labeled, separated by HILIC-UPLC chromatography, and detected by mass spectrometry. In some embodiments, the quality of the test library is further determined based on the quantification of total sialic acid. In some embodiments, sialic acid is released and measured under natural conditions using the AdvanceBio Total Sialic Acid Quantification Kit from Agilent. This kit uses enzymatic cleavage to release sialic acid, which is then converted to H2O2 and detected with a fluorescent reporter dye. In some embodiments, the quality of the test library is further determined based on hemagglutination assays.

[0119] 5.4. Methods for generating and preparing ABP libraries The analytical methods disclosed herein can be used to analyze or characterize libraries containing any of the ABPs disclosed herein. ABPs can be purified from host cells transfected with genes encoding antibodies. Purification can be performed by any method known in the art, such as using a heparinized HP column, using a salt gradient, or eluting the filtrate of the host cell culture medium with protein A resin.

[0120] The ABP described herein can be a naturally occurring antibody or an engineered antibody. The variable region domain of the ABP can be any naturally occurring variable domain or its engineered form. An engineered form refers to a variable region domain generated using recombinant DNA engineering techniques. Such engineered forms include, for example, those generated from the variable region of a specific antibody by insertion, deletion, or alteration of the amino acid sequence of the specific antibody or by alteration of the amino acid sequence of the specific antibody. Specific examples include engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a first antibody, and the remainder of the variable region domain from a second antibody.

[0121] The variable region domain can be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, V present in the variable region domain... H The domain can link to the immunoglobulin CH1 domain or fragments thereof. Similarly, V L The domain can be linked to the CK domain or a fragment thereof. In this way, for example, an antibody can be a Fab fragment, wherein the antigen-binding domain contains a related V domain covalently linked to the CH1 and CK domains at its C-terminus, respectively. H and V L Domains. The CH1 domain can be extended with further amino acids, for example to provide a hinge region or part of a hinge region domain as found in the Fab' fragment, or to provide further domains such as the antibody CH2 and CH3 domains.

[0122] Fully human monoclonal antibodies can be generated using any number of techniques familiar to those skilled in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) conversion of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization with human B cells, fusion of spleen cells from immunized transgenic mice carrying an inserted human immunoglobulin gene, isolation from a phage library of human immunoglobulin V region, or other procedures as known in the art and based on the disclosure herein. For example, fully human monoclonal antibodies can be obtained from transgenic mice engineered to produce specific human antibodies in response to antigenic stimulation. Methods for obtaining fully human antibodies from transgenic mice are described, for example, in Green et al. Nature Genet 7:13, 1994; Lonberg et al. Nature 368:856, 1994; Taylor et al., Int. Immun 6:579, 1994; US Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol 8:455-58; Jakobovits et al., 1995 Ann. NY Acad. Sci. 764:525-35. In this technique, elements of human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines containing targeted disruption of endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol 8:455-58 (1997). For example, human immunoglobulin transgenes can be small gene constructs or translocations on yeast artificial chromosomes that undergo B cell-specific DNA rearrangements and hypermutations in mouse lymphoid tissue. Fully human monoclonal antibodies can be obtained by immunizing transgenic mice, which can then produce human antibodies specific to one or more antigen targets. Lymphoid cells from immunized transgenic mice can be used to generate hybridomas that secrete human antibodies according to the methods described herein.

[0123] In some embodiments, the ABP comprises homologous pairs of the heavy and light chain CDR3 sequences disclosed herein. For example, the CDR can be incorporated into known antibody framework regions (IgG1, IgG2, etc.) or conjugated with a suitable carrier to enhance its half-life. Suitable carriers include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, transferrin, etc. These and other suitable carriers are known in the art. Such conjugated CDR peptides can be monomers, dimers, tetramers, or other forms. In one embodiment, one or more water-soluble polymers are bonded to one or more specific sites on the binder, such as the amino terminus.

[0124] The antigen-binding fragments of the ABP of the present invention can be generated using conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives generated by genetic engineering techniques are also considered.

[0125] Other embodiments include chimeric antibodies, such as humanized forms of non-human (e.g., mouse) monoclonal antibodies. Such humanized antibodies can be prepared using known techniques and offer the advantage of reduced immunogenicity when administered to humans. In one embodiment, the humanized antibody comprises a variable domain (or all or part of its antigen-binding site) of a mouse antibody and a constant domain derived from a human antibody. Alternatively, the humanized antibody fragment may comprise an antigen-binding site of a mouse antibody and a variable domain fragment derived from a human antibody (lacking the antigen-binding site). Procedures for generating chimeric and further engineered antibodies include those described in Riechmann et al., 1988, Nature 332:323; Liu et al., 1987, Proc. Nat. Acad. Sci. USA 84:3439; Larrick et al., 1989, Bio / Technology 7:934; and Winter et al., 1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDR-transplanted antibody. Techniques for humanized antibodies are discussed, for example, in U.S. Patent Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, 6,881,557; Padlan et al., 1995, FASEB J. 9:133-39; and Tamura et al., 2000, J. Immunol. 164:1432-41.

[0126] Another method for generating the human antibodies of the present invention includes immortalizing human peripheral blood cells via EBV transformation. See, for example, U.S. Patent No. 4,464,456. Such immortalized B cell lines (or lymphoblastoid cell lines) that generate ABPs specifically binding to one or more targets can be identified by immunoassay methods (e.g., ELISA) as provided herein, and then isolated by standard cloning techniques. According to methods known in the art, the stability of ABP-generating lymphoblastoid cell lines can be improved by fusing the transformed cell lines with mouse myeloma to generate mouse-human hybrid cell lines (see, for example, Glasky et al.). Hybridoma 8:377-89 (1989)). Another method for generating human ABP is in vitro immunization, which involves sensitizing human spleen B cells with an antigen target and then fusing the sensitized cells with a heterologous hybrid fusion partner. See, for example, Boerner et al., 1991. J. Immunol. 147:86-95.

[0127] In some embodiments, B cells that produce ABP are selected, and molecular biology techniques known in the art are employed (WO 92 / 02551; US ​​Patent 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996) and the light and heavy chain variable regions cloned from B cells as described herein. B cells from immunized animals can be isolated from spleen, lymph node, or peripheral blood samples by selecting cells that produce antibodies that specifically bind to antigen targets. B cells can also be isolated from humans, for example from peripheral blood samples.

[0128] Methods for detecting individual B cells that produce antibodies with desired specificity are well known in the art, such as plaque formation, fluorescence-activated cell sorting, and detection of specific antibodies after in vitro stimulation. Methods for selecting B cells that produce specific antibodies include, for example, preparing a single-cell suspension of B cells in soft agar containing the antigen target. The binding of the specific antibody produced by the B cell to the antigen leads to the formation of a complex, which may be visible as an immunoprecipitate.

[0129] In some implementations, B cells that produce specific antibodies are selected by using methods that allow the identification of naturally paired antibodies. For example, Adler et al., "A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library," can be employed. Mabs The method described in (9, 1282-1996, 2017), which is incorporated herein by reference in its entirety, combines microfluidics, molecular genomics, yeast single-stranded variable fragment (scFv) display, fluorescence-activated cell sorting (FACS), and deep sequencing. In short, B cells can be isolated from immunized animals and then merged. B cells are encapsulated in microdroplets along with oligomeric dT beads and lysis solution, and mRNA-binding beads are purified from the microdroplets and injected into a second emulsion containing an OE-RT-PCR amplification mixture to generate DNA amplicons encoding naturally paired scFvs with heavy and light chain Ig. The library of naturally paired amplicons is then electroporated into yeast for scFv display. FACS is used to identify high-affinity scFvs. Finally, deep antibody sequencing is used to identify all clones in the scFv library before and after sorting.

[0130] After selecting B cells that produce the desired antibody, the specific antibody gene can be cloned by isolating and amplifying DNA or mRNA in accordance with what is known in the art and the methods described herein.

[0131] In some embodiments, the ABP is generated by the methods described in Examples 1-5 of this disclosure. In some embodiments, the ABP is generated and disclosed in PCT / US2020 / 030878, filed April 30, 2020; PCT / US2021 / 037232, filed June 14, 2021; and PCT / US2021 / 044523, filed August 4, 2021, which are incorporated herein by reference in their entirety.

[0132] The method for obtaining the antibodies of this invention can also employ various phage display techniques known in the art. See, for example, Winter et al., 1994. Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. A library of human or mouse immunoglobulin variable region genes can be generated in a phage vector, and this library can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to ABP or its variants or fragments. See, for example, U.S. Patent No. 5,223,409; Huse et al., 1989. Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and the references cited therein. For example, a library containing multiple multinucleotide sequences encoding Ig variable region fragments can be inserted into the genome of a filamentous phage (such as M13 or a variant thereof), framed with a sequence encoding a phage coat protein. The fusion protein can be a fusion of the coat protein with a light chain variable region domain and / or with a heavy chain variable region domain. According to some embodiments, immunoglobulin Fab fragments can also be displayed on phage particles (see, for example, U.S. Patent No. 5,698,426).

[0133] In one embodiment, in a hybridoma, variable regions of genes expressing monoclonal antibodies of interest are amplified using nucleotide primers. These primers can be synthesized by those skilled in the art or can be purchased from commercially available sources. (See, for example, Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including V...) HaV Hb V Hc V Hd C H1 V L and C L Primers for the region. These primers can be used to amplify the variable region of the heavy or light chain, and then inserted into vectors such as ImmunoZAP™H or ImmunoZAP™L (Stratagene), respectively. These vectors can then be introduced into… E. coli Expression can be performed in yeast or mammal-based systems. Using these methods, large quantities of vitamin V can be produced. H and V L Single-chain proteins with fusion of domains (see Bird et al.) Science 242:423-426, 1988).

[0134] The ABP (e.g., antibody, antibody fragment, and antibody derivative) of the present invention may comprise any constant region known in the art. The light chain constant region may be, for example, a κ or λ type light chain constant region, such as a human κ or λ type light chain constant region. The heavy chain constant region may be, for example, an α-, δ-, ε-, γ-, or μ- type heavy chain constant region, such as a human α-, δ-, ε-, γ-, or μ- type heavy chain constant region. In one embodiment, the light chain or heavy chain constant region is a fragment, derivative, variant, or mutant protein of a naturally occurring constant region.

[0135] Techniques for deriving antibodies from different subclasses or isotypes of an antibody of interest are known, known as subclass conversion. Thus, for example, IgG antibodies can be derived from IgM antibodies, and... vice versa Such techniques allow for the preparation of novel antibodies that possess the antigen-binding properties of a given antibody (parental antibody) but also exhibit biological characteristics associated with antibody isotypes or subclasses different from the parent antibody. Recombinant DNA techniques can be employed. Cloned DNA encoding specific antibody peptides can be used in such procedures, for example, DNA encoding constant domains of an antibody of the desired isotype. See also Lantto et al., 2002. Methods Mol. Biol 178:303-16.

[0136] Single-chain antibodies (scFvs) can be formed by linking heavy and light chain variable domain (Fv region) fragments via amino acid bridges (short peptide linkers, e.g., synthetic sequences of amino acid residues), thereby producing a single polypeptide chain. Such single-chain Fvs (scFvs) have been developed by encoding polypeptides with two variable domains (V... L and V HThe peptides are prepared by fusing DNA encoding peptide linkers between two variable domains. The resulting peptides can fold back to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997). Prot. Eng 10:423; Kortt et al., 2001, Biomol. Eng 18:95-108; Bird et al., 1988, Science 242:423-26; and Huston et al., 1988, Proc. Natl. Acad. Sci USA 85:5879-83). By combining different components containing V L and V H The polypeptide can form polymers scFv that bind to different epitopes (Kriangkum et al., 2001). Biomol. Eng 18:31-40). Techniques for developing single-chain antibodies include those described in: U.S. Patent No. 4,946,778; Bird, 1988. Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci USA 85:5879; Ward et al., 1989, Nature 334:544; de Graaf et al., 2002, Methods Mol Biol 178:379-87.

[0137] In some aspects, the present invention includes ABPs generated from a library of antibody-encoded expression vectors. The library contains 10, 100, 1,000, 10,000, or more than 100,000 different antibody sequences. In some aspects, the ABPs are generated from mammalian cells recombinantly engineered with antibody sequences encoded by a single plasma cell or plasmablast. In some aspects, the ABPs are multivalent because they contain antibodies with different antigen-binding properties. In some embodiments, the ABP binds to multiple epitopes on a target antigen. In some embodiments, the ABP binds to multiple antigens.

[0138] Once cells producing the antibodies of the present invention are obtained using any technique known in the art, specific antibody genes can be cloned by isolating and amplifying DNA or mRNA from them according to the standard procedures described herein. The resulting antibodies can be sequenced and CDRs identified, and the DNA encoding the CDRs can be manipulated as previously described to generate other antibodies according to the present invention.

[0139] The ABP of the present invention is preferably active and / or binds to one or more of the domains described herein in the cell-based methods and / or in vivo methods described herein. Therefore, such binders can be identified using the methods described herein.

[0140] Other antibodies according to the invention can be obtained by conventional immunization and cell fusion procedures as described herein and known in the art.

[0141] The molecular evolution of the complementarity-determining region (CDR) at the antibody binding site center has also been used to isolate antibodies with increased affinity, such as antibodies with increased affinity for c-erbB-2, as described by Schier et al., 1996. J. Mol. Biol As stated in 263:551.

[0142] While human, partially human, or humanized antibodies will be suitable for many applications, particularly those involving the administration of antibodies to human subjects, other types of antigen-binding proteins will be suitable for certain applications. The non-human antibodies of the present invention can, for example, be derived from any antibody-producing animal, such as mice, rats, rabbits, goats, donkeys, or non-human primates (such as monkeys (e.g., cynomolgus monkeys or rhesus monkeys) or apes (e.g., chimpanzees)). The non-human antibodies of the present invention can be used, for example, in in vitro and cell culture-based applications, or any other application in which an immune response to the antibodies of the present invention does not occur, is insignificant, can be prevented, is not a problem, or is required. In one embodiment, the non-human antibody of the present invention is administered to a non-human subject. In another embodiment, the non-human antibody does not elicit an immune response in the non-human subject. In yet another embodiment, the non-human antibody is derived from the same species as the non-human subject, for example, the mouse antibody of the present invention is administered to mice. Antibodies from a specific species can be prepared, for example, by immunizing animals of that species with a desired immunogen or using an artificial system for generating antibodies of that species (e.g., a bacterial or phage display-based system for generating antibodies of a specific species), or by converting an antibody from one species into an antibody from another species (e.g., by replacing the constant region of an antibody with a constant region from another species, or by replacing one or more amino acid residues of the antibody to make it more closely resemble the sequence of an antibody from another species). In one embodiment, the antibody is a chimeric antibody that contains amino acid sequences derived from antibodies from two or more different species.

[0143] Antigen-binding proteins can be prepared and screened for desired properties using any of a number of conventional techniques. Some techniques involve isolating the nucleic acid of the polypeptide chain (or a portion thereof) encoding the ABP of interest and manipulating the nucleic acid using recombinant DNA techniques. For example, the nucleic acid can be fused with another nucleic acid of interest or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues. Furthermore, antigen-binding proteins can be purified from cells that naturally express them (e.g., antibodies can be purified from hybridomas that produce them) or generated in a recombinant expression system using any techniques known in the art. See, for example... Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual , Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0144] Any expression system known in the art can be used to prepare the recombinant polypeptides of the present invention. The expression systems have been fully described above. Typically, host cells are transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Suitable host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as… E. coli or Bacillus ( Bacilli Higher eukaryotic cells include insect cells and established mammalian-derived cell lines. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell line, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70), as described by McMahan et al., 1991, EMBO J. 10: 2821. (This information was provided by Pouwels et al.) Cloning Vectors: A Laboratory Manual Elsevier (New York, 1985) describes suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts.

[0145] Monoclonal antibody (mAb) production cell lines are typically generated by randomly inserting expression constructs into the genome of mammalian production cells (e.g., the CHO genome) (Rita Costa et al., 2010). However, this canonical approach produces cell lines with multiple copies of the mAb inserted into the CHO genome. If we randomly insert our polyclonal antibody construct library into the CHO genome, many clones will express multiple antibodies, leading to frequent non-natural pairing between heavy and light chain Ig. Furthermore, different genomic locations have different levels of transcriptional activity (Kito et al., 2002), which can result in heterogeneous, inconsistent, and / or unstable biological production. Therefore, in some aspects, this invention provides CHO cell lines with a stable Flp recombinase recognition target (FRT) landing pad engineered into the genome. Such site-directed genome-integrated cell lines can be used for stable expression of ABP.

[0146] It should be understood that the antibodies of the present invention may have at least one amino acid substitution, provided that the antibody retains its binding specificity. Therefore, modifications to the antibody structure are covered within the scope of the present invention. These may include amino acid substitutions, which may be conserved or non-conserved, and do not impair the binding ability of the antibody containing ABP. Conservative amino acid substitutions may cover amino acid residues that are not naturally occurring and are typically incorporated through chemical peptide synthesis rather than synthesis in a biological system. These include peptide mimics and other reversed or inverted forms of amino acid moieties. Conservative amino acid substitutions may also involve replacing native amino acid residues with standard residues such that the polarity or charge of the amino acid residue at that position has little or no effect.

[0147] Non-conservative substitution can involve exchanging a member of one class of amino acids or amino acid mimics for a member of another class with different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues can be introduced into homologous regions of human antibodies or into non-homologous regions of the molecule.

[0148] Furthermore, those skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. Variants can then be screened using activity methods known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if a change to a particular amino acid residue is found to result in disruptive, undesirably reduced, or unsuitable activity, variants with such changes can be avoided. In other words, based on information gathered from these routine experiments, those skilled in the art can readily determine which amino acids should be avoided, alone or in combination with other mutations.

[0149] Those skilled in the art will be able to use well-known techniques to identify suitable variants of the peptides described herein. In some embodiments, those skilled in the art can identify suitable regions in the molecule that can be altered without destroying activity by targeting regions considered unimportant to activity. In some embodiments, conserved residues and portions of the molecule in similar peptides can be identified. In some embodiments, conserved amino acid substitutions can be made even in regions that may be important for biological activity or for structure, without destroying biological activity or adversely affecting the peptide structure.

[0150] Furthermore, those skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on this comparison, the importance of amino acid residues in the protein corresponding to amino acid residues important for activity or structure in similar proteins can be predicted. Those skilled in the art can then choose chemically similar amino acids to replace such predicted important amino acid residues.

[0151] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence associated with that structure in similar peptides. Given this information, those skilled in the art can predict the alignment of the antibody's amino acid residues relative to its three-dimensional structure. In some embodiments, those skilled in the art may choose not to fundamentally alter the predicted amino acid residues located on the protein surface, as such residues may be involved in important interactions with other molecules.

[0152] Many scientific publications have dedicated themselves to predicting secondary structures. See Moult. J., Curr. Op. in Biotech ., 7(4):422-427 (1996); Chou et al., Biochem. , 13(2):222-245 (1974); Chou et al., Biochem. 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol ., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem ., 47:251-276; and Chou et al., Biophys. J ., 26:367-384 (1979). Furthermore, computer programs are currently available to assist in the prediction of secondary structures. One approach to predicting secondary structures is based on homology modeling. For example, two peptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. Recent developments in protein structure databases (PDBs) have enhanced the predictability of secondary structures, including the potential number of folds within the peptide or protein structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been proposed (Brenner et al., Curr. Op. Struct. Biol ., 7(3):369-376 (1997)), there is a finite number of folds in a given polypeptide or protein, and once the critical number of structures has been resolved, structure prediction becomes significantly more accurate.

[0153] Other methods for predicting secondary structures include "threading" (Jones, D., Curr. Opin. Struct. Biol .,7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), “Spectral Analysis” (Bowie et al., Science 253:164-170 (1991); Gribskov et al., Meth. Enzym ., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci ., 84(13):4355-4358 (1987)), and “evolutionary linkage” (see Holm, ibid. (1999), and Brenner, ibid. (1997)).

[0154] In some embodiments, antibody variants include glycosylated variants, wherein the number and / or type of glycosylation sites have been altered compared to the amino acid sequence of the parent polypeptide. In some embodiments, the variants contain more or fewer N-linked glycosylation sites than the native protein. N-linked glycosylation sites are characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue other than proline. Substituting amino acid residues to produce this sequence provides potential new sites for adding N-linked glycans. Alternatively, substitutions that eliminate this sequence will remove existing N-linked glycans. Rearrangements of N-linked glycans are also provided, wherein one or more N-linked glycosylation sites (typically those naturally present) are eliminated and one or more new N-linked sites are generated. Additional preferred antibody variants include cysteine ​​variants, wherein one or more cysteine ​​residues are missing or substituted with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be useful when the antibody must be refolded into a biologically active conformation, such as after the separation of insoluble inclusion bodies. Cysteine ​​variants typically have fewer cysteine ​​residues than the natural protein and are usually even in number to minimize interactions caused by unpaired cysteine ​​residues.

[0155] According to certain embodiments, preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter the binding affinity, and / or (4) impart or modify other physiological, chemical, or functional properties to such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in some embodiments, conserved amino acid substitutions) can be made in naturally occurring sequences (in some embodiments, in the polypeptide portion outside the domains forming intermolecular contacts). In some embodiments, conserved amino acid substitutions generally do not substantially alter the structural characterization of the parental sequence (e.g., the substituted amino acid should not tend to disrupt the helices present in the parental sequence or disrupt other types of secondary structures characterizing the parental sequence). Examples of recognized peptide secondary and tertiary structures in this field are described in the following: Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, ed., Garland Publishing, New York, NY (1991)); and Thornton. et al . Nature 354:105 (1991), these references are incorporated into this paper by reference.

[0156] In some embodiments, the antibodies of the present invention may be chemically bonded to polymers, lipids, or other components.

[0157] The binder may comprise at least one of the CDRs described herein incorporated into the biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support, framework, or scaffold capable of displaying one or more amino acid sequences (e.g., CDRs, variable regions, etc.) bound to an antigen in local surface regions. Such structures may be naturally occurring polypeptides or polypeptide “folds” (structural motifs), or may have one or more modifications relative to naturally occurring polypeptides or folds, such as the addition, deletion, or substitution of amino acids. These scaffolds may be derived from polypeptides of any species (or more than one species), such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0158] Typically, biocompatible framework structures are based on protein scaffolds or backbones other than immunoglobulin domains. For example, those based on fibronectin, ankylosing skeletal proteins, lipid transport proteins, neo-oncogenes, cytochrome b, CP1 zinc finger, PST1, coiled helices, LACI-D1, Z domains, and amylase aprotinin domains can be used (see, for example, Nygren and Uhlen, 1997). Curr. Opin. in Struct (Biol., 7, 463-469).

[0159] It should be understood that the antibodies of the present invention include the humanized antibodies described herein. Humanized antibodies (such as those described herein) can be produced using techniques known to those skilled in the art (Zhang, W., et al.). Molecular Immunology. 42(12):1445-1451, 2005; Hwang W. et al., Methods. 36(1):35-42, 2005; Dall'Acqua WF, et al. Methods 36(1):43-60, 2005; And Clark, M., I mmunology Today. 21(8):397-402, 2000)。

[0160] The peptides and proteins of the present invention can be purified using protein purification techniques well known to those skilled in the art. These techniques involve, to a certain extent, the coarse fractionation of protein and non-protein fractions. After separating the peptides from other proteins, the peptides or polypeptides of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogenization). As used herein, the term "purified peptide" is intended to refer to a composition that is separable from other components, wherein the peptide is purified to any degree relative to its naturally available state. Thus, a purified peptide also refers to a peptide removed from the environment in which it may naturally exist. Generally, "purified" will refer to a peptide composition that has been fractionated to remove various other components, and the composition substantially retains its expressed biological activity. When using the term "substantially purified," this expression will refer to a peptide or polypeptide composition in which the peptide or polypeptide forms the major component of the composition, such as about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0161] Various purification techniques will be well known to those skilled in the art at ABP. These include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), or by heat denaturation followed by centrifugation; chromatographic methods such as affinity chromatography (protein-A column), ion exchange, gel filtration, reversed-phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, isoelectric focusing, gel electrophoresis, and combinations of these techniques. As is generally known in the art, it is believed that the order of various purification steps can be altered, or certain steps can be omitted, and suitable methods for preparing substantially purified peptides can still be achieved. Exemplary purification steps are provided in the examples below.

[0162] In some embodiments, the purification process includes one or more steps selected from: clarification, affinity chromatography, virus inactivation and deep filtration, hydrophobic interaction chromatography or membrane filtration, multimode anion exchange chromatography or membrane filtration, multimode cation exchange chromatography, cation exchange chromatography, virus filtration, and UF / DF. In some embodiments, protein concentration is measured between each step.

[0163] According to this disclosure, various methods for quantifying the degree of purification of peptides will be known to those skilled in the art. These include, for example, determining the specific binding activity of the active fraction, or assessing the amount of peptides or polypeptides within the fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a peptide fraction is to calculate the binding activity of the fraction and compare it with the binding activity of the initial extract, thereby calculating the degree of purification, which is assessed herein by way of “purification fold”. Of course, the actual unit used to express the amount of binding activity will depend on the specific method technique chosen after purification and whether the peptide or polypeptide exhibits detectable binding activity.

[0164] The binding affinity of the ABP (e.g., antibody) according to the present invention to the antigen target can be less than or equal to 5 x 10⁻⁶. - 7 M, less than or equal to 1 x 10 -7 M, less than or equal to 0.5 x 10 -7 M, less than or equal to 1 x 10 -8 M, less than or equal to 1 x 10 -9 M, less than or equal to 1 x 10 -10 M, less than or equal to 1 x 10 -11 M is less than or equal to 1 x 10 -12 M.

[0165] The affinity of ABP and the degree of antibody-inhibitory binding can be determined by those skilled in the art using conventional techniques, such as those described by Scatchard et al. Ann. NY Acad. Sci.Those described in 51:660-672 (1949) or via surface plasmon resonance (SPR; BIAcore, Biosensor, Piscataway, NJ). For SPR, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase running along a flow cell. If binding of the ligand to the immobilized target occurs, a local refractive index change results in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to obtain the apparent rate constants of the association and dissociation phases of the binding reaction. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53:2560-65 (1993)).

[0166] 5.5. Pharmaceutical Compositions One aspect of this disclosure provides a pharmaceutical composition comprising an ABP library that is tested by the methods disclosed herein and / or is acceptable under the acceptance criteria disclosed herein. In some embodiments, the pharmaceutical composition comprises the test library disclosed above. In some embodiments, the pharmaceutical composition is tested by the methods disclosed herein.

[0167] Such pharmaceutical compositions comprise a therapeutically or preventively effective amount of ABP, as well as pharmaceutically acceptable and physiologically acceptable formulation materials. In some embodiments, the effective amount is determined based on results obtained from the methods provided herein.

[0168] Pharmaceutical compositions may contain formulation materials that are used to alter, maintain, or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition.

[0169] Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, and other organic acids); fillers (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); and complexing agents (such as caffeine, polyvinylpyrrolidone, and β-cyclohexane). ; fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight peptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbitol) Acids or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic acid, PEG, dehydrated sorbitol esters, polysorbates (such as polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyrosine); stability enhancers (sucrose or sorbitol); tensile enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride, mannitol)). Sorbitol; delivery medium; diluent; excipient and / or adjuvant. Neutral buffered saline or saline mixed with the same serum albumin is an example of a suitable diluent. Preservatives, such as benzyl alcohol, may also be added according to appropriate industry standards. A suitable excipient solution (e.g., sucrose) can be used as a diluent to formulate the composition into a lyophilized form. Suitable components are non-toxic to the recipient at the doses and concentrations used. Other examples of components that can be used in pharmaceutical formulations are presented in Remington's Pharmaceutical Sciences, 16th edition (1980) and 20th edition (2000), Mack Publishing Company, Easton, PA.

[0170] Optionally, the composition further comprises one or more physiologically active agents, such as anti-angiogenic substances, chemotherapeutic substances (such as capecitabine, 5-fluorouracil, or doxorubicin), analgesics, etc., non-exclusive examples of which are provided herein. In various specific embodiments, in addition to ABP, the composition comprises one, two, three, four, five, or six physiologically active agents.

[0171] In another embodiment of the invention, the compositions disclosed herein can be formulated into neutral or salt forms. Illustrative pharmaceutically acceptable salts include acid addition salts (forming with the free amino groups of proteins) and salts formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. After formulation, the solution will be administered in a dosage form compatible and in a therapeutically effective amount.

[0172] Carriers may also contain any or all solvents, dispersion media, catalysts, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption-retarding agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions should be considered unless any conventional media or reagent is incompatible with the active ingredient. Additional active ingredients may also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to humans.

[0173] The optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery method, and required dosage. See, for example, Remington's Pharmaceutical Sciences, ibid. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the peptide. For example, suitable compositions may be water for injection or physiological saline solutions for parenteral administration.

[0174] 5.5.1. Drug active ingredient content In some embodiments, the active ingredient (i.e., the protein and peptide of the present invention) is present in the pharmaceutical composition at a concentration of at least 0.01 mg / ml, at least 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml. In some embodiments, the pharmaceutical composition comprises at least 10 mg / ml, at least 100 mg / ml, at least 500 mg / ml, or at least 1000 mg / ml of ABP. In some embodiments, the pharmaceutical composition comprises from 10 mg / ml to 100 mg / ml of ABP or from 10 mg / ml to 500 mg / ml of ABP.

[0175] In some embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, or 500 mg / ml. In some embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml.

[0176] 5.5.2. Overview of the formulation The pharmaceutical composition may be in any form suitable for human or veterinary use, including liquids, oils, emulsions, gels, colloids, aerosols, or solids.

[0177] The pharmaceutical composition can be formulated for administration via any route of administration suitable for human or veterinary medicine, including enteral and parenteral administration.

[0178] In various embodiments, the pharmaceutical composition is formulated for administration by inhalation. In some of these embodiments, the pharmaceutical composition is formulated for administration via a vaporizer. In some of these embodiments, the pharmaceutical composition is formulated for administration via a nebulizer. In some of these embodiments, the pharmaceutical composition is formulated for administration via an aerosol nebulizer.

[0179] In various embodiments, the pharmaceutical composition is formulated for oral administration, oral administration, or sublingual administration.

[0180] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.

[0181] In some embodiments, the pharmaceutical composition is formulated for intrathecal or intraventricular administration.

[0182] In some implementations, the pharmaceutical composition is formulated for topical application.

[0183] In some embodiments, the pharmaceutical composition is formulated for injection or infusion.

[0184] 5.5.3. Pharmacological compositions suitable for injection For intravenous, subcutaneous, or subcutaneous injection, or injection at the site of pain, the active ingredient will be in a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using, for example, isotonic mediators (such as sodium chloride injection, Ringer's solution, lactated Ringer's solution). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0185] In various embodiments, the unit dosage form is a vial, ampoule, bottle, or pre-filled syringe. In some embodiments, the unit dosage form contains 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg, or 100 mg of the pharmaceutical composition. In some embodiments, the unit dosage form contains 125 mg, 150 mg, 175 mg, or 200 mg of the pharmaceutical composition. In some embodiments, the unit dosage form contains 250 mg of the pharmaceutical composition.

[0186] In typical embodiments, the unit dosage form of the pharmaceutical composition is in liquid form. In various embodiments, the unit dosage form contains 0.1 mL to 50 mL of the pharmaceutical composition. In some embodiments, the unit dosage form contains 1 mL, 2.5 mL, 5 mL, 7.5 mL, 10 mL, 25 mL, or 50 mL of the pharmaceutical composition. In some embodiments, the unit dosage form contains more than 50 mL of the pharmaceutical composition.

[0187] In certain embodiments, the unit dosage form is a vial containing 1 ml of a pharmaceutical composition with a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml. In some embodiments, the unit dosage form is a vial containing 2 ml of a pharmaceutical composition with a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml.

[0188] In some implementations, the unit dosage form of the pharmaceutical composition is in a solid form suitable for solubilization, such as a lyophilized product.

[0189] Unit dosage form implementations suitable for subcutaneous, intradermal, or intramuscular administration include pre-filled syringes, auto-injectors, and auto-injection pens, each containing a predetermined amount of the pharmaceutical composition described above.

[0190] In various embodiments, the unit dosage form is a pre-filled syringe, comprising a syringe and a predetermined amount of pharmaceutical composition. In some pre-filled syringe embodiments, the syringe is suitable for subcutaneous administration. In some embodiments, the syringe is suitable for self-administration. In a particular embodiment, the pre-filled syringe is a disposable syringe.

[0191] In various embodiments, the pre-filled syringe contains about 0.1 mL to about 0.5 mL of the pharmaceutical composition. In some embodiments, the syringe contains about 0.5 mL of the pharmaceutical composition. In a particular embodiment, the syringe contains about 1.0 mL of the pharmaceutical composition. In a specific embodiment, the syringe contains about 2.0 mL of the pharmaceutical composition.

[0192] In some embodiments, the unit dosage form is an auto-injection pen. An auto-injection pen includes an auto-injection pen containing a pharmaceutical composition as described herein. In some embodiments, the auto-injection pen delivers a predetermined volume of pharmaceutical composition. In other embodiments, the auto-injection pen is configured to deliver a volume of pharmaceutical composition set by the user.

[0193] In various embodiments, the auto-injector contains about 0.1 mL to about 5.0 mL of the pharmaceutical composition. In a specific embodiment, the auto-injector contains about 0.5 mL of the pharmaceutical composition. In a particular embodiment, the auto-injector contains about 1.0 mL of the pharmaceutical composition. In other embodiments, the auto-injector contains about 5.0 mL of the pharmaceutical composition.

[0194] 5.5.4. A mixture of plasma IVIg and recombinant hyperimmune agents In some embodiments, recombinant hyperimmune agents are incorporated into conventional plasma IVIg to increase the antipathogenic titer of IVIg. In some embodiments, several antipathogenic recombinant hyperimmune agents are incorporated into conventional plasma IVIg; for example, hyperimmune agents against Hib, pneumococcus, influenza A virus, and tetanus are simultaneously incorporated into plasma IVIg to treat patients with primary immunodeficiency. Incorporation of hyperimmune agents increases antibody titers against pathogens to which patients with primary immunodeficiency are particularly susceptible. Any number of additive formulations can be mixed with plasma IVIg to generate increased titers against any number of pathogens.

[0195] In some implementations, the additive recombinant hyperimmune agent is mixed with plasma IVIg by a pharmacist. In some implementations, the additive recombinant hyperimmune agent is mixed with plasma IVIg by the manufacturer.

[0196] 5.6. Instructions for Use The methods disclosed herein can be used to analyze or characterize ABP-containing libraries during or after library generation. The methods disclosed herein can be used to analyze or characterize cell lines used to generate ABP-containing libraries. In some embodiments, the method is used to select cell lines capable of generating high-quality ABP-containing libraries. In some embodiments, the method is used to confirm ABP generation and / or ensure the quality of ABP. In some embodiments, the method is used before, during, or after generating a pharmaceutical composition containing an ABP-containing library. In some embodiments, the method is used to test the pharmaceutical composition during or after distribution. In some embodiments, the method is used to test the pharmaceutical composition prior to therapeutic use of the composition. Therefore, this disclosure provides a method for treating a patient with a library containing ABP previously tested using the methods disclosed herein.

[0197] The terms “treatment”, “treating,” etc., are used herein to generally mean achieving a desired pharmacological and / or physiological effect. An effect may be preventative in relation to the complete or partial prevention of a disease, condition, or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease or condition and / or adverse reactions (such as symptoms) attributable to the disease or condition. As used herein, “treatment” encompasses any treatment of a disease or condition in mammals (particularly humans) and includes: (a) preventing the occurrence of a disease or condition in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing a disease or condition (e.g., preventing its development); or (c) alleviating a disease or condition (e.g., causing it to subside, providing improvement in one or more symptoms). Improvement in any condition can be readily assessed using standard methods and techniques known in the art. The population of subjects treated by disease-based methods includes subjects with an undesirable condition or disease, as well as subjects at risk of developing a condition or disease.

[0198] In vivo and / or in vitro methods may be optionally used to help determine the optimal dose range. The precise dose used in the formulation will also depend on the route of administration and the severity of the condition, and should be determined based on the practitioner's judgment and the individual subject's circumstances. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0199] In some embodiments, the methods disclosed herein are used to ensure the quality or potency of ABP in the library. In some embodiments, the methods disclosed herein are used to adjust the optimal dosage range for the therapeutic use of the library.

[0200] The actual amount administered, as well as the rate and timing of administration, will depend on the nature and severity of the disease being treated. Treatment prescriptions (e.g., decisions regarding dosage, etc.) fall within the responsibility of general practitioners and other physicians, and generally take into account the condition to be treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols described above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0201] In some embodiments, the pharmaceutical composition is administered by inhalation, oral administration, oral application, sublingual administration, injection, or local application.

[0202] In some implementations, the pharmaceutical composition is administered once daily, 2-4 times daily, 2-4 times weekly, once weekly, or once every two weeks.

[0203] 6. Example The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental error and deviation should certainly be allowed.

[0204] Unless otherwise stated, the practice of this invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are within the scope of the art. Such techniques are well explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., latest edition); Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (Edited by S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (Plenum Press) Volumes A and B (1992).

[0205] 6.1.1. Example 1: Generation of an ABP library with activity targeting human thymocytes or T cells Four libraries of ABP targeting human thymocytes or T cells were generated, namely recombinant human anti-thymocyte globulin (rhATG). In vitro and in vivo studies were conducted to demonstrate the functional similarity between this rhATG and commercially available rabbit-ATG (Thymoglobulin, Sanofi).

[0206] Commercially available anti-thymocyte globulin (ATG, (Sanofi)) can be used to induce transplant tolerance and is prepared by immunizing New Zealand rabbits with human thymocytes; blood is harvested from thousands of animals and antibodies are purified from plasma. The ABP library disclosed in this paper (i.e., rhATG) combines the efficacy advantages of polyclonal ATG with the safety advantages of a fully human recombinant ABP library.

[0207] First, transgenic mice carrying the inserted human immunoglobulin gene were immunized with human thymocytes or human T cells. Two Trianni mice were injected into their paw pads twice weekly for three weeks, followed by booster immunizations over the next two weeks. One to two million thymocytes were injected into each mouse at each time point. Before the final booster immunization, serum titers of thymocyte antibodies were assessed by flow cytometry using a series of serum dilutions from 1:200 to 1:145,000 per animal. We observed strong serum responses in both animals, with one showing a slightly stronger response. After sacrifice, lymph nodes (popliteal, groin, axillary, and mesentery) were surgically removed. Single-cell suspensions were prepared from each animal by manual disruption followed by passing through a 70µm filter. Next, B cells were isolated from each sample using the EasySep™ Mouse Pan-B Cell Isolation Kit (Stemcell Technologies) negative selection kit. Lymph node B cell populations were quantified by counting on a C-Chip hematology counter (Incyto) and viability was assessed using trypan blue. Cells were then diluted to 5,000–6,000 cells / mL in phosphate-buffered saline (PBS) containing 12% OptiPrep™ density gradient medium (Sigma). This cell mixture was used for microfluidic encapsulation. We ran approximately one million B cells from each of six animals using our emulsion droplet microfluidic platform.

[0208] DNA libraries encoding scFv derived from single-cell RNA are generated using emulsion droplet microfluidic platforms or vortex emulsions, where the native heavy-light Ig pairings are intact. Methods for generating the DNA libraries include 1) poly(A)+ mRNA capture, 2) multiple overlap extension reverse transcriptase polymerase chain reaction (OE-RT-PCR), and 3) nested PCR to remove artifacts and add adaptors for deep sequencing or yeast display libraries. The scFv libraries are generated from approximately one million B cells from each animal that achieved a positive titer.

[0209] For poly(A)+ mRNA capture, a custom-designed co-flow emulsion droplet microfluidic chip (Dolomite) made of glass was used. The microfluidic chip has two input channels for the fluorocarbon oil (Dolomite), one input channel for the aforementioned cell suspension mixture, and one input channel for oligomeric dT beads (NEBs) at a concentration of 1.25 mg / ml in cell lysis buffer (20 mM Tris pH 7.5, 0.5 M NaCl, 1 mM EDTA, 0.5% Tween-20, and 20 mM dithiothreitol). For most of the chip's length, the input channels were etched to 50 µm × 150 µm, narrowing to 55 µm at the droplet junction, and coated with hydrophobic Pico-Glide (Dolomite). The liquid was pumped through the chip using three Mitos P-Pump pressure pumps (Dolomite). Droplet size depended on the pressure, but droplets with a diameter of approximately 45 µm were generally optimally stable. The emulsion was collected into cooled 2 ml microcentrifuge tubes and incubated at 40°C for 15 minutes to capture mRNA. Beads were extracted from the droplets using Pico-Break (Dolomite). In some embodiments, vortexing was used to prepare similar single-cell separated emulsions.

[0210] For multiplex OE-RT-PCR, a glass Telos droplet emulsion microfluidic chip (Dolomite) was used. mRNA-binding beads were resuspended in the OE-RT-PCR mixture and injected into the microfluidic chip with a mineral oil-based surfactant mixture (available from GigaGen) under pressure to generate 27 µm droplets. The OE-RT-PCR mixture contained 2x one-step RT-PCR buffer, 2.0 mM MgSO4, SuperScript III reverse transcriptase, and Platinum Taq (Thermo Fisher Scientific), plus a mixture of primers targeting the IgK C region, IgG C region, and all V regions. The overlapping region was a DNA sequence encoding a Gly-Ser-rich scFv adapter sequence. DNA fragments were recovered from the droplets using a droplet rupture solution (available from GigaGen) and then purified using a QIAquick PCR purification kit (Qiagen). In some embodiments, a similar OE-RT-PCR emulsion was prepared using vortexing.

[0211] For nested PCR, the purified OE-RT-PCR product was first run on a 1.7% agarose gel at 150 V for 80 minutes. A 1200-1500 bp band corresponding to the ligation product was excised and purified using NucleoSpin Gel and the PCR Clean-up Kit (Macherey Nagel). PCR was then performed to add an adaptor for Illumina sequencing or yeast display; for sequencing, a seven-nucleotide random sequence was added to increase base calling accuracy in subsequent next-generation sequencing steps. Nested PCR was performed using 2x NEBNext high-fidelity amplification mixture (NEB) with primers containing the Illumina adaptor or primers for cloning into yeast expression vectors. The nested PCR product was run on a 1.2% agarose gel at 150 V for 50 minutes. An 800-1100 bp band was excised and purified using NucleoSpin Gel and the PCR Clean-up Kit (Macherey Nagel).

[0212] To convert the GigaLink™ scFv library into a full-length CHO expression library, nested external PCR primers were used to add adaptors with overhangs for Gibson assembly to the 5' and 3' ends of the scFv library. The scFv library was then inserted into a vector containing a single promoter, a secretory leader sequence of light chain Ig, and the remainder of the IgG1 constant region using the NEBuilder HiFi DNA assembly master mix (NEB, Ipswich, MA, USA), resulting in a cloned scFv library. This intermediate library was then transformed into… E. coli In this process, the plasmids were plated onto LB-ampicillin plates, and 0.5–1 million colonies were scraped and pooled for plasmid purification using the ZymoPURE II Plasmid Maxiprep kit (Zymo Research, Irvine, CA, USA). To generate a full-length antibody library, the product of GA1 was linearized with BamHI-HF (NEB, Ipswich, MA, USA) and used as a vector to insert a synthetic amplicon containing a portion of the light chain Ig constant region, the poly(A) signal of the light chain Ig, the promoter of the IgG gene, and the secretory leader sequence of the IgG gene for a second Gibson assembly. The full-length library was then transformed into… E. coli The plasmids were plated on LB-ampicillin plates, and more than 500,000 colonies were scraped off. The plasmids were then purified using the ZymoPURE IIPlasmid Maxiprep kit (Zymo Research) to prepare a full-length recombinant hyperimmune Maxiprep library for transfection.

[0213] Adherent Flp-In™-CHO cell lines with FRT sites for genome integration (Thermo Fisher Scientific, Waltham, MA, USA) were adapted to suspension culture. For all steps in the adaptation process, “Ham’s F-12” refers to Ham’s F-12 (containing L-glutamine, Thermo Fisher Scientific, Waltham, MA, USA) plus 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and “BalanCD” refers to BalanCD CHO Growth A (Irvine Scientific) containing 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA). To adapt this cell line to suspension, the cells were first passaged into a mixture of 50% Ham’s F-12 and 50% BalanCD in a T-flask. Next, the cells were passaged into a mixture of 25% Ham’s F-12 and 75% BalanCD and transferred to a shaken Erlenmeyer flask. The cells were then passaged into 10% Ham's F-12, 90% BalanCD + 0.2% anti-caking agent (Irvine Scientific, Santa Ana, CA, USA) and stored for future use.

[0214] Using Amaxa Nucleofector 4D (SG buffer, pulsed DU133; Lonza, Basel, Switzerland), 100 million adapted Flp-In CHO cells were transfected with each recombinant hyperimmune preparation library. These cells were seeded into shaken Erlenmeyer flasks and incubated at 37°C and 125 rpm for 48 hours. After 48 hours, cells were counted to determine viability, and cells were seeded at 1 million cells / mL and selection was initiated in fresh medium using 600 μg / mL hygromycin-B (Gemini Bio, West Sacramento, CA, USA). During the 7-day selection period, cells were counted and the medium was changed every 2–3 days. During expansion, the library was maintained at 600 μg / mL hygromycin-B (Gemini Bio, West Sacramento, CA, USA) until viability exceeded 95%. When cells were >95% viable and doubled every 24 hours, the cell lines were preserved for liquid nitrogen storage.

[0215] CHO cells stably expressing the antibody library were grown in a medium consisting of 90% BalanCD CHO Growth A medium (Irvine Scientific, Santa Ana, CA), 9% Ham's F-12 (Thermo Fisher Scientific, Waltham, MA, USA), 1% FBS (Thermo Fisher Scientific), 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and 0.2% anti-caking agent (Irvine Scientific, Santa Ana, CA, USA). For small-scale production, cells were grown at a density of 1 × 10⁶ cells / year. 6Cells / mL were seeded into 50 mL of medium in 250 mL Erlenmeyer flasks and grown at 37 °C, 5% CO2, and 125 rpm. Cells were continuously grown under these conditions, supplemented with 7.5 mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) on days 2, 4, and 7 of the production run. On day 8, the supernatant was harvested by centrifugation and subsequently filtered through a 0.22 μm 250 mL filter flask (EMD Millipore, Burlington, MA, USA) with a 1 μm pre-filter. The harvested cell culture medium (HCCF) was stored at 4 °C until protein A purification. For large-scale production of the plasma cell recombinant hyperimmune agent, cells were grown in the same medium, but with some modifications to the production conditions. Seed cultures were used to grow the culture from 2 × 10⁶ cells / mL at 37 °C. 7 The number of cells increased to 1.2 × 10⁻⁶. 10 Cells. Then the cells are divided into 1×10⁻⁶ cells. 6 Cells / mL were seeded at 2L in 5L flasks (triple, day 0). On day 2, the temperature was changed from 37°C to 33°C. On days 2, 4, 6, 8, 10, and 13, 300mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) was added to each flask. The supernatant was harvested on day 14.

[0216] After harvest, HCCF was purified using MabSelect SuRe protein A resin (GE Life Sciences, Marlborough, MA, USA) with the following buffers: equilibration buffer, top wash (Chase), wash buffer 2 (25 mM Tris, 150 mM NaCl, pH 7.4), wash buffer 1 (25 mM Tris, 1 M NaCl, pH 7.4), elution buffer (20 mM citric acid, pH 3.0), and neutralization buffer (100 mM Tris, pH 8.0 for small-scale; 1 M Tris, pH 9.0 for large-scale). The column was sterilized with 0.1 N NaOH before and after use. For large-scale production of recombinant hyperimmune plasma cell preparations, an additional wash buffer 3 consisting of 0.5 M arginine, pH 7.4 was used, followed by an additional wash with wash buffer 2 before elution. The purification steps were performed in the following order: equilibration, loading, top washing, wash 1, wash 2 (for large-scale production: wash 3, wash 2), elution, and neutralization (manually added to the tube used to collect the eluent fraction). Recombinant hyperimmune preparations (ABP) were concentrated using Vivaspin 20, 30 kDa molecular weight cutoff centrifuge tubes (Sartorius, Göttingen, Germany) and prepared in PBS (for small-scale production) or 0.2 M glycine, pH 4.5 (for large-scale production), followed by 0.22 μm filtration.

[0217] ELISA was used to test the binding of rhATG (i.e., anti-T cell and anti-thymocyte ABP) to antigens known to be expressed on the surface of T cells and thymocytes. ELISA showed binding to CD4, CD45, and CD81. Antigens were coated onto ELISA plates at 1 μg / mL. Titration curves were plotted starting at 100 μg / mL for each antibody with stepwise 1 / 3 dilutions to determine the EC50. Because different secondary detection antibodies were used, EC50 values ​​between rabbit-ATG and rhATG cannot be directly compared. However, it was determined that within each library, the antigens exhibited stronger binding than their respective background levels. For both rhATG and rabbit-ATG, antibody responses were broadly reactive to many T cell antigens, with both binding very strongly to CD45 and CD5, and weakly to CD4, CD11, and CD81 (data not shown).

[0218] In vivo validation studies were conducted. An in vivo model of GvHD (graft-versus-host disease) was used to demonstrate the functional efficacy of ATG treatment in delaying GvHD. One x 10^7 human PBMCs from a single donor were transplanted into NSG mice. The study used six mice per group, with IV infusions of the test drugs: rhATG (ABP), commercial rabbit-ATG, and a solvent control. Animals were treated at a single time point 7 days post-implantation (6 mg / kg). Additionally, a positive control group (8 mice) received abatacept, a drug commonly used for GvHD prophylaxis, administered intraperitoneally (IP) every other day from day 5 until the end of the study. Immune cell expansion, indicating progression to GvHD, was measured by flow cytometry, and the animals were monitored for weight loss and clinical manifestations of GvHD leading to death.

[0219] Forty-two days after PBMC implantation, any surviving animals were removed, and survival analysis was performed for each treatment group. No significant delay was observed with rhATG. p =0.2, Mantel-Cox), and only a slight delay in GvHD was observed with rabbit-ATG ( p =0.01, Mantel-Cox (data not shown). Flow cytometry was used to measure transplanted PBMCs before treatment, 2 days after treatment, and 9 days after treatment. rhATG and rabbit-ATG depleted CD45 + Cells, as observed 2 days after treatment, resulted in CD45... + Complete cell implantation was delayed; however, by day 9, there were no significant differences between any groups (data not shown).

[0220] The results showed that rhATG (ABP library) had an antigen-specific antibody binding profile similar to currently commercially available rabbit-ATG, although some differences were observed. Furthermore, rhATG also showed similar performance to commercial rabbit-ATG in delaying the progression to GvHD in mice using different dosing regimens.

[0221] 6.1.2. Example 2: An ABP library with activity against Haemophilus influenzae type b (Hib) from a human donor. The generation Conduct in vitro and in vivo studies to test for the effects against type b. Haemophilus influenzae The test used an active polyclonal antibody library (pAb) for Hib, specifically an ABP library. The tested antibodies were anti-Hib pAbs prepared from four different B-cell subtypes collected from donors vaccinated with the Pedvax-HIB conjugate. The four subtypes tested were CD43... + Plasma blasts, CD27+ memory B cells, peripheral CD138 + Plasma cells and pan-B cells (all B cells). All four pAbs were first tested in vitro. (By CD138) +pAb made from plasma cells is most effective in vitro, so this product was then tested relative to IVIG in an in vivo challenge model.

[0222] Two donors (Donor 1, a 26-year-old white female, and Donor 2, a 21-year-old Asian male) were vaccinated with the Pedvax Hib vaccine (Merck, Kenilworth, NJ, USA) using a CRO (BloodCenter Wisconsin, Milwaukee, WI, USA). Leukalysis was performed eight or nine days later to obtain PBMCs. Simultaneously, plasma was separated from separate blood samples on the day of leukalysis and prior to vaccination. ELISA (Alpha Diagnostics, San Antonio, TX, USA; see Methods below) of the Hib-resistant plasma samples confirmed a vaccine response compared to plasma from the same donor prior to vaccination. The sample collection protocol was approved by the Institutional Review Board (IRB) protocol #PRO00028063 (Medical College of Wisconsin / Froedtert Hospital IRB) for GigaGen. Informed consent was obtained from all participants, and samples were transported to GigaGen for deidentification.

[0223] To isolate pan-B cells, we used the Human EasySep pan-B cell enrichment kit (Stemcell #19554, Vancouver, BC, Canada). To isolate CD43... + For cells, we used pan-B cells and CD43 positive-selected beads (Miltenyi #130-091-333, Bergisch Gladbach, Germany). To isolate CD27... + We applied CD27-positive selection beads (Miltenyi #130-051-601, Bergisch Gladbach, Germany) to cells derived from CD43. +Negative fractions were selected. For plasma cells, we used the EasySep Human CD138 Positive Selection Kit (Stemcell #18357, Vancouver, BC, Canada) on PBMCs. After isolation, antibody-generating cells were cryopreserved using CryoStor® CS10 (Stemcell Technologies, Vancouver, BC, Canada). Immediately before generating paired heavy and light chain libraries, cells were thawed, washed in cold DPBS + 0.5% BSA, assessed viability with trypan blue on a Countess™ cell counter (Thermo Fisher Scientific, Waltham, MA, USA), and then resuspended at 5,000–6,000 cells / μl in 12% OptiPrep™ density gradient medium (Sigma, St. Louis, MO, USA). This cell mixture was then used for microfluidic encapsulation as described in the next section.

[0224] Generate scFv libraries from antibody-producing cells (Adler et al.) Mab 9, 1282-1996, 2017) comprises three steps: (i) poly(A)+ mRNA capture, (ii) multiple overlap extension reverse transcriptase polymerase chain reaction (OE-RT-PCR), and (iii) nested PCR to remove artifacts and add adaptor sequences for deep sequencing or yeast display libraries.

[0225] To convert the GigaLink™ scFv library into a full-length CHO expression library, we first used nested external PCR primers to add adaptors with overhangs for Gibson assembly to the 5' and 3' ends of the scFv library. Then, using the NEBuilder HiFi DNA assembly master mix (NEB, Ipswich, MA, USA), we inserted the scFv library into a vector containing a single promoter, a secretory leader sequence of light chain Ig, and the remainder of the IgG1 constant region, producing a cloned scFv library. This intermediate library was then transformed into… E. coliIn this study, plating was performed on LB-ampicillin plates, and 0.5–1 million colonies were scraped and pooled for plasmid purification using the ZymoPURE II Plasmid Maxiprep kit (Zymo Research, Irvine, CA, USA). To generate a full-length antibody library, we linearized the GA1 product with BamHI-HF (NEB, Ipswich, MA, USA) and used it as a vector to insert a synthetic amplicon containing a portion of the light chain Ig constant region, the poly(A) signal of the light chain Ig, the promoter of the IgG gene, and the secretory leader sequence of the IgG gene for a second Gibson assembly. The full-length library was then transformed into… E. coli The plasmids were then plated onto LB-ampicillin plates. We typically scrape off >500,000 colonies and purify the plasmids using the ZymoPURE II Plasmid Maxiprep kit (Zymo Research) to prepare a full-length recombinant hyperimmune Maxiprep library for transfection.

[0226] We adapted adherent Flp-In™-CHO cell lines with FRT sites for genome integration (Thermo Fisher Scientific, Waltham, MA, USA) to suspension culture. For all steps in the adaptation process, “Ham’s F-12” refers to Ham’s F-12 (containing L-glutamine, Thermo Fisher Scientific, Waltham, MA, USA) plus 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and “BalanCD” refers to BalanCD CHO Growth A (Irvine Scientific) containing 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA). To adapt this cell line to suspension, we first passaged the cells in a mixture of 50% Ham’s F-12 and 50% BalanCD in a T-flask. Next, the cells were passaged in a mixture of 25% Ham’s F-12 and 75% BalanCD and transferred to a shaken Erlenmeyer flask. The cells were then passaged into 10% Ham's F-12, 90% BalanCD + 0.2% anti-caking agent (Irvine Scientific, Santa Ana, CA, USA) and stored for future use.

[0227] Using Amaxa Nucleofector 4D (SG buffer, pulsed DU133; Lonza, Basel, Switzerland), 100 million adapted Flp-In CHO cells were transfected with each recombinant hyperimmune preparation library. These cells were seeded into shaken Erlenmeyer flasks and incubated at 37°C and 125 rpm for 48 hours. After 48 hours, cells were counted to determine viability, and cells were seeded at 1 million cells / mL and selection was initiated in fresh medium using 600 μg / mL hygromycin-B (Gemini Bio, West Sacramento, CA, USA). During the 7-day selection period, cells were counted and the medium was changed every 2–3 days. During expansion, the library was maintained at 600 μg / mL hygromycin-B (Gemini Bio, West Sacramento, CA, USA) until viability exceeded 95%. When cells were >95% viable and doubled every 24 hours, the cell lines were preserved for liquid nitrogen storage.

[0228] CHO cells stably expressing the antibody library were grown in a medium consisting of 90% BalanCD CHO Growth A medium (Irvine Scientific, Santa Ana, CA), 9% Ham's F-12 (Thermo Fisher Scientific, Waltham, MA, USA), 1% FBS (Thermo Fisher Scientific), 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and 0.2% anti-caking agent (Irvine Scientific, Santa Ana, CA, USA). For small-scale production, cells were grown at a density of 1 × 10⁶ cells / year. 6Cells / mL were seeded into 50 mL of medium in 250 mL Erlenmeyer flasks and grown at 37 °C, 5% CO2, and 125 rpm. Cells were continuously grown under these conditions, supplemented with 7.5 mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) on days 2, 4, and 7 of the production run. On day 8, the supernatant was harvested by centrifugation and subsequently filtered through a 0.22 μm 250 mL filter flask (EMD Millipore, Burlington, MA, USA) with a 1 μm pre-filter. The harvested cell culture medium (HCCF) was stored at 4 °C until protein A purification. For large-scale production of the plasma cell recombinant hyperimmune agent, cells were grown in the same medium, but with some modifications to the production conditions. Seed cultures were used to grow the culture from 2 × 10⁶ cells / mL at 37 °C. 7 The number of cells increased to 1.2 × 10⁻⁶. 10 Cells. Then the cells are divided into 1×10⁻⁶ cells. 6 Cells / mL were seeded at 2L in 5L flasks (triple, day 0). On day 2, the temperature was changed from 37°C to 33°C. On days 2, 4, 6, 8, 10, and 13, 300mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) was added to each flask. The supernatant was harvested on day 14.

[0229] After harvest, HCCF was purified using MabSelect SuRe protein A resin (GE Life Sciences, Marlborough, MA, USA) with the following buffers: equilibration buffer, top wash (Chase), wash buffer 2 (25 mM Tris, 150 mM NaCl, pH 7.4), wash buffer 1 (25 mM Tris, 1 M NaCl, pH 7.4), elution buffer (20 mM citric acid, pH 3.0), and neutralization buffer (100 mM Tris, pH 8.0 for small-scale; 1 M Tris, pH 9.0 for large-scale). The column was sterilized with 0.1 N NaOH before and after use. For large-scale production of recombinant hyperimmune plasma cell preparations, an additional wash buffer 3 consisting of 0.5 M arginine, pH 7.4 was used, followed by an additional wash with wash buffer 2 before elution. The purification steps were performed in the following order: equilibration, loading, top washing, wash 1, wash 2 (for large-scale production: wash 3, wash 2), elution, and neutralization (manually added to the tube used to collect the eluent fraction). The recombinant hyperimmune preparation was concentrated using Vivaspin 20, 30 kDa molecular weight cutoff centrifuge tubes (Sartorius, Göttingen, Germany) and prepared in PBS (for small-scale production) or 0.2 M glycine, pH 4.5 (for large-scale production), followed by 0.22 μm filtration.

[0230] Imaging capillary isoelectric focusing (iCIEF) was performed using a Maurice imaging cIEF analyzer (Protein Simple, San Jose, CA, USA). Capillary electrophoresis of sodium dodecyl sulfate (CE-SDS) was performed under both reducing and non-reducing conditions using a LabChip GX II Touch HT (Perkin Elmer, Waltham, MA, USA). Endotoxin levels were measured using Endosafe nexgen-PTS (Charles River, Wilmington, MA, USA).

[0231] We observed a 92.2% yield of HBV ABP in our protein A step. Under non-reducing conditions, we observed a single peak (>99%) at 166.2 kDa using CE-SDS. Under reducing conditions, ABP showed >99% pure IgG monomers and <1% other proteins, while plasma IVIg showed approximately 3.1% unknown proteins, suggesting that the recombinant hyperimmune agent can be produced with higher IgG purity than plasma IVIg. Analysis of the purified recombinant hyperimmune agent by iCIEF showed a broad spectrum of isoelectric material, although plasma IVIg showed a considerably wider range of isoelectric material. We speculate that plasma IVIg has a broader range of isoelectric material because it contains a wider range of antibody diversity and also includes different IgG isotypes (the recombinant hyperimmune agent contains only IgG1) and IgL. Finally, the endotoxin level was <0.5 endotoxin units (EU) / mg, which is a typical benchmark for recombinant mAb therapeutics.

[0232] The deep antibody sequencing library was quantified using the Illumina Library Quantification Kit (KAPA, Wilmington, MA, USA) and diluted to 17.5 pM. The library was sequenced on MiSeq (Illumina, San Diego, CA, USA) using a 500-cycle MiSeq kit v2 according to the manufacturer's instructions. To prepare the sequencing library, Illumina sequencing adaptors were added to the 5' and 3' ends of the constructs of interest using tail PCR. We then obtained 340 cycles of forward reads and 162 cycles of reverse reads. This produced forward and reverse reads overlapping at portions of the CDR3-H and VH- genes, increasing the confidence in nucleotide calls. The sequencing library was then sequenced using our previously reported bioinformatics workflow (Adler et al.). Mab Sequence analysis was performed (9, 1282-1996, 2017). Pearson correlation analysis was performed using the cor function in R version 3.4.2.

[0233] Each of the four HBV ABPs was derived from 1.12–1.39 million input cells. After library processing via our library generation pipeline, the clonal diversity of the recombinant hyperimmune formulations was less than 2,000 antibody clones (range: 880 to 1,659), capturing a significant portion of the input antibody diversity. All four recombinant hyperimmune formulations exhibited 93% median germline IgHV identity, indicating that no cell type produced antibodies with significantly higher affinity, consistent with previous analyses of Hib-inoculated individuals (Truck et al., 2015). Clonal diversity did not show a strong bias towards the most common antibody in any mixture. The most common antibody was present at a frequency of 3.5% (plasma cell hyperimmune formulation). The pan-B recombinant hyperimmune formulation showed the least skewed clonal diversity (the top 20 antibodies accounted for 12.7% of all antibodies), while the plasma cell recombinant hyperimmune formulation showed the most skewed clonal diversity (the top 20 antibodies accounted for 26.6% of all antibodies).

[0234] We examined the genetic diversity of four recombinant hyperimmune agent libraries. Overlap analysis showed that no more than 11.8% of clones were shared between any two given recombinant hyperimmune agent libraries. Pearson correlation analysis was not significant between any two pairwise comparisons. p <0.01). All four recombinant hyperimmune preparation libraries contained multiple IgGV-J gene pairs, including high-frequency antibodies with IgHV3-23 and IgHJ4 genes, which have been observed elsewhere in anti-Hib libraries (Silverman & Lucas, 1991; Adderson et al., 1993; Lucas et al., 2003; Trück et al., 2015). Other common IgHV genes included IgHV3-30, IgHV1-69, and IgHV3-7. All libraries also included a complementarity-determining region (CDR) 3 sequence containing either the peptide GYGFD or GYGMD previously observed in anti-Hib libraries (Lucas et al., 2003; Trück et al., 2015). We conclude that all four libraries contain canonical anti-Hib sequences and similar levels of phylogenetic differences and genetic diversity. However, these four libraries do contain different antibody mixtures that may have different functional characteristics.

[0235] Human anti-Hib-PRP IgG ELISA kit (Alpha Diagnostics #980-100-PHG, San Antonio, TX, USA) was used for anti-Hib ELISA titers. Serial dilutions of the antibody preparations were performed in a low NSB (non-specific binding) sample diluent. Quantification was performed at 450 nm on a microplate reader (Molecular Devices, Fremont, CA, USA). EC50 values ​​were calculated using SoftMax Pro (Molecular Devices, Fremont, CA, USA). We also determined anti-Hib PRP antibody titers from plasma banks of two donors before and after vaccination with the Hib active vaccine and IVIg. Recombinant hyperimmune preparations from plasma cells, pan-B, and plasmablasts produced significantly higher Hib-binding titers than IVIg (range: 160× to 2,323×), with the plasma cell hyperimmune preparation producing the highest titers. Post-vaccination plasma showed an anti-Hib titer of only 3.7 × IVIg, and no anti-Hib titer was detected in recombinant hyperimmune memory B cells under the test conditions. In summary, these data demonstrate that our manufacturing process can significantly increase anti-Hib titers simply by selecting appropriate cell types from vaccine-treated donors.

[0236] In vitro neutralization studies were conducted at a CRO (ImQuest Frederick, MD, USA). Type b Haemophilus influenzae The Eagan strain was obtained as a frozen glycerol strain from Zeptometrix (#0801679, Buffalo, NY, USA) and stored at -80°C. flow Haemophilus influenzae Strain ATCC 10211 was obtained as the lyophilized primary culture from the American Type Culture Collection (ATCC, Frederick, MD, USA) and propagated according to the supplier's recommendations. Colonies incubated overnight on chocolate agar plates were inoculated into growth media (brain heart infusion or BHI broth, BD BBL 299070, San Jose, CA, USA; enrichment broth containing 2% Fildes, Remel #R45037, San Diego, CA, USA) and allowed to reach an optical density (OD) of approximately 0.4 at 625 nm. 625 Adjust the culture to OD. 625 It is 0.15, which is equivalent to approximately 5 × 10 8 Colony forming units (CFU) / mL. The culture was further diluted to 5 × 10⁻⁶ in dilution buffer (Hanks balanced salt solution, Gibco, Waltham, MA, USA #14025-092, containing 2% Fildes enrichment broth).4 CFU / mL. By using 50 μL of 5 × 10⁻⁶ CFU / mL... 3 and 5×10 2 Two copies of the diluent were plated on chocolate agar and incubated at 37°C / 5% CO2 for 24 hours. Colony counts were then performed to confirm the density of the bacterial culture used in this method.

[0237] Dilute the test sample three-fold in dilution buffer, starting at 200 μg / mL, to evaluate a total of ten dilutions. Add 10 μL of each dilution in duplicate to a 96-well microtiter plate. Then, adjust the concentration to approximately 5 × 10⁻⁶. 4 Eagan or ATCC 10211 bacteria at a concentration of 1 CFU / mL were added to the plate in a volume of 20 μL to achieve a total bacterial density of 1 × 10⁻⁶ CFU / mL in the wells. 4 20 μL CFU / well. After incubation at 37°C / 5% CO2 for 15 minutes, add 25 μL of baby rabbit complement (Pel-Freez #31061-1, Rogers, AR, USA) and 25 μL of dilution buffer to each well. Incubate the plate at 37°C / 5% CO2 for 60 minutes. After incubation, dilute 5 μL of each reaction mixture in 45 μL of dilution buffer and plate the entire 50 μL onto chocolate agar plates. Incubate the plate at 37°C / 5% CO2 for approximately 16 hours. After incubation, count the bacterial colonies. The test concentration that kills >50% of bacteria is SBI.

[0238] As expected from the ELISA data, the memory B cell recombinant hyperimmune agent failed to neutralize Hib strains at any tested concentration. The plasma cell recombinant hyperimmune agent again produced the highest titers, with SBIs of 81 and 243 for Eagan and ATCC 10211 strains, respectively. The pan-B and plasmablastic cell recombinant hyperimmune agents were 1 / 9 the potency of the plasma cell recombinant hyperimmune agent. Neutralization of IVIg was not detected at any tested concentration. We conclude that the plasma cell recombinant hyperimmune agent is the most potent among the four cell types tested.

[0239] All vertebrate experiments were conducted under the supervision and approval of the Institutional Animal Care and Use Committee of Sinclair Research Center, LLC, Missouri (USA) in accordance with the standards incorporated in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies, 8th Edition), or under the supervision and approval of the National Committee of Animal Ethics, Denmark, in accordance with EU Directive 2010 / 63 / EU (License No.: 2014-15-0201-00171).

[0240] For acute toxicity, Balb / cJ mice (Charles River, Wilmington, MA, USA) were randomly assigned to seven groups of six animals each by the CRO (Sinclair Research, Auxvasse, MO, USA). Three groups received a single dose of the recombinant hyperimmune agent at 30 mg / kg, 100 mg / kg, or 300 mg / kg. The negative control group received a single dose of saline solution. The remaining three groups received a single dose of plasma IVIg (Gammagard; Grifols, Sant Cugat, Catalonia) at 30 mg / kg, 100 mg / kg, or 300 mg / kg. The test sample was diluted in 0.2 M glycine, pH 4.5. The test sample was administered intravenously via the tail vein. The dose volume was calculated based on the most recent body weight of each animal. Mice were then observed twice daily for 8 days for general health, response at the test application site, morbidity and mortality, body weight, and gross physical examination (skin, mucous membranes, eyes, ears, nose, and respiration). Three days later, the animals were euthanized with CO2 gas, and terminal serum chemistry was performed, including albumin, globulin, glucose, total protein, blood urea nitrogen, and several other indicators.

[0241] For any test group, we observed no findings related to the test article. We conclude that the no-observed adverse reaction dose (NOAEL) for a single intravenous dose of the recombinant plasma cell hyperimmune agent is 300 mg / kg. IVIg is typically administered in immunocompromised patients at approximately 300 mg / kg for protection against Hib and other pathogens, and the Hib hyperimmune product is thousands of times more effective; therefore, we conclude that the recombinant plasma cell hyperimmune agent does not have observable toxicity at the minimum effective dose.

[0242] For pharmacokinetics, the CRO (Sinclair Research, Auxvasse, MO, USA) administered a 100 mg / kg intravenous tail vein dose of recombinant plasma cell hyperimmune agent to twenty male Balb / cJ mice (Charles River, Wilmington, MA, USA). A sparse blood collection procedure was followed, ensuring that no mouse received more than two of the planned seven pharmacokinetic (PK) blood collections. Serum human IgG was then measured using a sandwich ligand binding method (LBA) and Meso Scale Discovery (MSD; Rockville, MD, USA) electrochemiluminescence (ECL) technology. A capture antibody (SouthernBiotech #2049-01, Birmingham, AL, USA) was coated onto 96-well plates (MSD, Rockville, MD, USA). Serum samples were diluted to the minimum desired dilution (MRD) of 1:100 in PBS / T containing 1% BSA (PBS / T / BSA). The diluted samples were then added to the designated wells. Following another washing step, wells were inoculated with PBS / T / BSA containing 1 mg / mL biotinylated goat anti-human IgG (SouthernBiotech #2049-08, Birmingham, AL, USA). After incubation, streptavidin-SULFO-TAG was added, followed by 2× read buffer T (MSD, Rockville, MD, USA). ECL units were measured using an MSD QuickPlex SQ 120 instrument. Standard curves were additionally generated for each run using a plasma cell-based recombinant hyperimmune preparation. Data were fitted using Discovery Workbench software (MSD, Rockville, MD, USA) using a four-parameter logic (4-PL) curve fitting of mean ECL units against nominal IgG standard values. Under the assumption of failed intravenous administration, two animals with readings of 1100 ng / mL or lower at the 1-hour time point were removed from further analysis. We then used the PKNCA package in R (Denney et al., 2015) to apply non-compartmental analysis to the concentration-time data to estimate the observed maximum plasma concentration (C0). 最大 ), the time to reach the observed maximum plasma concentration (T) 最大 ) and half-life (t 1 / 2 ).

[0243] One hour after administration (T) 最大 The highest observed plasma concentration was 12,360 ng / mL (C). 最大 Half-life (t) of recombinant hyperimmune agents 1 / 2The time was approximately 34.5 hours. Combining these data with ELISA titer data, we estimated that the maximum anti-Hib trough level for a single intravenous dose of 100 mg / kg was 861 IU / mL.

[0244] Haemophilus influenzae Strain strain ATCC10211 was grown overnight on chocolate agar plates at 35°C and 5% CO2. Single overnight colonies were resuspended in sterile saline to a final concentration of 1.5 × 10⁻⁶. 8 CFU / mL. Dilute this suspension in BHI broth containing 5% mucin and 2% hemoglobin to approximately 1×10⁻⁶ CFU / mL. 6 One CFU / mL, and further diluted 10-fold to 10 CFU / mL.

[0245] Use 10 4 10 5 Or 10 6 Balb / cJ mice (Taconic, Denmark; n=6 / group) were inoculated with a single 0.5 mL intraperitoneal dose of 1 CFU / mL Hib bacteria (strain ATCC 10211). Approximately 1 hour prior to inoculation, mice were orally treated with 45 μL of norrofen (20 mg ibuprofen / mL, corresponding to approximately 30 mg / kg) for pain relief. Twenty-four hours prior to inoculation, mice were administered 300 mg / kg of recombinant Hib hyperimmune preparation, 300 mg / kg of plasma IVIg, or saline. One hour after inoculation, mice were administered 20 mg / kg of ciprofloxacin antibiotic as a positive control. Clinical signs of infection were assessed in mice every 2–6 hours, and mice severely affected by the infection were euthanized. After another 72 hours, any live animals were anesthetized with Zoltil mixture, and blood was collected via axillary incision. Mice were euthanized by cervical dislocation, 2 mL of sterile saline was injected intraperitoneally, and the abdomen was gently massaged before opening and sampling with a pipette. Dilute each sample 10-fold in saline and apply 20 µL of each solution to a chocolate agar plate. Incubate all agar plates at 35°C in ambient air for 18–22 hours.

[0246] In the solvent control group, Hib infection was lethal to all but one mouse at all inoculation doses. In contrast, in the recombinant hyperimmune agent treatment group (at 10... 6 In the CFU inoculation group, only one out of 18 mice was severely affected. IVIg showed significantly lower protective efficacy than the recombinant hyperimmune agent, with 10 of them... 5 CFU and 10 6 5 / 6 mice and 10 mice in the CFU inoculation group 4Two-sixths of the mice in the CFU inoculation group were severely affected by the infection. Analysis of the bacterial load in the blood showed that the recombinant hyperimmune agent eliminated Hib from the bloodstream in all animals, while IVIg treatment resulted in a significantly lower bacterial load than the solvent control only in one inoculation group, and no significant reduction was observed in either inoculation group (Dunnett's multiple comparison test). p <0.05). In peritoneal lavage fluid, compared with the solvent control group, the recombinant hyperimmune agent again significantly reduced the bacterial load (Dunnett's multiple comparison test). p <0.05). However, although Hib bacteria were not detected in the peritoneal lavage fluid of surviving animals treated with ciprofloxacin, they were detected in the peritoneal lavage fluid of 6 / 17 surviving animals treated with the recombinant hyperimmune agent (range: 23-77 CFU / mL). This suggests a difference in efficacy of the recombinant hyperimmune agent between the peritoneum and blood, which may be due to the bioavailability of the drug or complement in the peritoneum.

[0247] In some embodiments, a Hib hyperimmune agent is incorporated into regular plasma IVIg to increase the anti-Hib titer of the IVIg. In some embodiments, several antipathogen hyperimmune agents are incorporated into regular plasma IVIg, for example, hyperimmune agents against Hib, pneumococcus, influenza A virus, and tetanus are incorporated into plasma IVIg to treat patients with primary immunodeficiency. Incorporation of a hyperimmune agent increases the titer of antibodies against pathogens that patients with primary immunodeficiency are particularly susceptible to. Any number of additive formulations can be mixed with plasma IVIg to generate increased titers against any number of pathogens.

[0248] Using a series of in vitro and in vivo experiments, the following were determined. For Hib, plasma cells produced the most potent ABP after inoculation. The potency of plasma cell Hib ABP was >2,300x that of plasma IVIG (by ELISA). In an in vivo challenge model, plasma cell Hib ABP strongly protected against Hib infection. The use of plasmablasts and pan-B cells also resulted in in vitro potent ABP, although with lower potency than plasma cells. For this antigen, ABP prepared from memory B cells had undetectable potency levels in in vitro methods.

[0249] 6.1.3. Example 3: Generation of an ABP library with activity against Streptococcus pneumoniae capsular polysaccharide Streptococcus pneumoniae causes pneumococcal pneumonia. A recombinant polyclonal antibody (pAb) active against Streptococcus pneumoniae was generated, namely an ABP library, "GG-Pnc". In vitro testing of GG-Pnc demonstrated its effectiveness against Streptococcus pneumoniae. Streptococcus pneumoniaeThe in vitro functional efficacy and activity of capsular polysaccharides (GG-Pnc) were investigated. A library was analyzed using large-scale pneumococcal polysaccharide ELISA, serotype-specific ELISA, and serotype-specific opsonization methods.

[0250] GG-Pnc, or ABP library, was prepared using the recombinant technology described in Examples 1 and 2. This library was prepared from three donors vaccinated with Pneumovax-23. Pneumovax-23 consists of capsular polysaccharides from 23 pneumococcal serotypes. As measured by ELISA, all three donors showed increased titers of pneumococcal capsular polysaccharides after vaccination. rpAb was prepared from a mixture of all B-cell subtypes isolated from the donors.

[0251] The Alpha Diagnostics ELISA measures the overall polysaccharide-specific antibody response to 23 pneumococcal polysaccharides found in the Pneumovax-23 vaccine and is used to measure the EC50 of the ABP library. An 8-step, 3-fold dilution series and a 4-point logistic analysis were performed to calculate the EC50. The potency of the ABP library GG-PNC is approximately 100 times that of IVIG.

[0252] Serotype multiplex ELISA was performed to assess antibody diversity in the GG-Pnc ABP library compared to IVIG. Twenty pneumococcal serotypes were measured by ELISA. Antibody-specific responses in GG-Pnc and IVIG (Gamunex) were measured using international standards for pneumococcal-specific responses. For all serotypes except serotype 6A, GG-Pnc showed similar or higher concentrations than IVIG.

[0253] Serotype-specific opsonization methods were employed to assess antibody-induced killing function. Fourteen pneumococcal serotypes were measured by opsonization of phagocytic responses using GG-Pnc and IVIG (Gamunex). Consistent with multiplex ELISA, GG-Pnc was similar to or more effective than IVIG for all serotypes except 6A.

[0254] A serotype 2-specific ELISA was performed to determine the ability of GG-Pnc to bind to this serotype, as it was not included in previous analyses but is an available option for in vivo mouse models. An 8-step, 3-fold dilution series was performed, and a 4-point logistic analysis was used to calculate the EC50; only GG-Pnc had a value because IVIG binds minimally to serotype 2, even at very high concentrations.

[0255] The GG-Pnc ABP library strongly binds to a group of different pneumococcal serotypes and is able to neutralize all serotypes as tested using in vitro opsonization phagocytosis methods. Without performing serotype-specific enrichment procedures and using all B cells isolated from vaccinated donors, GG-Pnc is similar to or more effective than IVIG for all serotypes except one (for binding and killing). GG-Pnc also binds strongly to serotype 2.

[0256] 6.1.4. Example 4: Generation of an ABP library with activity against influenza A antigen The recombinant method described herein was used to generate an ABP library that is active against influenza A antigen (ABP1).

[0257] 6.1.5. Example 5: Generation of an ABP library with activity against hepatitis B virus antigen Two ABP libraries with activity against hepatitis B virus antigen were generated using the recombinant method described herein. CHO cells stably expressing antibody libraries with activity against hepatitis B virus antigen were used to generate the CHO master cell bank (MCB).

[0258] A vial from the MCB was thawed for seed culture expansion. EX-CELL Advanced CHO fed-batch medium (MilliporeSigma, Burlington, MA) was used for cell expansion and passage, thawed from the cell bank vial and used in the shake-flask and shake-batch (RM) bioreactor. EX-CELL Advanced CHO fed-batch medium was used as the basal medium for both the 200L SUB seed culture (N-1) and the 200L fed-batch production bioreactor. EX-CELL Advanced CHO F1 feed (MilliporeSigma) and CellVento 4Feed COMP (MilliporeSigma) were added. Glucose levels were monitored daily starting from day 2 and maintained above 4 g / L by supplementing with 500 g / L glucose stock solution (prepared from powder, MilliporeSigma) to increase the concentration to 6 g / L as needed. Antifoaming agents were added as needed to reduce any foam buildup after day 2. The bioreactor was harvested when cell viability was ≤75% or on day 16.

[0259] The large quantities of harvested material are clarified and then downstream processed through a series of standard chromatographic purification and virus reduction steps: (i) affinity chromatography, (ii) low pH virus inactivation, (iii) hydrophobic interaction chromatography or membrane filtration, (iv) multimode anion exchange chromatography or membrane filtration, (v) multimode cation exchange chromatography, (vi) anion exchange chromatography or membrane filtration, (vii) cation exchange chromatography, (viii) virus filtration, and / or (ix) ultrafiltration and / or percolation.

[0260] 6.1.6. Example 6: Fingerprinting method for ABP library The ABP library with binding specificity to hepatitis B virus antigen (rHBIG) contains 2306 unique V antigens. H Sequence and 147 unique V L The library contains over 2000 unique IgG clones. Therefore, conventional methods for analyzing single antibodies (e.g., sequencing) cannot be used to test compositions containing thousands of antibodies. A new method is needed to characterize and analyze the identity and consistency of compositions across multiple batches and / or over time.

[0261] To develop a method, the sequences of antibody clones in the library were analyzed, and computational bioinformatics analysis was performed to understand the charge state (isoelectric point) distribution of the clones. The charge state distribution of the library, weighted by RNA sequence abundance, provides insights into... Figure 1 middle.

[0262] The charge distribution was also determined experimentally. A novel CEX-HPLC (cation exchange high-performance liquid chromatography) method using a pH gradient was employed to characterize the charge distribution of the antibody population in the recombinant pAb product. The CEX-HPLC spectra of the rHBIG library showed multiple peaks, consistent with its polyclonal nature.

[0263] IgG antibodies in a polyclonal antibody library have different charge states and can therefore be separated by cation exchange-high performance liquid chromatography (CEX-HPLC), a chromatographic method involving cation exchange chromatography with a pH gradient from low pH (pH 5.6) mobile phase A to high pH (pH 10.2) mobile phase B. Figure 2 As shown, the polarity of the IgG charge changes depending on the pH of the buffer solution.

[0264] The following materials and equipment are used in CEX-HPLC.

[0265] Method conditions are obtained by testing various conditions to maximize resolution and minimize variations in results (e.g., the number of peaks over the gradient length). Specifically, various flow rates, gradients of %B, and gradient runtimes are tested to achieve reproducibility, including inter-column reproducibility and intra-method reproducibility.

[0266] To test the reproducibility of this method, the same batch of polyclonal libraries was tested three times under the following conditions, and the results are provided in Figure 3 middle.

[0267] Figure 3 The results (e.g., peak area and height as a function of retention time (min)) were significantly consistent across the three separate runs, demonstrating high intra-method reproducibility.

[0268] The CEX-HPLC method was performed using three different column batches of Proteomix SCX-NP1.7 (4.6 x 100 mm) columns: S / N 2A54701 (LN DW054), S / N 0A60382 (LN DW166), and S / N 9A60383 (LN430794). These three columns were installed at different ports and run on the same day. Results are provided in... Figure 4 In this study, it demonstrated high intercolumn reproducibility.

[0269] To find the optimal flow rate for resolution, the CEX-HPLC method was performed under the following conditions at three different flow rates (0.5 mL / min, 0.75 mL / min, and 1.0 mL / min). Figure 5 The results provided indicate that flow rates of 0.75 mL / min and 1.0 mL / min offer comparable resolution. A slightly lower flow rate (0.75 mL / min) was chosen to maintain the pressure within the optimal range.

[0270] The CEX-HPLC method was performed under the following conditions using three different gradients (15-100% B, 5-100% B, and 10-100% B). Figure 6 The results provided indicate that a narrower range tends to provide higher resolution. The effect of sample pH was occasionally observed. A 10-100% B solution was selected to reduce the sample matrix effect (pH).

[0271] The CEX-HPLC method was performed under the following conditions at three different gradient times (35 min, 45 min, and 60 min). Figure 7The results provided indicate that longer gradient times tend to provide higher resolution. A gradient time of 45 min was chosen to achieve good resolution while maintaining a reasonable runtime.

[0272] From a series of experiments, the following conditions were selected for use in the CEX-HPLC method.

[0273] To test the specificity of the CEX-HPLC method, different samples were used: anti-HBV plasma hyperimmune preparation (HyperHEP), ABP library with binding specificity to hepatitis B virus antigen (rHBIG), ABP library with binding specificity to CoV antigen (rCIG), and recombinant monoclonal antibody (anti-CTLA-4). Figure 8 The results provided demonstrate that CEX-HPLC chromatograms are specific to each sample and represent their unique characteristics. These results indicate that this method can be used to characterize and differentiate different samples.

[0274] The ABP library with binding specificity to hepatitis B virus antigen (rHBIG) and six individual antibodies (PN-6103.02, 6104.02, 6105.02, 6115.02, 6116.02, and 6117.02) in the library were subjected to CEX-HPLC. Results are provided in... Figure 9 The results showed that the HPLC signal from rHBIG represented a combination of signals from individual monoclonal antibodies in the library.

[0275] To understand the comparability of HPLC chromatograms collected using columns from different brands, rHBIG was analyzed using two different brands of columns under the conditions provided below. Results are provided in... Figure 10 The HPLC signals from the Proteomix SCX NP-1.7 show resolution comparable to the MabPac SCX-10. They have generally similar spectra, but differ in detail when using columns from different suppliers.

[0276] These studies demonstrate that the CEX-HPLC method can distinguish rHBIG from anti-HBV hyperimmune products (HyperHEP), monoclonal antibodies (anti-CTLA-4), or various recombinant polyclonal antibodies (anti-CoV-2; rCIG). This analysis provides reproducible characterization for the same samples using the same method as well as for the same samples from different batches from the same column. Together, these findings demonstrate that the CEX-HPLC method is reliable and suitable for fingerprinting ABP libraries.

[0277] 6.1.7. Example 7: Size heterogeneity measured by SEC and SEC-MALS A high-resolution SEC-HPLC method has been developed to characterize high molecular weight substances (HMW), polyclonal antibody monomers (pAb peak), and low molecular weight substances (LMW) in ABP libraries. This method can be used for process development, formulation development, in-process testing, and release and stability studies.

[0278] The high-resolution SEC-HPLC method involves the following steps: • Device settings - Prepare the HPLC system according to the system protocol.

[0279] - Connected to an HPLC column.

[0280] • Data acquisition method parameters:

[0281] • Example HPLC sequence:

[0282] Increase the flow rate slowly to the desired value to avoid a rapid increase in pressure, which could damage the column. Equilibrate the column for at least 30 minutes before starting the sequence.

[0283] After every 3 sample injections, including 1 blank injection of the mobile phase.

[0284] The SEC method used in this paper incorporates several improvements to conventional processes to achieve high resolution for the analysis of polyclonal antibodies. A hybrid chemistry termed BEH (ethylene-bridged hybridization) is employed to reduce residual silanol groups on the stationary phase. The mobile phase (1X PBS) is adjusted to pH 7.0, and the NaCl concentration is increased to 500 mM (10 mM PBS containing 500 mM NaCl, pH 7.0) to further reduce charge-based protein-column interactions. A column packed with small-particle-size resin (e.g., particle size less than 2.7 µm) is selected to improve separation efficiency. The column temperature is increased to 30 °C to reduce antibody-column interactions. Monoclonal antibodies elute with a narrow main peak on this column. This method can be used to characterize the size heterogeneity of polyclonal antibody drug substances, drug products, in-process, and stable samples.

[0285] Because the library contains more than 1000 antibodies with diverse biophysical properties, the presence of multiple monomer peaks is consistent with the polyclonal nature of the product, as measured using this high-resolution method specifically developed for polyclonal products. This high-resolution method can clearly distinguish HMW, polyclonal monomer peaks, and LMW substances present in the library. Tox and GMP batches have similar spectra ( Figure 11 (Table 1), and both exhibited approximately 98% monomeric pAb peaks, along with less than approximately 2% HMW and LMW components overall. Although the instrument software identified a certain number of peaks within the retention time window corresponding to the antibody monomers, many overlapping peaks (more than two or three) were actually present. The resolvable peaks were numbered according to their elution order. Due to the overlapping nature of the product spectra, some variability in the abundance of individually resolved pAb peaks could be observed.

[0286] To further characterize and limit the ability of this method to distinguish HMW and LMW substances from monomeric pAb substances, forced degradation studies were conducted. Figure 12 The image shows a set of example chromatograms of Tox materials incubated at 40°C for up to 2 weeks. The chromatograms show that the HMW, LMW, and pAb peaks are well resolved, and the SEC-HPLC method can detect the increase in LMW under this stress condition. The pAb peak, clearly distinguishable between the HMW and LMW peaks, is qualified to elute within a retention time of 7.5–9.5 min. As confirmed by SEC-Multi-Angle Light Scattering (MALS), the pAb peaks within this acceptable retention range (e.g., ...) Figure 12 The image shown is a polyclonal antibody monomer (a full-length antibody of approximately 150 kDa). SEC-MALS data also show that the results for Tox and GMP batches are comparable. Figure 13 ).

[0287] Characterization data demonstrate that the uniquely developed method can distinguish HMW and LMW substances from full-length antibody material.

[0288] 6.1.8. Example 8: Size heterogeneity measured by CE-SDS CE-SDS (sodium dodecyl sulfate capillary electrophoresis) was used as an orthogonal method to characterize the size heterogeneity of rHBIG under both reducing and non-reducing conditions. In short, antibody samples were denatured with SDS containing or without a reducing agent (dithiothreitol, DTT) and then electrokinetically introduced into fused silica capillaries filled with molecular sieve reagents. Once a high-voltage electric field was applied, the negatively charged antibody molecules passing through the SDS were mobilized and separated according to their size. Two key parameters for protein size determination and quantification were migration time and absorbance. Migration time is related to protein size: larger proteins migrate more slowly due to the sieving effect of the gel within the chip channels. Absorbance intensity, in turn, is related to the concentration of these proteins. Protein abundance was calculated as a percentage of the peak area corrected for migration time.

[0289] The results show that the method is stability-indicative, with (HC+LC)% and pAb%.

[0290] 6.1.9. Example 9: Dynamic Light Scattering (DLS) Characterization Dynamic light scattering (DLS) is a technique used to study protein size heterogeneity. This technique measures the time-dependent intensity of light scattered by diffused protein particles. Based on the fluctuating light intensity, an autocorrelation function can be calculated. The shape of the autocorrelation function is related to the size and size distribution of the particles in the sample.

[0291] 6.1.10. Example 10: Characterization of protein melting temperature (Tm) Melting temperature (T) m (T) is an indicator of the thermal stability of a protein's tertiary structure. High T m Associated with high structural stability, the Tm of rHBIG was measured using two methods: fluorescence-based protein thermal shift assay (PTS) and differential scanning calorimetry (DSC). In the PTS method, a mixture of protein and fluorescent dye was subjected to a thermal ramp from 25°C to 95°C. Upon protein melting, the interaction between the fluorescent dye and the thermally exposed hydrophobic residues enhanced fluorescence. Monitoring the fluorescence intensity as a function of temperature provided information on the phase transition of the protein domains upon temperature elevation.

[0292] DSC is another technique used to characterize antibody Tm. In short, the protein sample is subjected to a thermal ramp from 20°C to 100°C. The heat flux during the temperature ramp is recorded, and after subtracting the contribution from the formulation buffer, the heat capacity as a function of temperature is calculated from the heat flux. Protein melting events manifest as changes in heat capacity.

[0293] The results show that DSC and PTS can be used to detect protein melting events.

[0294] 6.1.11. Example 11: Anticomplement assay (ACA) To test the potential of HMW substances (especially aggregates) to trigger nonspecific activation of complement, an anticomplement assay (ACA) was performed.

[0295] In short, immunoglobulins were incubated with guinea pig complement. The nonspecific binding of the immunoglobulin preparation to complement led to the depletion of complement in the sample. Following this incubation step, the sample was titrated with a combination of hemolysin (a rabbit antibody against sheep erythrocytes) and sensitized sheep erythrocytes. Sheep erythrocytes were sensitized by loading hemolysin. The degree of lysis of sheep erythrocytes was spectrophotometrically determined by measuring the released hemoglobin at 541 nm, and the results were expressed as complement activity units (CH). 50 )Report.

[0296] Due to the biological reagents used, ACA assays exhibit high complexity and potential variability. Therefore, these samples were also tested in an in vitro human system that measures nonspecific complement activation of C5a by ELISA after incubation with human serum.

[0297] All tested rHBIG samples met the European Pharmacopoeia Monographs requirement of ≤1 CH for IVIG. 50 The acceptance criteria for rHBIG / mg IgG do not include any adjustments to the significantly lower dose expected compared to the dose used for IVIG.

[0298] 7. Incorporate by reference This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 519,815, filed August 15, 2023, the entire contents of which are incorporated herein by reference.

[0299] All publications, patents, patent applications and other documents cited in this application are hereby incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application or other document were individually indicated as being incorporated herein by reference for all purposes.

[0300] 8. Equivalent solution Although various specific embodiments have been shown and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the invention. Many changes will become apparent to those skilled in the art after reading this specification.

Claims

1. A method for analyzing a test library containing antigen-binding protein (ABP), comprising: (a) Measure the charge state distribution of the test library, wherein the test library contains at least 100 ABPs; (b) Compare the charge state distribution with a reference distribution, wherein the reference distribution is the charge state distribution of a reference library; and (c) Determine the quality of the test library based on the comparison.

2. The method of claim 1, wherein the charge state distribution is measured in step (a) by cation exchange-high performance liquid chromatography (CEX-HPLC).

3. The method of claim 2, wherein the CEX-HPLC is performed with a pH gradient from mobile phase A to mobile phase B, wherein mobile phase A has a low pH from pH 5 to pH 7, and mobile phase B has a high pH from pH 9 to pH 11.

4. The method of claim 3, wherein the mobile phase A has a low pH from pH 5 to pH 6 or from pH 5.5 to pH 6, and / or the mobile phase B has a high pH from pH 10 to pH 11 or from pH 10 to pH 10.

5.

5. The method of any one of claims 2 to 4, wherein the CEX-HPLC is performed using a column containing a strong cation exchanger or a weak cation exchanger.

6. The method of any one of claims 2 to 5, wherein the CEX-HPLC is performed at a flow rate between 0.25 mL / min and 2 mL / min.

7. The method of claim 6, wherein the CEX-HPLC is performed at a flow rate between 0.5 mL / min and 1.5 mL / min, between 0.75 mL / min and 1 mL / min, or between 0.75 mL / min and 0.85 mL / min.

8. The method of claim 7, wherein the CEX-HPLC is performed at a flow rate of 0.5 mL / min, 0.75 mL / min, or 1.0 mL / min.

9. The method according to any one of claims 2 to 8, wherein the CEX-HPLC is performed at 20-40°C, 25-35°C, 25°C, 30°C, or 35°C.

10. The method of any one of claims 2 to 9, wherein the CEX-HPLC is performed in a 5-100% B gradient, a 10-100% B gradient, a 15-100% B gradient, or a 20-100% B gradient.

11. The method according to any one of claims 2 to 10, wherein the CEX-HPLC is performed at gradient times of 30-60 min, 30-45 min, 30-40 min, or 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

12. The method of any one of claims 1 to 11, wherein the CEX-HPLC is performed using a column packed with a resin having a particle size of less than 3 µm, less than 2.9 µm, less than 2.8 µm, less than 2.7 µm, less than 2.6 µm, or less than 2.5 µm, optionally wherein the resin is a porous resin or a non-porous resin.

13. The method of any one of claims 1 to 12, wherein the reference distribution is measured by cation exchange-high performance liquid chromatography (CEX-HPLC).

14. The method of claim 13, wherein the reference distribution is measured by cation exchange-high performance liquid chromatography (CEX-HPLC) under the same conditions as those used to measure the charge state distribution in (a).

15. The method of any one of claims 1 to 14, wherein the reference library contains the same ABP as the test library.

16. The method of any one of claims 1 to 15, wherein the test library and the reference library are generated from the same production cell line or its progeny.

17. The method of any one of claims 1 to 16, wherein the reference library has been analyzed by sequencing.

18. The method of claim 15 or 17, wherein the reference library is a different batch from the test library.

19. The method of any one of claims 1 to 18, wherein the reference library comprises an antibody.

20. The method of any one of claims 1 to 18, wherein the reference library comprises a variety of antibodies.

21. The method of any one of claims 1 to 20, wherein the reference library has been generated by mixing multiple monoclonal antibodies.

22. The method of any one of claims 1 to 21, wherein the reference library comprises a subset of the at least 100 ABPs in the test library.

23. The method of any one of claims 1 to 22, wherein the test library and the reference library have been generated separately.

24. The method of any one of claims 1 to 23, wherein the test library comprises at least 500 ABPs, at least 1,000 ABPs, at least 2,000 ABPs, at least 3,000 ABPs, at least 4,000 ABPs, at least 5,000 ABPs, at least 6,000 ABPs, at least 7,000 ABPs, at least 8,000 ABPs, at least 9,000 ABPs, or at least 10,000 ABPs.

25. The method of any one of claims 1 to 24, wherein in step (b), the charge state distribution is compared with a reference distribution based on the peak retention time.

26. The method of any one of claims 1 to 25, wherein in step (b), the charge state distribution is compared with a reference distribution based on peak area, peak height, or number of peaks.

27. The method of any one of claims 1 to 26, wherein in step (c), the test library is determined to have better quality when the charge state distribution of the test library is closer to the reference distribution.

28. The method of any one of claims 1 to 27, wherein in step (c), the test library is determined to have good quality when the charge state distribution of the test library is at least 50%, 60%, 70%, 80%, 90%, 96%, 97%, 98%, or 99% identical to the reference distribution in terms of peak size, peak retention time, or number of peaks.

29. The method of any one of claims 1 to 28, wherein in step (c), the quality of the test library is further determined based on the effectiveness of the test library as measured by ELISA.

30. The method of any one of claims 1 to 29, wherein in step (c), the quality of the test library is further determined based on an analysis of the size heterogeneity of the test library.

31. The method of claim 30, wherein the size heterogeneity is determined by size exclusion chromatography (SEC)-HPLC.

32. The method of claim 31, wherein the SEC-HPLC is performed using an SEC column comprising small-particle resin, wherein the particle size is less than 2.9µm, 2.8µm, 2.7µm, 2.6µm or 2.5µm.

33. The method of claim 31 or 32, wherein the SEC-HPLC is performed using a BEH stationary phase.

34. The method of any one of claims 31 to 33, wherein the SEC-HPLC is performed using a mobile phase with a pH between 6.7 and 7.3, between 6.8 and 7.2, between 6.9 and 7.1, or about 7.

0.

35. The method of any one of claims 31 to 34, wherein the SEC-HPLC is performed using a mobile phase containing a NaCl concentration between 450 mM and 550 mM, between 480 mM and 520 mM, or about 500 mM.

36. The method of any one of claims 1 to 35, wherein in step (c), the quality of the test library is further determined based on at least one of the following: (a) The amino acid sequences of the IgG1 and IgK frameworks, optionally verified by LC-MS peptide mapping; (b) Disulfide bond linkage between IgG1 and the IgK constant region, optionally verified by LC-MS non-reducing peptide mapping; (c) The size heterogeneity of the test library is optionally characterized by multi-angle light scattering (MALS) technique; (d) The melting temperature of the test library may optionally be measured by differential scanning calorimetry (DSC); (e) The glass transition temperature of the test library may optionally be measured by differential scanning calorimetry (DSC); (f) Analysis of released N-glycans; (g) Quantitative analysis of total sialic acid; and (h) Blood coagulation assay.

37. The method of any one of claims 1 to 36, wherein the test library is a pharmaceutical composition comprising the ABP and a pharmaceutically acceptable excipient.

38. The method of any one of claims 1 to 37, wherein the test library has been prepared by a process comprising: (a) Generating the at least 100 types of ABP by culturing production cell lines; and (b) Purify the at least 100 types of ABP.

39. The method of claim 38, wherein the purification is performed by at least one of the following steps: (i) Affinity chromatography, (ii) Low pH virus inactivation, (iii) Hydrophobic interaction chromatography or membrane filtration, (iv) Multimode anion exchange chromatography or membrane filtration, (v) Multimode cation exchange chromatography, (vi) Anion exchange chromatography or membrane filtration, (vii) Cation exchange chromatography, (viii) Virus filtering, and (ix) Ultrafiltration and / or percolation.

40. The method of claim 39, wherein the purification is performed by two, three, four, five, six, seven, eight, or all nine steps thereof: (i) Affinity chromatography, (ii) Low pH virus inactivation, (iii) Hydrophobic interaction chromatography or membrane filtration, (iv) Multimode anion exchange chromatography or membrane filtration, (v) Multimode cation exchange chromatography, (vi) Anion exchange chromatography or membrane filtration, (vii) Cation exchange chromatography, (viii) Virus filtering, and (ix) Ultrafiltration and / or percolation.

41. The method of any one of claims 1 to 40, wherein the ABP is an antibody.

42. The method of claim 41, wherein the ABP is an antibody specific to the antigen.

43. The method of claim 42, wherein the antigen is a viral or bacterial antigen.

44. The method of any one of claims 1 to 43, further comprising selecting the test library for preparing a pharmaceutical composition if the test library meets the acceptance criteria.

45. The method of claim 34, wherein the test library meets the acceptance criteria when it has a charge distribution that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 96%, 97%, 98%, or 99% the same as the reference distribution in terms of peak size, peak retention time, and / or number of peaks.

46. ​​The method of claim 44 or 45, wherein the acceptance criteria further comprises one or more factors selected from the following: a. The SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to the polyclonal antibodies accounted for 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more of the entire peak. b. The SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to high molecular weight (HMW) accounted for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak; and c. The SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to low molecular weight (LMW) accounted for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak.

47. The method of any of the preceding claims, further comprising preparing a pharmaceutical composition constituting the test library.

48. A pharmaceutical composition comprising the test library and prepared by the method of claim 47.

49. A method for analyzing a test library containing antigen-binding protein (ABP), comprising: (a) Measure the size heterogeneity of the test library, wherein the test library contains at least 100 ABPs; (b) Compare the peaks corresponding to high molecular weight substances (HMW), polyclonal antibody monomers (pAb peak), and low molecular weight substances (LMW); and (c) Determine the quality of the test library based on the comparison.

50. The method of claim 49, wherein the size heterogeneity of the test library is measured by size exclusion chromatography (SEC)-HPLC.

51. The method of claim 50, wherein the SEC-HPLC is performed using an SEC column comprising small-particle resin, wherein the particle size is less than 2.9 µm, 2.8 µm, 2.7 µm, 2.6 µm, or 2.5 µm.

52. The method of claim 50 or 51, wherein the SEC-HPLC is performed using a BEH stationary phase.

53. The method of any one of claims 50 to 52, wherein the SEC-HPLC is performed using a mobile phase with a pH between 6.7 and 7.3, between 6.8 and 7.2, between 6.9 and 7.1, or about 7.

0.

54. The method of any one of claims 50-53, wherein the SEC-HPLC is performed using a mobile phase with a NaCl concentration between 450 mM and 550 mM, between 480 mM and 520 mM, or about 500 mM.

55. The method of any one of claims 50 to 54, wherein the SEC-HPLC is performed at a temperature between 28°C and 32°C, between 29°C and 31°C, or about 30°C.

56. The method of any one of claims 49 to 55, wherein the test library meets the acceptance criteria in the following condition: a. The SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to the polyclonal antibodies accounted for 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more of the entire peak. b. The SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to high molecular weight (HMW) accounted for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak; and / or c. The SEC (size exclusion chromatography)-HPLC of the test library showed that the peaks corresponding to low molecular weight (LMW) accounted for less than 5.0%, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% of the total peak.

57. The method of any one of claims 49 to 56, further comprising preparing a pharmaceutical composition comprising the test library.

58. A pharmaceutical composition comprising the test library prepared by the method of claim 57.

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