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〈129〉 ANALYTICAL PROCEDURES FOR RECOMBINANT THERAPEUTIC MONOCLONAL ANTIBODIES

Jan 08 , 2025

INTRODUCTION

This chapter provides analytical procedures for murine, chimeric, and humanized IgG isotype monoclonal antibodies and subtypes (e.g., IgG1 and IgG2). Subclasses show differences in amino acid sequence and in the number of disulfide bonds, and in some cases they require subclass-specific analysis. This chapter applies to monoclonal antibodies for therapeutic and prophylactic use and for use as in vivo diagnostics. It does not apply to monoclonal antibodies used as reagents in the manufacture of medicinal products, for which applicable requirements are determined by the appropriate regulatory agency.

Naturally occurring polyclonal antibodies in serum or plasma can bind to many different targets as part of the immune system. In contrast, therapeutic monoclonal antibodies for human use are preparations of an immunoglobulin (or a fragment of an immunoglobulin) that have specificity for a target and are derived from a single clone of cells. Therapeutic monoclonal antibodies can bind to and neutralize soluble antigens, and their mechanism of action (MOA) often involves blocking of the ligand from binding to its cognate receptor. Alternatively, therapeutic monoclonal antibodies can recognize and bind to cell-bound antigens and in these cases may also engage the immune system through Fc (crystallizable fragment) mediated effector functions as part of their MOA. Monoclonal antibodies that have a single defined binding specificity are monospecific, whereas recombinantly engineered bispecific monoclonal antibodies can bind to two different targets (e.g., antigens). IgG-type monoclonal antibodies have a molecular weight of approximately 150 kDa. Each molecule consists of two heavy and two light polypeptide chains that have a molecular weight of approximately 50 and 25 kDa, respectively. Antibodies can be schematically represented as a Y joined by disulfide bridges, where each arm of the Y is called the Fab domain, and the stem of the Y is called the Fc domain. Monoclonal antibodies are glycoproteins that have a glycosylation site in the Fc portion located on each of the heavy chains and have possible additional glycosylation sites in the Fab domain, depending on the molecule.

The specificity of a monoclonal antibody is based on its antigen binding site, which is located in the Fab part of the molecule. The Fc domain contains receptor binding sites that are associated with antibody effector functions that have subclass-specific applications such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Pharmacokinetic effects via binding to the neonatal Fc receptor also are present in this domain.

Monoclonal antibodies are produced in cell culture using a seed lot system with master and working cell banks. After harvest, purification steps ensure that product- and process-related impurities are reduced to an acceptable level.

Currently licensed monoclonal antibody therapeutics include those involved in the activation of effector cells, cell killing, cross-linked induced apoptosis, antagonism against several targets, and agonist antibodies.

This chapter includes purity assessments by size-exclusion chromatography (SE-HPLC), capillary electrophoresis, and analysis of oligosaccharides, and it provides validated procedures for each of these.

Although this chapter is focused on IgG-type monoclonal antibodies, the principles of the tests included can apply to other related molecules, such as IgM or other isotype antibodies, antibody fragments, and Fc-fusion proteins. When the active substance is a conjugated antibody, such tests can be performed on the purified antibody before modification or conjugation.

Alternative methods and/or procedures may be used if they provide advantages in terms of accuracy, sensitivity, precision, selectivity, or adaptability to automation or computerized data reduction, or in other special circumstances. Such alternative procedures and methods shall be validated as described in Validation of Compendial Procedures 〈1225〉 and must be shown to give equivalent or better results.

Source from USP:https://online.uspnf.com/uspnf/document/2_GUID-AEE677A4-5F8C-46B3-B949-D5D3DF112DB8_20101_en-US?source=TOC



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