Methods of Separating and Studying Human Antiviral Monoclonal Antibodies

Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B lymphocytes against a particular antigen. Monoclonal antibodies are identical in several properties such as protein sequence, antigen-binding site region, binding affinity for their targets, and identical downstream functional effects. Antiviral mAbs can be used to blunt viral propagation through direct effects. They can also engage the host's immune system, leading to the induction of long-lasting protective effects. Because of long development timeframes, antiviral antibodies are limited in rapid deployment and use. Nevertheless, the commercial development of human antiviral mAb therapies will likely surge.

Separation Techniques and Methods

For nearly all mAbs, techniques employing capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) or ultra-high pressure liquid chromatography (UHPLC or UPLC) are among the routine analytical methods that are commonly used for the quantification and characterization of antibody purity. These techniques are most frequently used to separate and quantify product size and charge variants.

  • Physicochemical properties
  • For most antibody molecules the standard separation-based methods that are used as part of product quality testing are size-exclusion chromatography (SEC); CE sodium dodecyl sulfate (CE-SDS), under both reducing and nonreducing conditions; and charge variant separation, using one of three dominant techniques: cation-exchange chromatography (CEX), capillary zone electrophoresis (CZE), or isoelectric focusing (IEF). In addition, other analytical separation methods that are used during characterization studies or in support of process development include the following modes of separation: hydrophobic interaction chromatography (HIC), reversed-phase UHPLC, anion-exchange chromatography (AEX), and boronate affinity chromatography (BAC).

  • Specific affinity
  • Immobilized biological ligands (proteins, lectins, etc.), such as protein A/G, have a specific affinity to immunoglobulins and capture them. Antigen affinity purification is used to purify those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains.

Typical platform separation (downstream) process for mAbs. Fig.1 Typical platform separation (downstream) process for mAbs. (Yoshimoto, 2019)

Detection of Monoclonal Antibodies

Methods for detection of antibodies include immunoprecipitation assay, in which antigen-antibody (Ag-Ab) complex aggregates are detected, often by hemagglutination; immunocytochemistry, for in situ Ab detection in tissue slices; immunoblotting (dot blot technique) whereby Ag-Ab aggregates are trapped on membranes and then detected with a secondary Ab to yield spots; and immunosorbent assays, which are similar to immunoblotting but by using a tagged secondary antibody, allow the primary antibody to be quantified.

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Reference

  1. Yoshimoto, N.; et al. A method for designing flow-through chromatography processes. InMATEC Web of Conferences. EDP Sciences. 2019, 268: 01004.
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