Search

CN-121986264-A - Method for analyzing recombinant polyclonal protein

CN121986264ACN 121986264 ACN121986264 ACN 121986264ACN-121986264-A

Abstract

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

Inventors

  • YUE HONGJUN
  • R.A. Mizrahi
  • A. R. Nidken
  • Y.Y.Jiang

Assignees

  • 吉加根公司

Dates

Publication Date
20260505
Application Date
20240814
Priority Date
20230815

Claims (20)

  1. 1. A method of analyzing a test library comprising Antigen Binding Proteins (ABPs), comprising: (a) Measuring the charge state distribution of the test library, wherein the test library comprises at least 100 ABPs; (b) Comparing the charge state distribution with a reference distribution, wherein the reference distribution is a charge state distribution of a reference library, and (C) Determining the quality of the test library based on the comparison.
  2. 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. 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 the mobile phase a has a low pH from pH 5 to pH 7 and the mobile phase B has a high pH from pH 9 to pH 11.
  4. 4. A process according to 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 11 or from pH 10 to 10.5.
  5. 5. The process of any one of claims 2 to 4, wherein the CEX-HPLC is performed with a column comprising a strong or weak cation exchanger.
  6. 6. The method of any one of claims 2 to 5, wherein the CEX-HPLC is performed at a flow rate between 0.25mL/min and 2 mL/min.
  7. 7. The method of claim 6, wherein the CEX-HPLC is performed at a flow rate between 0.5mL/min and 1.5mL/min, between 0.75mL/min and 1mL/min, or between 0.75mL/min and 0.85 mL/min.
  8. 8. The method of claim 7, wherein the CEX-HPLC is performed at a flow rate of 0.5mL/min, 0.75mL/min, or 1.0 mL/min.
  9. 9. The process of any one of claims 2 to 8, wherein the CEX-HPLC is performed at 20-40 ℃, 25-35 ℃,25 ℃, 30 ℃, or 35 ℃.
  10. 10. The method of any one of claims 2 to 9, wherein the CEX-HPLC is performed with a 5-100% B gradient, a 10-100% B gradient, a 15-100% B gradient, or a 20-100% B gradient.
  11. 11. The method of any one of claims 2 to 10, wherein the CEX-HPLC is performed with a 30-60min gradient time, a 30-45min gradient time, a 30-40min gradient time, or a 35min gradient time, a 40min gradient time, a 45min gradient time, a 50min gradient time, a 55min gradient time, or a 60min gradient time.
  12. 12. The process of any one of claims 1 to 11, wherein the CEX-HPLC is performed with a column packed with a resin having a particle size of less than 3 μιη, less than 2.9 μιη, less than 2.8 μιη, less than 2.7 μιη, less than 2.6 μιη, or less than 2.5 μιη, optionally wherein the resin is a porous resin or a non-porous resin.
  13. 13. The method of any one of claims 1 to 12, wherein the reference profile is measured by cation exchange-high performance liquid chromatography (CEX-HPLC).
  14. 14. The method of claim 13, wherein the reference profile is measured by cation exchange-high performance liquid chromatography (CEX-HPLC) under the same conditions as the conditions under which the charge state profile was measured in (a).
  15. 15. The method of any one of claims 1 to 14, wherein the reference library comprises the same ABP as the test library.
  16. 16. The method of any one of claims 1 to 15, wherein the test library and the reference library are produced by the same producer cell line or progeny thereof.
  17. 17. The method of any one of claims 1 to 16, wherein the reference library has been analyzed by sequencing.
  18. 18. The method of claim 15 or 17, wherein the reference library is a different lot than the test library.
  19. 19. The method of any one of claims 1 to 18, wherein the reference library comprises an antibody.
  20. 20. The method of any one of claims 1 to 18, wherein the reference library comprises a plurality of antibodies.

Description

Method for analyzing recombinant polyclonal protein 1. Technical field Provided herein is a method of analyzing a library comprising at least 100 Antigen Binding Proteins (ABPs). Various uses and applications of the methods related to ABP production, manufacture, distribution, storage, and therapeutic uses are also provided. 2. Background art 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 the plasma of thousands of human donors. IVIg is used as an antibody replacement therapy for patients suffering from humoral immunodeficiency (Lucas et al, 2010; resnick et al, 2012). However, IVIg has low titers of antibodies against many common pathogens, which results in significant morbidity and mortality in immunodeficient patients (Orange et al, 2010). To increase anti-pathogen titers, some groups developed high titers of plasma-derived antibodies, commonly referred to as hyperimmune formulations (Bozzo & Jorquera, 2017). Hyperimmune formulations are typically derived from donor plasma shortly after administration of the active vaccine, such as HyperHEP B (Grifols), which has a high titer against hepatitis b virus. Hyperimmune formulations derived from the donor to which the active vaccine was recently administered are excellent candidates for passive immunization, but commercially expanding such products is a challenge (Kreil et al 2012). Importantly, it can be difficult to identify strong responders willing to vaccinate and repeat donation of plasma. Thus, hyperimmune formulation manufacturing batches must be derived from different groups of donors, resulting in batch-to-batch variability. The anti-pathogen titers vary widely in hyperimmune formulations, from as low as 2 to 3-fold (SCHAMPERA et al, 2017) to as high as 50-fold (Kreil et al, 2012). Thus, in some cases, the physician may simply administer a larger dose of IVIg (Polilli et al, 2012). Doctors and patients would benefit from more consistent, higher titer hyperimmune formulations that are easier to manufacture on a large scale. Many of these problems can be addressed by the use of recombinant DNA technology in combination with microfluidic and molecular genomic strategies to generate multivalent hyperimmune globulins. The general strategy was previously used to generate recombinant multivalent hyperimmune globulins against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or Zika virus, as described in PCT/US2020/030878 submitted at 30.4.2020, PCT/US2021/037232 submitted at 14.6.2021, and PCT/US2021/044523 submitted at 4.8.2021, which are incorporated herein by reference in their entirety. Multivalent hyperimmunes contain thousands of Antigen Binding Proteins (ABPs) with binding specificity for therapeutic target antigens. RPP shows greater therapeutic efficacy against viral infections compared to IVIg, demonstrating their potential as new therapeutic agents. To facilitate the broad therapeutic use of Antigen Binding Protein (ABP) libraries, it is critical to produce high quality compositions with thousands of ABPs in substantial yields and maintain their properties and quality. Thus, 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 modalities. The control of such mixtures is complex. Some variation between batches is expected and slight variations in population distribution have been noted without any impact on key quality attributes (CQAs). It is important that the product be consistently manufactured, for example, by using the same seed culture, regardless of batch size, to maintain consistent cell age from batch to batch. In addition, it is necessary to analyze the manufactured product to ensure the safety, identity, strength, quality and efficacy of the mixture. Provided herein is a novel method for testing ABP libraries to assess and ensure quality and identity of ABPs. The method can be used in the production, distribution, storage and therapeutic use of the library. Accordingly, one aspect of the present disclosure provides a method of analyzing a test library comprising Antigen Binding Proteins (ABPs), comprising measuring a charge status distribution of the test library, wherein the test library comprises at least 100 ABPs, comparing the charge status distribution to a reference distribution, wherein the reference distribution is the charge status distribution of the reference library, and determining the quality of the test library based on the comparison. In some embodiments, the charge state distribution is measured in step (a) by cation exchange-high performance liquid chromatography (CEX-HPLC). In some embodiments, CEX-HPLC is performed with a pH gradient