Heterogeneity of Monoclonal Antibodies

doi:10.1002/jps.21180

Journal of Pharmaceutical Sciences

Volume 97, Issue 7, July 2008, Pages 2426-2447

https://doi.org/10.1002/jps.21180 Get rights and content

Abstract

Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules. This article reviews the current knowledge of enzymatic and nonenzymatic modifications of monoclonal antibodies including the common ones such as incomplete disulfide bond formation, glycosylation, N-terminal pyroglutamine cyclization, C-terminal lysine processing, deamidation, isomerization, and oxidation, and less common ones such as modification of the N-terminal amino acids by maleuric acid and amidation of the C-terminal amino acid. In addition, noncovalent associations with other molecules, conformational diversity and aggregation of monoclonal antibodies are also discussed. Through a complete understanding of the heterogeneity of monoclonal antibodies, strategies can be employed to better identify the potential modifications and thoroughly characterize the molecules.

Section snippets

INTRODUCTION

The primary structure of an antibody from a single cell clone is homogeneous. However, all the monoclonal antibodies studied are heterogeneous in nature. It has been generally recognized that heterogeneity, especially charge heterogeneity, is a common feature of monoclonal antibodies including monoclonal antibodies of different species such as human,1., 2., 3., 4., 5., 6., 7., 8., 9., 10., 11. rabbit,¹²^,¹³ mouse,14., 15., 16., 17., 18., 19., 20., 21., 22., 23., 24., 25., 26., 27., 28. chimeric

Disulfide Bonds

IgG molecules are composed of two heavy chains and two light chains. Each light chain is connected to each heavy chain by one disulfide bond. Each heavy chain is connected to the other heavy chain by two to four disulfide bonds depending on the subtypes of the antibodies. Each IgG has 12 domains (one variable (V_L) and one constant domain (C_L) of each light chain, one variable (V_H) and three constant domains (CH1, CH2, and CH3) of each heavy chain), and each domain contains one intrachain

NONCOVALENT INTERACTION

In addition to covalent modifications, monoclonal antibodies can also associate with other molecules noncovalently. For example, immunoglobulin is one of the major proteins that can bind to riboflavin.²²¹ Two IgG2 antibodies that can bind riboflavin and its derivatives tightly were found in patients of multiple myeloma.¹⁵⁵^,²²²^,²²³ As riboflavin is bound to the antigen combining site, it is still arguable whether the riboflavin is the actual antigen of the IgG molecules or just a cofactor.²²⁴

CONFORMATIONAL HETEROGENEITY

Protein exists as an ensemble of different conformations in equilibrium instead of a single rigid structure.¹¹⁹ By studying the complex binding kinetics of hapten at various concentrations to three specific antibodies, Foote and Milstein²²⁹ proposed that monoclonal antibodies exist as more than one conformation, at least at the combining site, in equilibrium. Different conformations of a monoclonal IgE were observed by X-ray crystallography and kinetics studies.²³⁰ In the study of a murine

AGGREGATION

Monoclonal antibodies like all other proteins are prone to aggregation. Aggregates are formed mainly because of intermolecular interactions of hydrophobic regions as a result of partial or transient unfolding of the proteins.233., 234., 235. Conditions such as incubation at high temperature, contact with hydrophobic surfaces, exposure to extreme pH, and exposure to shear forces that induce protein unfolding and promote protein–protein interactions will most likely increase the amount of

SUMMARY

Heterogeneity is common for monoclonal antibodies including recombinant monoclonal antibodies due to modifications of various natures. Different orthogonal techniques are required to understand the structure and stability of the molecules thoroughly. Characterization experience of one antibody can certainly help to understand the heterogeneity of other antibodies, however the information cannot be generalized even though monoclonal antibodies of either humanized or fully human share more than

Acknowledgements

The authors would like to thank Lyman G. Armes for critical review of the manuscript, and Czeslaw H. Radziejewski, Gary J. Welch and Peter Moesta for their support.

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ReviewsHeterogeneity of Monoclonal Antibodies

Abstract

Section snippets

INTRODUCTION

Disulfide Bonds

NONCOVALENT INTERACTION

CONFORMATIONAL HETEROGENEITY

AGGREGATION

SUMMARY

Acknowledgements

FEBS Lett

J Chromatogr

J Chromatogr

Mol Immunol

J Chromatogr

J Pharm Biomed Anal

J Chromatogr A

J Chromatogr A

Anal Biochem

Anal Biochem

J Chromatogr A

J Chromatogr B Biomed Sci Appl

J Pharm Biomed Anal

J Chromatogr A

J Chromatogr A

J Chromatogr B Analyt Technol Biomed Life Sci

Anal Biochem

Anal Biochem

Biochem Biophys Res Commun

J Pharm Biomed Anal

J Chromatogr A

J Biochem Biophys Methods

J Pharm Biomed Anal

J Chromatogr A

Anal Biochem

Mol Immunol

Anal Biochem

Mol Immunol

Anal Biochem

Immunochemistry

Anal Biochem

Arch Biochem Biophys

Mol Immunol

J Biol Chem

J Immunol Methods

Mol Immunol

A study on globulin formation by plasma-cell neoplasm (5563) transplantable in mice

Biochem J

The homogeneous-gamma-G-immunoglobulin produced by mouse plasmacytoma 5563 and its subsequent heterogeneity in serum

Biochem J

One cell-one immunoglobulin. Origin of limited heterogeneity of myeloma proteins

Biochem J

Michroheterogeneity of serum myeloma immunoglobulins revealed by a technique of high resolution two-dimensional electrophoresis

Electrophoresis

Two-dimensional gel electrophoresis of serum specimens from patients with monoclonal gammopathies

Clin Chem

Two-dimensional electrophoretic analysis of immunoglobulin patterns in monoclonal gammopathies

Electrophoresis

Pattern variations of polyclonal and monoclonal immunoglobulins of different isotypes analyzed by high-resolution two-dimensional electrophoresis

Electrophoresis

Influence of cell- and Media-derived factors on the integrity of a human monoclonal antibody after secretion into serum-free cell culture supernatants

Biotechnol Bioeng

Complete separation of anti-hapten antibodies by two-dimensional affinity electrophoresis

Electrophoresis

Studies on the heterogeneity of anti-hapten antibodies by means of two-dimensional affinity electrophoresis

Electrophoresis

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

Hybridoma

Scaleup of monoclonal antibody purification using radial streaming ion exchange chromatography

Biotechnol Bioeng

Production and characterization of monoclonal antibodies against the novel neutrophil activating peptide NAP/IL-8

J Immunol

Micro isoelectric point heterogeneity of a murine monoclonal antibody(L6) originating from cell cultivation conditions

Biotechnol Tech

Reviews
Heterogeneity of Monoclonal Antibodies