Immunoglobulin-binding proteins and methods of use thereof

Abstract

Disclosed herein is a separation matrix comprising an immunoglobulin binding protein and methods of using the same.

Description

[0001] Docket No.11708-002WO1 IMMUNOGLOBULIN-BINDING PROTEINS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 622,915, filed January 19, 2024, which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT CLAUSE This invention was made with government support under Grant No.1R61AI174294-01 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on January 17, 2025, as an .XML file entitled “11708- 002WO1_ST26.xml” created on January 15, 2025, and having a file size of 1,803,365 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND Antibodies and related molecules are now most of the top ten highest-selling drugs in the world, and they continue to become increasingly important. Essentially all antibodies are purified using an antibody-binding protein to selectively capture them from the culture medium of the cells which produce them, allowing contaminants to be washed away. These antibody- binding proteins, typically used to bind immunoglobins, have diverse applications, including antibody purification, diagnostics, therapeutic imaging, and molecular biology research. The most common antibody-binding proteins used for this purpose are protein A of Staphylococcus aureus and Streptococcal protein G of because of its high affinity and selectivity. However, despite their clinical relevance, protein A and protein G are often difficult and costly to obtain, and their stability and selectivity are not optimal. As such, there exists a need for alternative antibody-binding proteins. These needs and others are at least partially satisfied by the present disclosure. ^ Docket No.11708-002WO1 SUMMARY Disclosed herein is a collection of immunoglobulin-binding proteins. These proteins can be used as alternatives to protein A and / or protein G for antibody purification and other applications. These proteins are able to bind to immunoglobulins with higher affinity, better selectivity, better stability and reusability, and faster binding kinetics than the conventional alternatives. In one aspect, provided herein is a separation matrix comprising: an immunoglobulin binding protein which binds at a site other than the antigen-recognition site of an antibody, Fc fragment (e.g., CH2 domain and / or CH3 domain), Fc fusion protein, scFv, Fab (e.g., CH1 domain, CL domain, VL domain, VH domain, or any combination thereof), antibody-drug conjugate, or other antibody or antibody derivative, wherein the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-9, SEQ ID NOS: 19-26, SEQ ID NOS: 27-120, or SEQ ID NOS: 186-191; and a solid support; wherein the immunoglobulin binding protein is coupled to the solid support. In another aspect, provided is a method for the purification of an antibody, the method comprising: preparing any of the disclosed separation matrices; contacting a sample comprising an antibody with the separation matrix; washing unbound proteins and non-protein material from the separation matrix using a wash buffer; and eluting the antibody using a low pH buffer. Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS FIGURES 1A-1B depict 16.5% tris tricine SDS-PAGE. Lane numbers in FIG.1A are as follows: 1. low range molecular weight marker; 2. IgG binder of SEQ ID NO: 1; 3. IgG binder of SEQ ID NO: 2; 4. IgG binder of SEQ ID NO: 3; 5. IgG binder of SEQ ID NO: 4; 6. IgG binder of SEQ ID NO: 5; 7. IgG binder of SEQ ID NO: 6; 8. IgG binder of SEQ ID NO: 7; 9. IgG binder of SEQ ID NO: 8; 10. IgG binder of SEQ ID NO: 1400. Lane numbers in FIG. 1B are as follows: 1. low range molecular weight marker; 2. IgG binder of SEQ ID NO: 9. FIGURES 2A-2J depict MALDI spectra for IgG binder of SEQ ID NO: 1 (FIG. 2A); IgG binder of SEQ ID NO: 2 (FIG.2B); IgG binder of SEQ ID NO: 3 (FIG.2C); IgG binder of SEQ ID NO: 4 (FIG. 2D); IgG binder of SEQ ID NO: 5 (FIG.2E); IgG binder of SEQ ID ^ Docket No.11708-002WO1 NO: 6 (FIG.2F); IgG binder of SEQ ID NO: 7 (FIG.2G); IgG binder of SEQ ID NO: 8 (FIG. 2H); IgG binder of SEQ ID NO: 1400 (FIG.2I); IgG binder of SEQ ID NO: 9 (FIG.2J). FIGURE 3 depicts an ELISA procedure for screening IgG binders. FIGURE 4 depicts a comparison of Rituximab, Human IgG and alpaca IgG binding with binder. FIGURES 5A-5G depict ITC integrated heat of titration as a function of the molar ratio of IgG binders: rituximab (top) and raw data of the sequential titration (bottom) for IgG binder of SEQ ID NO: 2 (FIG. 5A); IgG binder of SEQ ID NO: 3 (FIG.5B); IgG binder of SEQ ID NO: 4 (FIG.5C); IgG binder of SEQ ID NO: 7 (FIG.5D); IgG binder of SEQ ID NO: 8 (FIG. 5E); IgG binder of SEQ ID NO: 1400 (FIG.5F); IgG binder of SEQ ID NO: 9 (FIG.5G). FIGURES 6A-6D depict IgG binder of SEQ ID NO: 9 (FIG. 6A), IgG binder of SEQ ID NO: 3 (FIG. 6B), protein A Z domain (SEQ ID NO: 1400) (FIG. 6C), and IgG binder of SEQ ID NO: 7 (FIG.6D) binding to Rituximab. FIGURES 7A-7D depict IgG binder of SEQ ID NO: 3 (FIG. 7A), IgG binder of SEQ ID NO: 9 (FIG. 7B), IgG binder of SEQ ID NO: 7 (FIG. 7C), and protein A Z domain (SEQ ID NO: 1400) (FIG.7D) binding to human polyclonal IgG. FIGURE 8 depicts rat IgG binding of IgG binders 75 (SEQ ID NO: 9), 404, (SEQ ID NO: 6) 405 (SEQ ID NO: 7), 406 (SEQ ID NO: 8), 957 (SEQ ID NO: 1), 965 (SEQ ID NO: 2), 974 (SEQ ID NO: 3), 978 (SEQ ID NO: 4), 987 (SEQ ID NO: 5), and protein A Z domain (SEQ ID NO: 1400). FIGURES 9A-9C depict protein A inhibition using IgG binders 75 (SEQ ID NO: 9) (FIG.9A), 974 (SEQ ID NO: 3) (FIG.9B), and 405 (SEQ ID NO: 7) (FIG.9C). FIGURES 10A-10E show MALS analysis plots. FIG.10A shows the MALS analysis plot for Rituximab. FIGS.10B-10E show the MALS analysis plots for IgG binder of SEQ ID NO: 9 (FIG. 10B), IgG binder of SEQ ID NO: 3 (FIG. 10C), IgG binder of SEQ ID NO: 7 (FIG. 10D), and protein A Z domain (SEQ ID NO: 1400) (FIG. 10E) in a ratio of 20:1 IgG binder:Rituximab. FIGURE 11 is a flowchart for the experimental setup used to conduct protein A Z domain surface display binding. FIGURE 12 depicts confocal microscope images of surface displayed protein A Z domain. FITC-IgG binds with the surface displayed protein A Z domain. FIGURES 13A-13B depict confocal microscope images of surface displayed protein A Z domain. ^ Docket No.11708-002WO1 FIGURES 14A-14C depict cytometry for surface displayed IgG binders. FIG. 14A shows expression of AIDA (adhesin involved in diffuse adherence) alone, and FIG.14B shows expression of protein A Z domain fused to AIDA. FIG.14C shows a 90:10 mixture of AIDA and protein A Z domain fused to AIDA. The box around the FITC+ data highlights that the percentage population observed (11%) in the FITC filter is closely matching with the expected value. DETAILED DESCRIPTION It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. DEFINITIONS In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings: Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and are not intended to exclude, for example, other additives, segments, integers, or steps. Furthermore, it is to be understood that the terms comprise, comprising, and comprises as they relate to various aspects, elements, and features of the disclosed invention also include the more limited aspects of “consisting essentially of” and “consisting of.” As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” includes aspects having two or more such cells unless the context clearly indicates otherwise. Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It should be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. ^ Docket No.11708-002WO1 As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, a human fetus or cell, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., a physician. SEPARATION MATRIX In some aspects, disclosed herein are immunoglobulin-binding proteins that can be used as an alternative to protein A. In one aspect, provided herein is a separation matrix comprising: an immunoglobulin binding protein, wherein the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-9, SEQ ID NOS: 19-26, SEQ ID NOS: 27-120, or SEQ ID NOS: 186-191; and a solid support; wherein the immunoglobulin binding protein is coupled to the solid support. In some aspects, the amino acid sequence is at least 80% identical to any one of SEQ ID NOS: 1-9, SEQ ID NOS: 19-26, SEQ ID NOS: 27-120, or SEQ ID NOS: 186-191, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 1-9, SEQ ID NOS: 19-26, SEQ ID NOS: 27-120, or SEQ ID NOS: 186-191. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 1-9, SEQ ID NOS: 19- 26, SEQ ID NOS: 27-120, or SEQ ID NOS: 186-191. In some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-9, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least ^ Docket No.11708-002WO1 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 1-9. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 1-9. In some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 1. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 2. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 3. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 4. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 7. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 8. In some aspects, the immunoglobulin binding protein comprises SEQ ID NO: 9. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 1. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 2. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 3. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 4. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 7. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 8. In some aspects, the immunoglobulin binding protein consists of SEQ ID NO: 9. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 1. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 201-241, or at least 81% ^ Docket No.11708-002WO1 identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 201-241. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 201-241. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 2. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 242-281, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 242-281. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 242-281. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 3. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 282-321, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 282-321. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 282-321. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 4. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 322-361, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, ^ Docket No.11708-002WO1 or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 322-361. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 322-361. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 5. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 362-401, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 362-401. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 362-401. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 6. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 402-431, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 402-431. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 402-431. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 7. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 432-469, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 432-469. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 432-469. ^ Docket No.11708-002WO1 In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 8. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 470-503, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 470-503. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 470-503. In some aspects, the immunoglobulin binding protein comprises a fragment of SEQ ID NO: 9. For example, in some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 504-528, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 504-528. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 504-528. In some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 191-200, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 191-200. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 191-200. In some aspects, the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 529-1399, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least 91%, or at least 92% identical, or at least ^ Docket No.11708-002WO1 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 529-1399. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 529-1399. In some aspects, the immunoglobulin-binding protein has increased alkaline tolerance compared to a reference control immunoglobulin-binding protein. For example, in some aspects, the binding ability of the immunoglobulin-binding protein (for example, to the Fc or Fab domain of an immunoglobulin) may not be impacted by exposure to a hydroxide salt (for example, NaOH or KOH) at a concentration of from about 0.01M to about 3 M, or from about 0.05 M to about 2.5 M, or from about 0.1 M to about 2 M, or from about 0.15 M to about 1.5 M, or from about 0.2 M to about 1 M, or from about 0.25 M to about 0.75 M, or from about 0.01 M to about 1 M, or from about 0.05 M to about 0.75 M, or from about 0.05 M to about 0.5 M. In other aspects, the binding ability of the immunoglobulin-binding protein (for example, to the Fc or Fab domain of an immunoglobulin) may not be impacted by exposure to a solution above about pH 9.0, or above about pH 9.25, or above about pH 9.5, or above about pH 9.75, or above about pH 10, or above about pH 10.25, or above about pH 10.5, or above about pH 10.75, or above about pH 11. In some such aspects, the binding protein can be reused after exposure to the elevated pH solution. In some aspects, the solid support comprises iron oxide, styrene-based polymers, agarose, alginate, sepharose, dextran, polyacrylamide, starch, cellulose, nitrocellulose, glass, electrospun polymer nanofibers, other polymer nanofibers, silica, magnetic nanoparticles, or any combination thereof. In some aspects, the immunoglobulin binding protein is coupled to the solid support using N-hydroxy succinimide, thiol or epoxy chemistry. Directed immobilization can be carried out using cysteine at the N or C terminal of the protein, or via amines on the protein N- terminus or side chains. In one aspect, provided herein is a separation matrix comprising: an immunoglobulin binding protein, wherein the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 121-185; and a solid support; wherein the immunoglobulin binding protein is coupled to the solid support. In some aspects, the amino acid sequence is at least 80% identical to any one of SEQ ID NOS: 121-185, or at least 81% identical, or at least 82% identical, or at least 83% identical, or at least 84% identical, or at least 85% identical, or at least 86% identical, or at least 87% identical, or at least 88% identical, or at least 89% identical, or at least 90% identical, or at least ^ Docket No.11708-002WO1 91%, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. In some aspects, the immunoglobulin binding protein comprises any one of SEQ ID NOS: 121-185. In some aspects, the immunoglobulin binding protein consists of any one of SEQ ID NOS: 121-185. In some aspects, multiple repeating units of immunoglobulin binding proteins (including identical units or distinct domains with variations in affinity, selectivity, or binding properties) can also be used for immunoglobulin binding and antibody purification. In some embodiments, the number of these repeating units can be up to 5 to 10 or 20 or more. In some embodiments, repeating units can be separated by linker sequences of 1-50 or more amino acids for steric reach and charge modification and can differ in affinity and selectivity. In some embodiments, smaller molecules of 10-50 amino acids can be derived from them which still retain useful binding activities. In some aspects, such a molecule comprises a helical domain comprising a sequence at least 80% identical to the closest matching sequence in one of the sequences disclosed herein. In some aspects, immunoglobulin binding proteins are used to bind other types of proteins that comprise Fc or Fab domains, such as Fc fusions and VH3 molecules. In some aspects, the immunoglobulin-binding protein disclosed herein can be used in analytical applications, including binding assays and lateral-flow chromatography. In some aspects, the immunoglobulin-binding proteins can be fused or conjugated to fluorescent reporters for detection of other types of proteins that comprise Fc or Fab domains, especially Fc fusions and VH3 molecules (e.g., CH2 domain, CH3 domain, CH1 domain, ilCL domain, VL domain, VH domain, or any combination thereof), by changes in fluorescence intensity, anisotropy, or lifetime, or in binding assays. The immunoglobulin-binding proteins can be used in this way either in soluble form or immobilized, e.g., on a fiber optic probe or solid support such as nitrocellulose, polystyrene, gold, silica or agarose. The immunoglobulin- binding proteins can be used in lateral-flow assays and in affinity chromatography. The immunoglobulin-binding proteins also can be fused to reporter / signal amplifying enzymes for use in assays. These enzymes include soybean or horseradish peroxidase, alkaline phosphatase, and biotin ligase, including the bacterial biotin ligase BirA* and the R118G mutant thereof. The immunoglobulin-binding proteins disclosed herein can further be used as the basis of “affibodies” or other derivatives for purification, industrial, analytical, vaccine, diagnostic, imaging, or therapeutic purposes. The immunoglobulin-binding proteins can be fused together, ^ Docket No.11708-002WO1 potentially along with other proteins. The immunoglobulin-binding proteins can be displayed on phage, bacteria, yeast, or ribosomes, mutated and / or shuffled and selected. The immunoglobulin-binding proteins can be fused and / or conjugated to particles or polymers such as PEG, starch or dextran or proteins such as elastin or other conditionally- soluble moieties to confer separation, filter-retention, partitioning (especially between phases comprising at least about 80% water by mass, or ionic liquids), or precipitation properties. Particles conjugated to the immunoglobulin-binding proteins can be used to retain antibodies which otherwise would pass through a filter. Such constructs can be used in continuous manufacturing of antibodies or related molecules including scFv, Fc fusions, bispecific antibodies, etc. In some aspects, one or more immunoglobulin-binding proteins are fused with zwitterionic polypeptides with, for example, a repetitive (VPX1X2G)n motif, where X1 and X2 are cationic and anionic amino acids, respectively, and n is the number of repeats. In some aspects, one or more immunoglobulin-binding proteins are fused with a peptide or protein responsive to temperature or ionic strength and used for separation of proteins to which the immunoglobulin-binding protein can bind. In some aspects, the immunoglobulin-binding protein has a higher affinity for murine IgG1compared and is immobilized on a solid support or conjugated to a reporter and used for purification or detection of murine IgG. In some aspects, one or more immunoglobulin-binding proteins are prepared in immobilized, fusion-protein, or conjugated form and contacted with von Willebrand factor (VWF) or its single or multiple domains, or human blood, serum, or plasma. In some aspects, one or more immunoglobulin-binding proteins or an affibody derived therefrom is contacted with mammalian B cells or administered to a mammal. In some aspects, an N-terminal methionine added to or deleted from the immunoglobulin-binding protein. The immunoglobulin-binding proteins can be recombined (potentially along with other proteins, such as domains of currently-used Proteins A or G) by methods including gene shuffling to derive new molecules with improved stability, expression level, selectivity, kinetics, or affinity. The immunoglobulin-binding proteins can be cyclized, e.g., by intein methods. The immunoglobulin-binding proteins can be mutated for greater stability to base, both by rational design and by selection. In some aspects, the separation matrix comprises at least one of the immunoglobulin- binding proteins coupled to the solid support via an arginine, aspartatic acid, glutamic acid, ^ Docket No.11708-002WO1 tyrosine, histidine, cysteine, or lysine, wherein the coupling comprises a thioether or amide bond. In some aspects, the separation matrix comprises at least one of the immunoglobulin- binding proteins coupled to the solid support via a negatively charged linker. In some aspects, the solid support is porous and either nanofibrous, membranous, roughly spherical, or monolithic. In some aspects, the solid support is cleaned with at least about 50 mM benzoate or benzyl alcohol, or both. In some aspects, at least one cysteine residue is added to the C-terminal helical region of the immunoglobulin-binding protein, conferring additional stability upon the immunoglobulin-binding protein. In some aspects, a synthetic DNA encoding one or more immunoglobulin-binding proteins is inserted into a plasmid or into the genome of a microorganism or plant and expressed in the microorganism or plant. In some aspects, the expressing microorganism is E. coli, yeast, fungus, or filamentous. In some aspects, a synthetic DNA encoding one or more immunoglobulin-binding proteins includes codons used by the intended expression host organism. In some aspects, the synthetic DNA encoding one or more of the proteins expressed here additionally encodes N-terminal or C-terminal sequences useful in coupling or adsorbing the immunoglobulin-binding proteins to the solid support. In some aspects, the immunoglobulin-binding protein is purified using a bioreactor, plant, centrifugation, filtration, and / or precipitation. In some aspects, the immunoglobulin- binding proteins is purified using a protein affinity tag encoded in the same DNA as encodes the immunoglobulin-binding proteins itself. In some aspects, the immunoglobulin-binding protein is purified by processes involving heating above 50°C and / or exposure to pH higher than about 9 or lower than about 5. In some aspects, the immunoglobulin-binding protein is purified using its affinity for antibody or Fc fragment. In some aspects, the immunoglobulin- binding protein is purified using ion exchange or hydrophobic interaction chromatography. In some aspects, the immunoglobulin-binding protein is modified with one or more amino acids or amino acid sequences suitable for coupling to the solid support. In some aspects, the immunoglobulin-binding protein is modified at more than one amino acid position in such a way as to make it more negatively charged at a pH of about 6.5. In some aspects, the immunoglobulin-binding protein is modified at more than one amino acid position in such a way as to make it have a higher density of total charge (sum of positive and negative charges) at a pH of about 6.5 (see U.S. Patent No. 11,660,550, hereby incorporated by reference in its entirety). In some aspects, the immunoglobulin-binding protein is modified with one or more polymer chains, including PEG and zwitterionic polymers. ^ Docket No.11708-002WO1 In some aspects, the immunoglobulin-binding protein is derived from or most closely related to a sequence found in an organism other than Staphylococcus aureus. In some aspects, the immunoglobulin-binding protein is derived from or most closely related to a sequence found in an organism not in the genus Staphylococcus. In some aspects, proteins are produced which comprise two or more immunoglobulin- binding proteins connected by linker sequences of amino acids. In some aspects, the linker sequences of amino acids include the sequence PKA, A(EAAAK)nA (where n = 2–5), GSS, or SGS. In some aspects, the linker sequences of amino acids include at least two of proline, glycine, alanine or serine. In some aspects, the linker sequences of amino acids include at least about 30% aspartic or glutamic acid. In some aspects, the linker sequences of amino acids comprise at least three amino acids. In some aspects, at least one linker sequence of amino acids comprises at least seven amino acids. In some aspects, at least one linker sequence of amino acids comprises at least 15 amino acids. In some aspects, at least one linker sequence of amino acids comprises at least 25 amino acids. In some aspects, the separation matrix comprises one or more immunoglobulin-binding proteins with three, four or five consecutive amino acids deleted from the N terminus of the immunoglobulin-binding protein, wherein the ligand exhibits reduced fragmentation, relative to a counterpart without the deletion, following exposure to about 0.5 M NaOH for at least about 5 hours. In some aspects, the separation matrix comprises an immunoglobulin-binding protein as a ligand coupled to the solid support, wherein at least one asparagine residue of the ligand has been mutated to an amino acid other than glutamine. In some aspects, the immunoglobulin-binding proteins are further engineered to enhance their stability in detergents and chaotropic agents such as Urea and Guanidium Hydrochloride or similar chemicals, and these proteins, which are less prone to aggregation, are utilized in the chromatographic purification of Fc-proteins and antibodies. In some aspects, the immunoglobulin-binding proteins are further stabilized by designing disulfide-bonded intact or partial Fc-binding domains, demonstrating enhanced stability compared to their ancestral domain scaffolds. In some aspects, the immunoglobulin-binding proteins are further modified with loops that accommodate divalent cation coordination motif(s) and release Fc- proteins and antibodies in the presence of EDTA and / or EGTA. In some aspects, the immunoglobulin-binding proteins induce minimal or reduced binding stress to the Fc-proteins and antibodies during purification, thereby resulting in reduced nucleation of Fc-proteins and antibodies in the subsequent formulation step(s). In some aspects, ^ Docket No.11708-002WO1 the immunoglobulin-binding proteins are fused to peptides or other protein domains, enabling their covalent or noncovalent attachment to an adsorbent or resin for more efficient purification of Fc-proteins or antibodies. In some aspects, the immunoglobulin-binding proteins are fused to form homo- or hetero-multimers via linkers of varying lengths. These multimeric fusion species aim to prevent intermolecular steric effects between Fc-proteins and antibodies during purification step(s) and to achieve dynamic binding capacity (DBC) values closer to the static binding capacities (SBC). In some aspects, the immunoglobulin-binding proteins are incorporated into chimeric domains, biomolecular scaffolds artificially designed from two or more naturally occurring Fc- binding domains via conformation-dependent residue grafting that are utilized to purify Fc- proteins and poly- and monoclonal antibodies. In some aspects, the immunoglobulin-binding proteins are additionally modified to contain a decreased number or absence of asparagine (Asn) due to the tendency of Asn to undergo deamidation or backbone cleavage reactions at high pH during the Cleaning in Place (CIP) process with agents like NaOH. In some aspects, the immunoglobulin-binding proteins are modified with added histidines to facilitate the elution of Fc antibodies or Fc-fusion biomolecules at milder pH ranges (pH > 4.5). In some aspects, the immunoglobulin-binding proteins are engineered to carry complex, mannosylated or glucosylated glycans, or are glycan-deficient, exhibiting reduced or enhanced structural stability. In some aspects, the immunoglobulin-binding proteins are further engineered to exhibit faster association or higher or reduced binding kinetics (konand koff), and binding affinity (Kd) to Fc-proteins and antibodies, or greater resistance to digestion by protease. In some aspects, disclosed herein are immunoglobulin-binding proteins that can be used as an alternative to protein A when fused or shuffled with Protein A, other proteins disclosed here, or other proteins. In further aspects, disclosed herein are immunoglobulin-binding proteins that can be used as an alternative to protein G. METHODS In one aspect, provided herein is a method for the purification of an antibody, the method comprising: preparing any of the disclosed separation matrices; contacting (e.g., incubating) a sample comprising an antibody with the separation matrix; washing unbound proteins and non-protein material from the separation matrix using a wash buffer; and eluting the antibody using a low pH buffer. ^ Docket No.11708-002WO1 In some aspects, preparing the separation matrix comprises rinsing the separation matrix with an equilibration buffer. In some aspects, the equilibration buffer has a neutral pH and / or a low conductivity. In some aspects, the equilibration buffer comprises a tris and / or phosphate buffer at a concentration of from about 20 mM to about 100 mM, or from about 30 mM to about 90 mM, or from about 40 mM to about 80 mM, or from about 50 mM to about 70 mM, or from about 20 mM to about 60 mM, or from about 30 mM to about 50 mM, or from about 60 mM to about 100 mM, or from about 70 mM to about 90 mM. In some aspects, the equilibration buffer has a pH of from about 6.5 to about 7.5, or from about 6.6 to about 7.4, or from about 6.7 to about 7.3, or from about 6.8 to about 7.2, or from about 6.9 to about 7.1, or from about 6.5 to about 7, or from about 6.6 to about 6.9, or from about 6.7 to about 6.8, or from about 7 to about 7.5, or from about 7.1 to about 7.4, or from about 7.2 to about 7.3. In some aspects, the sample is a cell culture supernatant. In some aspects, the sample is a biological fluid obtained from a subject (e.g., blood, urine, sweat, etc.). In some aspects, the antibody is an immunoglobulin G. In some aspects, the antibody is a monoclonal antibody. In some aspects, the antibody is rituximab. In some aspects, the wash buffer has a neutral pH and / or a high conductivity. In some aspects, the wash buffer comprises a tris and / or phosphate buffer at a concentration of from about 20 mM to about 100 mM, or from about 30 mM to about 90 mM, or from about 40 mM to about 80 mM, or from about 50 mM to about 70 mM, or from about 20 mM to about 60 mM, or from about 30 mM to about 50 mM, or from about 60 mM to about 100 mM, or from about 70 mM to about 90 mM. In some aspects, the wash buffer further comprises NaCl at a concentration of from about 0.2 M to about 1 M, or from about 0.3 M to about 0.9 M, or from about 0.4 M to about 0.8 M, or from about 0.5 M to about 0.7 M, or from about 0.2 M to about 0.6 M, or from about 0.3 M to about 0.5 M, or from about 0.6 M to about 1 M, or from about 0.7 M to about 0.8 M. In some aspects, the low pH buffer has a pH of from about 2 to about 5, or from about 2.2 to about 4.8, or from about 2.4 to about 4.6, or from about 2.6 to about 4.4, or from about 2.8 to about 4.2, or from about 3 to about 4, or from about 3.2 to about 3.8, or from about 3.4 to about 3.6, or from about 2 to about 3.5, or from about 2.2 to about 3.4, or from about 2.4 to about 3.2, or from about 2.6 to about 3, or from about 3.5 to about 5, or from about 3.6 to about 4.8, or from about 3.8 to about 4.6, or from about 4 to about 4.4. In some aspects, the low pH buffer comprises acetic acid, glycine, citric acid, phosphoric acid, or any combination thereof at a concentration of about 0.1 M, or about 0.08 M, or about 0.06 M, or about 0.05 M, or about 0.15 M, or about 0.2 M, or about 0.25 M, or ^ Docket No.11708-002WO1 about 0.3 M, or about 0.35 M, or about 0.4 M, or about 0.45 M, or about 0.5 M, or about 0.55 M, or about 0.6 M, or about 0.65 M, or about 0.7 M, or about 0.75 M, or about 0.8 M, or about 0.85 M, or about 0.9 M, or about 0.95 M, or about 1 M. In some aspects, the method further comprises: contacting (e.g., incubating) the separation matrix with a high pH alkaline solution; and storing the separation matrix in an ethanol solution. In some aspects, the high pH alkaline solution comprises NaOH at a concentration of from about 0.1 M to about 0.5 M, or from about 0.15 M to about 0.45 M, or from about 0.2 M to about 0.4 M, or from about 0.25 M to about 0.35 M, or from about 0.1 M to about 0.3 M, or from about 0.12 M to about 0.28 M, or from about 0.14 M to about 0.24 M, or from about 0.16 M, to about 0.22 M, or from about 0.18 M to about 0.2 M, or from about 0.3 M to about 0.5 M, or from about 0.32 M to about 0.48 M, or from about 0.34 M to about 0.46 M, or from about 0.36 M to about 0.44 M, or from about 0.38 M to about 0.42 M. In some aspects, the ethanol solution comprises about 20% ethanol, or about 18%, or about 16%, or about 14%, or about 12%, or about 10%, or about 8%, or about 6%, or about 5%, or about 22%, or about 24%, or about 26%, or about 28%, or about 30%, or about 32%, or about 34%, or about 36%, or about 38%, or about 40%, or about 42%, or about 44%, or about 46%, or about 48%, or about 50%. In some aspects, the separation matrix can be washed using a solution comprising an ammonium sulfate buffer having a pH of about 3, an arginine buffer, and a buffer containing the detergent LDAO (N,N-dimethyldodecylamine N-oxide) or LDAO derivatives that have head group, charge, hydrophobic chain length, and / or functionalized alkyl chain (e.g., MDOA, myristyldimethylamine oxide) to inactivate viruses. In some aspects, the ammonium sulfate buffer has a concentration of from about 0.5 M to about 2 M, or from about 0.6 M to about 1.8 M, or from about 0.8 M to about 1.6 M, or from about 1 M to about 1.4 M, or from about 0.5 M to about 1.2 M, or from about 0.6 M to about 1 M, or from about 1 M to about 2 M, or from about 1.2 M to about 1.8 M, or from about 1.4 M to about 1.6 M. In some aspects, the arginine buffer has a concentration of from about 0.1 M to about 1 M, or from about 0.2 M to about 0.9 M, or from about 0.3 M to about 0.8 M, or from about 0.4 M to about 0.7 M, or from about 0.5 M to about 0.6 M, or from about 0.1 M to about 0.6 M, or from about 0.2 M to about 0.5 M, or from about 0.3 M to about 0.4 M, or from about 0.5 M to about 1 M, or from about 0.6 M to about 0.9 M, or from about 0.7 M to about 0.8 M. The separation matrix can be washed with a neutral pH arginine buffer at a concentration of from about 0.1 M to about 2 M, or from about 0.2 M to about 1.8 M, or from ^ Docket No.11708-002WO1 about 0.4 M to about 1.6 M, or from about 0.6 M to about 1.4 M, or from about 0.8 M to about 1.2 M, or from about 0.1 M to about 1 M, or from about 0.2 M to about 0.8 M, or from about 0.4 M to about 0.6 M, or from about 1 M to about 2 M, or from about 1.2 M to about 1.8 M, or from about 1.4 M to about 1.6 M to reduce the aggregates of Fc containing proteins bound to the immunoglobulin-binding proteins. The separation matrix can be washed with high salt neutral pH buffer to remove the host cell proteins nonspecifically bound to the immunoglobulin-binding proteins. The separation matrix disclosed herein can be useful in continuous manufacturing processes, multicolumn chromatography, and separations based on filtration, partitioning and precipitation. The separation matrix can be of particular use in larger-scale processes, e.g., with liquid flow rates of more than about 20 mL per minute or about 100 mL per minute or about 2 L per minute, and in chromatographic processes with bed volumes over about 10 L. The separation matrix can be useful in processing clarified cell culture fluid, particularly in volumes over about 1000 L. The separation matrix can be advantageously used in environments with low levels of illumination, especially light in the shorter-wavelength third of the visible spectrum. In some aspects, the separation matrix can be used in purification in which elution occurs at a temperature below about 35°C with an elution buffer comprising a chaotropic agent, a sugar alcohol, and at least one amino acid (see Australian Patent Application Publication No. 2023222915, herby incorporated by reference in its entirety). In some aspects, the separation matrix can be used to retain antibodies which otherwise would pass through a filter, to be used in (semi-)continuous manufacturing of antibodies or related molecules including scFv, Fc fusions, bispecific antibodies, etc. In some aspects, the separation matrix is used in the purification of an Fc or Fab protein expressed in a bacterium, a yeast or filamentous fungus, a plant, a cell-free synthesis system, mammalian cells or in the milk of a mammal. In some aspects, the Fc or Fab protein to be purified is IgG monoclonal antibody, Fab ' fragment, F (ab ')2 Fragment, scFv, di-ScFv, bi-scFv, series connection (di, tri)-scFv, Fv, sdAb, three-function antibodies, BiTE, bispecific antibody or trivalent antibodies. In some aspects, the separation matrix is loaded with Fab, scFv or Fc protein that has already passed through an adsorbent (e.g., an ion-exchanger) in flowthrough mode. In some aspects, the separation matrix is contacted with Fab or Fc protein at a pH from about 7.5 to about 10 and containing a combination of monovalent cations and polybasic ^ Docket No.11708-002WO1 anions, optionally including glycine, tris, borate, or phosphate, at a concentration of from about 0.6 M to about 1.75M. In some aspects, the separation matrix is overloaded, or contacted with more than its static or dynamic capacity for Fab or Fc protein. In some aspects, the separation matrix is loaded with Fab or Fc protein and is washed before elution with a salt solution having a pH of from about 4.7 to about 5.7 containing sodium chloride, potassium chloride, and / or potassium thiocyanate at a concentration of from about 0.8 M to about 3.3M, and then optionally washed a second time with less than about 2 M salt. In some aspects, the separation matrix is loaded with Fab or Fc protein and is washed before elution with a buffer solution selected from: (i) a buffer solution containing 0.5 M NaCl, 25 mM sodium acetate, at pH 5.5; (ii) a buffer solution containing 0.5 M NaCl, 25 mm sodium acetate, at a pH of 5.5; and (iii) a buffer solution containing 0.5 M NaCl, 20 mm citric acid / sodium citrate at pH 6. In some aspects, the separation matrix is loaded with Fab or Fc protein and is eluted at a pH of from about 4 to about 5 with a solution containing at least about 50 mM acetyl arginine, agmatine, L-arginine acid, N-^-butyryl-L-arginine, and / or N-^-pivaloyl arginine. In some aspects, eluate from an adsorbent comprising one of the molecules disclosed herein is applied to IEX (ion exchange chromatography) or HIC (hydrophobic interactions chromatography) or MM (mixed mode or multi-mode) columns after filtration, and / or dilution. In some aspects, the eluate from a separation involving one of the proteins disclosed here is loaded onto an ion exchange, hydrophobic interaction chromatography, or mixed mode adsorbent column after incubation, centrifugation, filtration, pH adjustment, salt addition, or dilution by not more than two-fold. In some aspects, the eluate from a separation involving one of the proteins disclosed here is loaded onto an ion exchange, hydrophobic interaction chromatography, or mixed-mode adsorbent column after holding at pH below about 5.0 for at least about 30 minutes. In some aspects, a temperature-sensitive-binding variant of one of the immunoglobulin- binding proteins disclosed herein is identified by display library screening at varied temperatures, and used in affinity separations of an Fc or Fab or scFv protein, by contacting a solution comprising the protein and one or more impurities with a temperature-responsive protein material at a temperature at which the protein binds to the material; and eluting the protein from the material at a temperature below about 35°C with an elution buffer having a pH of from about 6.5 to about 7.5 and comprising a chaotropic agent, a sugar alcohol, an apolar amino acid, and a basic amino acid, wherein the protein is purified from one or more impurities ^ Docket No.11708-002WO1 in the solution and the chaotropic agent is urea, guanidinium chloride, sodium thiocyanate, potassium thiocyanate, or ammonium thiocyanate. In some aspects, the separation matrix comprises an immunoglobulin-binding protein disclosed herein which comprises at least one two- or three-helix bundle domain with an off- rate for dissociation of human IgG-1 slower than that of Staphylococcus aureus Cowan strain 1 protein A domain B in standard phosphate buffered saline solution at pH 7.4, or in 10 millimolar HEPES, 150 mM sodium chloride, with 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4, or in 20 mM Tris, pH 7.25. In some aspects, the separation matrix comprises an immunoglobulin-binding protein disclosed herein which comprises at least one two- or three-helix bundle domain with an on- rate for association of human IgG-1 faster than that of Staphylococcus aureus Cowan strain 1 protein A domain B in standard phosphate buffered saline solution at pH 7.4, or in 10 millimolar HEPES, 150 mM sodium chloride, with 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4, or in 20 mM Tris, pH 7.25. In some aspects, the microbial bioburden of the separation matrix is reduced by contacting the separation matrix with a composition comprising about 0.1 M acetic acid and about 20% ethanol for about 1 hour to about 4 hours. In some aspects, the separation matrix is sterilized by gamma-irradiation in a tube closed at both ends in the presence of benzyl alcohol, acetate or ethanol. In some aspects, the separation matrix is contacted with a mixture of an alkaline salt, one or more surfactants and, optionally, a chelator, for cleaning and / or sanitization. In some aspects, the separation matrix is prepared by immobilizing one or more immunoglobulin-binding proteins on the solid support prepared by shrinking a polysaccharide porous bead by at least about 10% by volume, wherein the shrinking comprises contacting the polysaccharide porous beads into contact with a water-soluble organic solvent and alkaline water. In some aspects, the separation matrix is contacted with clarified cell culture fluid in a first periodic counter current chromatography system (PCCS1) and a recombinant therapeutic protein is captured on the adsorbent. An eluate from PCCS1 comprising the recombinant therapeutic protein is continuously or intermittently fed into a second periodic counter current chromatography system (PCCS2) for further purifying and polishing of the recombinant therapeutic protein using an adsorbent in the PCCS2 that is different from that used in PCCS1. In some aspects, a protein of interest is eluted from interacting with the separation matrix, and then contacted in flow-through mode with a carbonaceous material (e.g., activated ^ Docket No.11708-002WO1 carbon or charcoal) and then contacted with one or more matrices selected from anion exchange media, cation exchange media, and virus filtration media. In some aspects, a monoclonal antibody or antigen binding fragment thereof is purified from a solution by (a) contacting said solution with the separation matrix, and eluting a crude protein eluent from the adsorbent using a first elution buffer; and (b) then contacting the crude protein eluent obtained at the end of step (a) with a second adsorbent and recovering purified monoclonal antibody or antigen binding fragment thereof from the second adsorbent using a second elution buffer, wherein each of the buffers comprises Bis Tris, acetic acid, sodium chloride (NaCI) and water. EXAMPLES Example 1: Production of the Binders Purification of antibodies and fragment crystallizable (Fc)-based recombinant therapeutic proteins utilizes affinity chromatography as a high-throughput and cost-effective capture step. The application of affinity chromatography in an economical fashion requires the inclusive removal of the contaminants and impurities, including process-related impurities (host cell protein, host cell DNA, lipids, upstream and downstream additives) and product- related impurities (modified, aggregated, and degraded variants of target protein) [1]. The most widely-used affinity ligands are based on Staphylococcus aureus protein A (SpA), a cell wall- anchored Ig-binding protein that aids in virulence. Proteins G and L are also widely used although less popular than protein A. During multiple chromatographic cycles in downstream operation, protein A degrades, liberating fragments that are problematic for subsequent unit operations. The design and production of stable affinity proteins are of great technological significance and economic importance. Column fouling, ligand leaching, carryover between subsequent cycles, bioburden control, and lower resin utilization are some of the issues solved by efficient cleaning in place (CIP). CIP uses NaOH (0.1- 0.5 M) to remove such contaminants from the matrix of the affinity ligand. As affinity capture agents, IgG-binding proteins need to have the stability to sustain many regeneration cycles with a harsh alkaline wash to ensure the safety and efficacy of products. Among the five domains of SPA (Staphylococcal protein A), the Fc^-binding B domain has been engineered to produce the hydroxylamine and CNBr-resistant Z domain (Ala1Val and Gly29Ala) [2]. Other derivatives of protein A and the Z domain include forms with increased avidity [3] and resistance to high-pH sanitization [4,5,6,7,8,9], and calcium- ^ Docket No.11708-002WO1 dependent affinity domains for milder elution

[0010] . Although significant progress has been made in the field of protein-ligand stability, it is still an area of active research. The emergence of high-throughput, inexpensive next-generation sequencing technologies has allowed the continuation of the exponential growth rate of sequence databases. The radical decrease in sequencing cost has facilitated the broad applications of bioinformatics tools in metagenomics data analysis. Metagenomics is the study of all the genomes or environmental DNA of a microbial community or all the organisms together in each environment. Vast sequence and structure data is invaluable to protein research and fueling the growth of protein discovery. Popular techniques like sequence-based homology detection, tertiary structure prediction, phylogenetic analysis, and experimental domain annotations are increasingly being used in protein design and discovery. Structural and biophysical information is needed to pinpoint specific protein families or functions in the annotated genome, metagenome, or protein database. For decades, homology search has remained a powerful tool and utilized as the core strategy in finding new members in existing protein families and discovering proteins with biotechnological utility. The search subject / database may be a new species' genome or an unexpected co-occurrence with other proteomic pathways. A FASTA database of protein A domains was created and used as the query in the TBLASTN procedure. A FASTA database was built by aligning the protein sequences of the 5 domains of protein A by the MUSCLE algorithm in the MEGA X platform as the query sequence for homology search. Afterward, the similarity-based genes were recovered from the metagenome through the TBLASTN procedure. TBLASTN is a modified BLAST search for finding significant homology between amino acid query sequences and a nucleotide database of subject sequences. It operates by translating the genomic / cDNA nucleotide database sequences in all six reading frames and aligning them with amino acid query sequences. Conserved sequences were collected with a threshold filter of 28%-70% (FIG. 1, TABLE 1) sequence identity and 50%- 85% query coverage as references and processed manually. Protein candidates meeting these criteria were chosen for further analysis. The Z domain of protein A used as a reference was VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAP K (SEQ ID NO: 1400). Gene synthesis: Synthetic nucleic acids were obtained from Genewiz (South Plainfield, NJ, USA). The synthesized gene fragments were ligated in the pET28 vector and transformed into the E. coli BL21 (DE3) host. The transformed bacteria were screened on ^ Docket No.11708-002WO1 antibiotic-resistant agar plates. The transformed bacterial cells were used in IgG binders' fermentative production. Fermentation and purification: A 5 mL aliquot of freshly transformed E. coli BL- 21(DE3) cells in terrific broth medium was incubated for 4–5 h at 37 °C and then used to inoculate to 2.8 L baffled Fernbach flasks containing 500 mL Terrific Broth (20 g tryptone, 24 g yeast extract, 4 ml of glycerol, 0.017 M KH2PO4, and 0.072 K2HPO4) with 50 ^g / mL kanamycin. When the cultures reached an OD600 of 0.6-0.8, protein expression was induced with 0.5 mM IPTG and grown for an additional 5 h. Bacterial cells were then harvested in an Avanti J-E High-Speed Centrifuge (Beckman Coulter, CA, USA) at 6000× g for 20 min and stored at −80 °C until further use. The bacterial pellet was mixed with half of its volume of a lysis buffer (50 mM Tris- HCl buffer, pH 8.0, 100 µg / mL lysozyme, 0.1% Triton X-100, 10 mM spermidine tetrahydrochloride, and 10% glycerol). The mixture was incubated at 4 ^ for 30 min, and the cells were lysed using a Biologics 150VT Ultrasonic Homogenizer at 40 kHz for three 15 s cycles. The cell lysate was centrifuged at 20,000× g for 20 min using an Eppendorf Centrifuge 5424 R. The supernatant was filtered using a 0.22 ^m syringe filter, loaded on a Ni Sepharose 6 FF column, and eluted using a linear gradient of 0-500 mM of Imidazole. The eluate was collected in small fractions and analyzed on an SDS-PAGE for purity testing. The pure fractions from Ni-NTA were loaded on the IgG Sepharose column to evaluate the binding with human IgG. The bound proteins were eluted using 0.1 M acetic acid, pH 3.5. The eluate was pH adjusted to pH 7.4 using a 2 M Tris solution. Mass analysis of purified candidates using MALDI (Matrix-assisted Laser Desorption / Ionization): Purified IgG binders were analyzed using MALDI-ToF-MS (Bruker MALDI TOF / TOF ultrafleXtreme mass spectrometer) at University of Houston Core facilities. SA (sinapinic acid) Double Layer, Ground Steel Targets was used for sample preparation. Matrix solubilization procedure: 0.5 ^L matrix solution A (saturated solution of SA in ethanol) was added onto the MALDI target and allowed it to dry. One part matrix solution B (saturated solution of SA in TA30 solvent (30:70 [v / v] acetonitrile: 0.1% TFA in water)) was mixed with 1 part analyte solution and deposited 0.5 ^L of the matrix / analyte mixture onto the matrix spot and allow to dry. All samples were analyzed using linear run mode. ^ Docket No.11708-002WO1 TABLE 1. Molecular weight confirmation. Use of the binder candidates: The IgG binding candidates can be immobilized on a solid support such as iron oxide, styrene-based polymers, agarose, alginate, sepharose, dextran, polyacrylamide, starch, cellulose, nitrocellulose, glass, electrospun polymer nanofibers, other polymer nanofibers, silica, magnetic nanoparticles, or any combination thereof. The binders on solid support (commonly known as affinity resin) can be used for the purification of the antibodies. Typical IgG capture from cell culture supernatant involves following steps: 1) Equilibration of the affinity resin using neutral pH low conductivity buffer 2) Loading of the clarified cell culture supernatant on the equilibrated resin 3) Washing of unbound proteins and non-protein material using a wash buffer, typically of the same composition as an equilibration buffer 4) Elution of the antibodies using a low pH (3) buffer 5) Regeneration of the resin using high-pH alkali solutions (0.1 to 0.5 M NaOH) to remove strongly and nonspecifically bound impurities ^ Docket No.11708-002WO1 6) Re-equilibration using an equilibration buffer and storage in 20 % ethanol Example 2: Screening of IgG binders using IgG Sepharose The IgG binders were expressed in E. coli and purified using Ni-NTA column chromatography. The Ni-NTA column eluate for each binder was loaded on the IgG Sepharose column, washed with phosphate-buffered saline solution, and eluted using 0.1 M acetic acid, pH 3.5. The eluate was pH adjusted to pH 7.4 using a 2 M tris solution. The IgG Sepharose column was immobilized with human polyclonal IgG. The binders that exhibited the elution peak in IgG Sepharose column chromatography were classified as having IgG binding ability, and conversely, binders that did not show an elution peak in the IgG Sepharose column were classified as non-binders. The results obtained from IgG Sepharose chromatography screening are given in TABLE 2 and TABLE 3 below. TABLE 2. Screening IgG binders using IgG Sepharose column chromatography. ^ Docket No.11708-002WO1 TABLE 3. DNA sequences of IgG binders in TABLE 2. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 The above IgG binders can optionally be tagged for purification, for example, His- tagged. TABLE 4 shows examples of the above sequences with His tags. TABLE 4. His-tagged IgG binders. ^ Docket No.11708-002WO1 TABLE 5, TABLE 6, and TABLE 7 provide additional protein A- and protein G-like sequences, respectively, which can also be used as binders. As above, these binders can also optionally be tagged (e.g., His-tagged). ^ Docket No.11708-002WO1 TABLE 5. Protein A-like sequences. * = stop codon. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 TABLE 6. Protein G C2 domain-like sequences. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 TABLE 7. More experimentally studied proteins. ^ Docket No.11708-002WO1 TABLE 8 provides specific binding region sequences for specific experimentally studied proteins. TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, TABLE 17, and TABLE 18 show fragments of experimentally studied proteins. As above, any of these sequences can also optionally be tagged (e.g., His- tagged). TABLE 8. Binding region sequences for the experimentally studied proteins. ^ Docket No.11708-002WO1 TABLE 9. Fragments of SEQ ID NO: 1. ^ Docket No.11708-002WO1 TABLE 10. SEQ ID NO: 2 fragments. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 TABLE 11. SEQ ID NO: 3 fragments. ^ Docket No.11708-002WO1 TABLE 12. SEQ ID NO: 4 fragments. ^ Docket No.11708-002WO1 TABLE 13. SEQ ID NO: 5 fragments. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 TABLE 14. SEQ ID NO: 6 fragments. ^ Docket No.11708-002WO1 TABLE 15. SEQ ID NO: 7 fragments. ^ Docket No.11708-002WO1 TABLE 16. SEQ ID NO: 8 fragments. ^ Docket No.11708-002WO1 TABLE 17. SEQ ID NO: 9 fragments. ^ Docket No.11708-002WO1 TABLE 18. Select fragments. ^ Docket No.11708-002WO1 TABLE 19 and TABLE 20 show additional sequences by phylogeny and phylogeny tree analysis, respectively. TABLE 19. Sequences by phylogeny. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 111 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 TABLE 20. Phylogeny tree analysis for the Pfam and TBLASTN search results search. Select 33 candidates which are away from all five protein A domains, Z domain, TBLASTN search results. ^ Docket No.11708-002WO1 ^ Docket No.11708-002WO1 Example 3: Screening of IgG binders using Enzyme-Linked Immunosorbent Assay IgG binders were purified using Ni-NTA and Superdex 200 size exclusion column chromatography (mobile phase 20 mM tris pH 7.25). The purified binders were analyzed on SDS-PAGE (FIGS.1A-1B) and MALDI-ToF (TABLE 1, FIGS.2A-2J). The binding of IgG binders with the human monoclonal antibody rituximab, human IgG, and alpaca IgG was tested using the ELISA method (FIG.3). The analyses were carried out at various concentrations of IgG binders. The absorbance values were divided with the absorbance from the blank sample (PBS) for normalization. The binders were ranked according to the average normalized absorbance (FIG.4). Example 4: Isothermal Titration Calorimetry (ITC) ITC studies were performed using a MicroCal PEAQ-ITC instrument (Malvern). The IgG binders and Rituximab (human IgG1 monoclonal antibodies) were buffer-exchanged in 20 mM tris of pH 7.25 using a SEC column (Cytiva Superdex-75 and Superdex-200) and degassed. A stirring speed of 750 rpm, a temperature of 25°C, a reference power of 10 µcal / s, high feedback, and an initial delay of 60 s was used for all the experiments. The first injection was 0.4 µL, and the rest were 2 µL with 150 s intervals between injections. The results for this analysis are given in TABLE 21 and TABLE 22 below and additionally shown in FIGS.5A- 5G. TABLE 21. Thermodynamic parameters obtained from ITC analysis. ^ Docket No.11708-002WO1 TABLE 22. Thermodynamic parameters obtained from ITC analysis. Example 5: Additional Experimental Data FIGS. 6A-6D and TABLE 23 show the results of a rituximab binding kinetics study using IgG binders of SEQ ID NOS: 3, 7, and 9. Protein A Z domain (SEQ ID NO: 1400) was used as a positive control. SPR experiments were conducted using the Biacore X100 system (Cytiva, Marlborough, MA, USA). Rituximab was covalently immobilized on flow cell 2 of a CM5 sensor chip, while flow cell 1 served as a reference channel. Immobilization was initiated by activating the carboxymethyl groups on the sensor chip with 70 µL of a mixture containing 0.05 M N-hydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC). Multiple injections of 70 µL rituximab (50 µg / mL) in HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4) were performed to facilitate covalent coupling, with buffer washing between injections. After achieving baseline stabilization, unreacted activated groups were blocked by injecting 70 µL of 1 M ethanolamine-HCl, pH 8.5. Flow cell 1 (reference channel) was prepared in parallel using the same blocking protocol, with all preparation steps conducted at a flow rate of 10 µL / min. For the binding kinetics study, serial dilutions of purified, buffer- exchanged IgG binder samples (3 nM to 400 nM) were freshly prepared in HBS-EP+ running buffer. Each dilution was injected at a flow rate of 30 µL / min for an association phase lasting 180 seconds. The dissociation phase was monitored over 300 seconds using HBS-EP+ buffer. ^ Docket No.11708-002WO1 Surface regeneration between samples was performed by injecting 100 mM sodium citrate containing 500 mM NaCl at pH 3.5 for 60 seconds. All experiments were conducted in triplicate, and data analysis was performed using Biacore X100 BIAevaluation software version 2.0.1 (Cytiva, Marlborough, MA, USA). The IgG binder of SEQ ID NO: 9 binds faster than protein A Z domain (SEQ ID NO: 1400), and the IgG binder of SEQ ID NO: 7 has an association rate similar to protein A Z domain (SEQ ID NO: 1400). TABLE 23. Binding kinetics study using SPR. A kinetics study was conducted to explore binding of the IgG binders with human polyclonal IgG using surface plasmon resonance (SPR) using IgG binders of SEQ ID NOS: 3, 7, and 9. Protein A Z domain (SEQ ID NO: 1400) was used as a positive control. A Biacore X100 was used for the study. Protein L immobilized on the Protein L sensor chip (Cytiva, lot 10351189) serves as the capture molecule. Human polyclonal IgG, at a concentration of 5 ^g / ml for 60 s onto the Protein L sensor chip and acts as the ligand. The IgG binders were loaded onto a human polyclonal IgG loaded chip at concentrations of 100 nM, 50 nM, 25nM, 12.5 nM, and 6.25 nM with a 60 second association time and 180 second dissociation time. The running buffer was HBS-EP+. The human polyclonal IgG was regenerated using 0.1 M glycine HCl at pH 1.7. FIGS.7A-7D and TABLE 24 show the results of kinetics model fitting. The IgG binder of SEQ ID NO: 3 showed faster association rate and same order dissociation as protein A Z domain (SEQ ID NO: 1400). The IgG binder of SEQ ID NO: 9 showed one log higher association rate and one log lower dissociation rate as compared to protein A Z domain (SEQ ID NO: 1400). The IgG binder of SEQ ID NO: 7 showed the same order of association and ^ Docket No.11708-002WO1 dissociation rate as protein A Z domain (SEQ ID NO: 1400), but the Rmax value was significantly smaller. TABLE 24. Kinetics model fitting. Next, a rat IgG binding screening was conducted using a protein L chip (Cytivia, lot 10351189).. Protein L immobilized on the Protein L sensor chip (Cytiva, lot 10351189) serves as the capture molecule. Rat polyclonal IgG, at a concentration of 20 ^g / ml for 60 s onto the Protein L sensor chip and acts as the ligand. All binders were loaded onto the chip at a 500 nM concentration and contacted with rat polyclonal IgG with a 60 second association time and 120 second dissociation time. FIG. 8 shows the results. IgG binders of SEQ ID NOS: 3 and 9 showed stronger binding with rat IgG compared to protein A Z domain (SEQ ID NO: 1400). Another study was conducted to explore protein A binding inhibition using IgG binders of SEQ ID NOS: 3, 7, and 9. Protein A Z domain (SEQ ID NO: 1400) was used as a positive control. Various concentrations of the IgG binders (~8 to 9 kDa) and 100 nm of protein A (46 kDa) were loaded onto a protein L chip (Cytivia, lot 10351189) preloaded with human IgG (5 µg / mL for 60 second). The association phase was conducted for 60 s time and dissociation was conducted for 120 second. FIGS. 9A-9C show thew results. For all three IgG binders tested, increase in the concentration of the IgG binder reduced the response of protein A, thus they are competing to bind at the same binding site on human IgG. A MALS analysis was then conducted to determine binding stoichiometry of IgG binders of SEQ ID NOS: 3, 7, and 9. Protein A Z domain (SEQ ID NO: 1400) was used as a positive control. Samples were analyzed on an Agilent 1290 Infinity liquid chromatography system (Agilent Technologies, Santa Clara, CA) using a 100 ^L injection volume onto a Superdex 20010 / 300 GL analytical SEC column (GE Healthcare, Piscataway, NJ), followed ^ Docket No.11708-002WO1 by a UV–Vis diode array detector (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, CA), multi-angle light scattering (MALS) detector (HELEOS II, Wyatt Technology, Santa Barbara, CA), and differential refractive index (dRI) detector (Optilab T-rEX, Wyatt Technology, Santa Barbara, CA). The mobile phase was PBS, and the flow rate was 0.5 mL / min. The UV–Vis detector was set to monitor the 280 nm wavelength. Molecular weight was estimated on the samples using the dRI detector for concentration with Zimm analysis on the MALS data collected at 10 scattering angles from 37.5° to 120.1°. The protein molecular weight was calculated using the dn / dc of proteins in water at 660^nm of 0.185^mL / g, the molecular weight of the Rituximab and IgG binders observed matching with the Expassy estimated molecular weight values. TABLE 25 shows these results. TABLE 25. MALS analysis. FIGS. 10A-10E show MALS analysis plots. Each of the IgG binders and protein A Z domain (SEQ ID NO: 1400), when mixed with Rituximab, showed two peaks. The first peak is a complex of the IgG binder with Rituximab, and second peak is free IgG binder. The molecular weight estimation using Astra 7.3.2 showed that two IgG binders of SEQ ID NO: 3, or one IgG binder of SEQ ID NO: 7, two IgG binders of SEQ ID NO: 9, or two protein A Z domains (SEQ ID NO: 1400) bind to one Rituximab. TABLE 26 shows the raw data for molecular weight estimation. ^ Docket No.11708-002WO1 TABLE 26. Molecular weight estimation using MALS. ^ Docket No.11708-002WO1 Finally, a study was conducted to explore surface display of the protein A Z domain (SEQ ID NO: 1400). The plasmid pAIDA1 (which expresses the “adhesin involved in diffuse adherence” (AIDA) protein, which is an outer membrane transporter) was used as the basis to surface display protein A Z domain (SEQ ID NO: 1400). E. coli transformed with the plasmid pMNCWZ1 (SEQ ID NO: 1400 fused with AIDA into the plasmid pAIDA1) were labeled with ^ Docket No.11708-002WO1 FITC conjugated human polyclonal IgG. The FITC labeled polyclonal human IgG binds with surface displayed IgG binders and can be sorted using FACS. FIG. 11 shows a flowchart of this experimental setup. TABLE 27 shows fluorescence measurements of the samples using a Tecan plate reader (Ex: 485, Em: 535). Three washes reduced fluorescence significantly for all three controls and the test sample. Resuspended cells in the test samples showed ~9x higher fluorescence than resuspended cells in the control samples. TABLE 27. Fluorescence measurements. FIG.12 shows confocal microcopy images of resuspended cells. The resuspended cells were diluted 1:20 fold using PBS and imaged under confocal microscopy in bright field and under FITC filter. The control sample was cells transformed with pAIDA1 plasmid. The test sample was cells transformed with pMNCWZ1 plasmid. The confocal microscope was set with a 63X oil lens at 6000 Hz. FIGS. 13A-13B show that E. coli cells displaying protein A Z domain (SEQ ID NO: 1400) showed FITC signal, indicating that surface-displayed protein A Z domain (SEQ ID NO: 1400) was binding with human polyclonal IgG. FIGS.14A-14C show cytometry for surface displayed IgG binders. The FITC-labeled polyclonal human IgG binds with surface-displayed IgG binders and can thus be sorted using FACS. The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein. ^ Docket No.11708-002WO1 Reference List 1. Wang L., et al. (2013). A safe, effective, and facility compatible cleaning in place procedure for affinity resin in large-scale monoclonal antibody purification. Journal of Chromatography A, 1308, 86-95. 2. Nilsson B., et al. (1987). A synthetic IgG-binding domain based on staphylococcal protein A. Protein Engineering, Design and Selection, 1:2, 107–113. 3. Andrew D., et al. (2016). Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity, mAbs, 8:4, 828- 838. 4. WO 2008 / 039141 5. US20200079878A1 6. WO 2012 / 083425 7. WO2012 / 087230 8. US10766933B2 9. US20060194950A1 10. WO2018046475 ^

Claims

Docket No.11708-002WO1 CLAIMS What is claimed is:

1. A separation matrix comprising: an immunoglobulin binding protein, wherein the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-9, SEQ ID NOS: 19-26, SEQ ID NOS: 27-120, or SEQ ID NOS: 186-191; and a solid support; wherein the immunoglobulin binding protein is coupled to the solid support.

2. The separation matrix of claim 1, wherein the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-9.

3. The separation matrix of claim 2, wherein the immunoglobulin binding protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

4. The separation matrix of any one of claims 1-3, wherein the solid support comprises iron oxide, styrene-based polymers, agarose, alginate, sepharose, dextran, polyacrylamide, starch, cellulose, nitrocellulose, glass, electrospun polymer nanofibers, other polymer nanofibers, silica, magnetic nanoparticles, or any combination thereof.

5. A method for the purification of an antibody, the method comprising: preparing the separation matrix of any one of claims 1-4; contacting a sample comprising an antibody with the separation matrix; washing unbound proteins and non-protein material from the separation matrix using a wash buffer; and eluting the antibody using a low pH buffer (acidic buffer).

6. The method of claim 5, wherein preparing the separation matrix comprises rinsing the separation matrix with an equilibration buffer.

7. The method of claim 6, wherein the equilibration buffer has a neutral pH and / or a low conductivity. ^Docket No.11708-002WO1 8. The method of any one of claims 5-7, wherein the sample is a cell culture supernatant.

9. The method of any one of claims 5-8, wherein the antibody is an immunoglobulin G.

10. The method of any one of claims 5-9, wherein the antibody is a monoclonal antibody.

11. The method of any one of claims 5-10, wherein the wash buffer has a neutral pH and / or a low conductivity.

12. The method of any one of claims 5-11, wherein the low pH buffer comprises acetic acid, glycine, citric acid, phosphoric acid, or any combination thereof at a concentration of about 0.1 M.

13. The method of any one of claims 5-12, further comprising: contacting the separation matrix with a high pH alkaline solution; and storing the separation matrix in an ethanol solution.

14. The method of claim 13, wherein the high pH alkaline solution comprises NaOH at a concentration of from about 0.01 M to about 0.5 M.

15. The method of any one of claims 13-14, wherein the ethanol solution comprises about 20% ethanol. ^

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