Minireview
Antibody Structure, Instability, and Formulation

https://doi.org/10.1002/jps.20727Get rights and content

ABSTRACT

The number of therapeutic monoclonal antibody in development has increased tremendously over the last several years and this trend continues. At present there are more than 23 approved antibodies on the US market and an estimated 200 or more are in development. Although antibodies share certain structural similarities, development of commercially viable antibody pharmaceuticals has not been straightforward because of their unique and somewhat unpredictable solution behavior. This article reviews the structure and function of antibodies and the mechanisms of physical and chemical instabilities. Various aspects of formulation development have been examined to identify the critical attributes for the stabilization of antibodies.

Section snippets

INTRODUCTION

Protein therapies are entering a new era with the influx of a significant number of antibody pharmaceuticals. Generally, protein drugs are effective at low concentrations with less side effects relative to small molecule drugs, even though, in rare cases, protein-induced antibody formation could be serious.1 Therefore, this category of therapeutics is gaining tremendous momentum and widespread recognition both in small and large drug firms. Among protein drug therapies, antibodies play a major

ANTIBODY STRUCTURE

Antibodies (immunoglobulins) are roughly Y-shaped molecules or combination of such molecules (Fig. 1). Their structures are divided into two regions—the variable (V) region (top of the Y) defining antigen-binding properties and the constant (C) region (stem of the Y), interacting with effector cells and molecules. Immunoglobulins can be divided into five different classes −IgA, IgD, IgE, IgM, and IgG based on their C regions, respectively designated as α, δ, ε, µ, and γ (five main heavy-chain

ANTIBODY INSTABILITY

Antibodies, like other proteins, are prone to a variety of physical and chemical degradation pathways, although antibodies, on the average, seem to be more stable than other proteins. Antibody instabilities can be observed in liquid, frozen, and lyophilized states. The glycosylation state of an antibody can significantly affect its degradation rate.40 In many cases, multiple degradation pathways can occur at the same time and the degradation mechanism may change depending on the stress

ANTIBODY FORMULATION

As mentioned above, antibodies, like other proteins, may generate a variety of degradants during production, processing, and storage both in liquid and solid states. Their tendency to generate such degradants depends very much on their individual sequence, pI, hydrophobicity, and carbohydrate content.23 The antibody degradation products may have reduced activity and more importantly, increased immunogenicity.14,87 A strong immune response (antibody formation) may lead to severe neutralization

SUMMARY

Antibodies have similar tertiary structures. The presence of a significant number of disulfide bonds and intimate domain–domain interactions in antibodies make them relatively stable and more resistant to moderate thermal stress compared to other proteins. Nevertheless, antibodies do experience a variety of instabilities similar to most proteins. These physical and chemical instabilities include denaturation, aggregation, surface adsorption, deamidation, oxidation, isomerization, fragmentation,

REFERENCES (115)

  • S. Sheriff et al.

    X-ray structure of the uncomplexed anti-tumor antibody br96 and comparison with its antigen-bound form

    J Mol Biol

    (1996)
  • E.D. Lobo et al.

    Antibody pharmacokinetics and pharmacodynamics

    J Pharm Sci

    (2004)
  • R.J. Harris et al.

    Identification of multiple sources of charge heterogeneity in a recombinant antibody

    J Chromatogr B Biomed Sci Appl

    (2001)
  • W. Dall'Acqua et al.

    Antibody engineering

    Curr Opin Struct Biol

    (1998)
  • K. Welfle et al.

    Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments

    Biochim Biophys Acta

    (1999)
  • W. Wang

    Instability, stabilization, and formulation of liquid protein pharmaceuticals

    Int J Pharm

    (1999)
  • N. Taschner et al.

    Modulation of antigenicity related to changes in antibody flexibility upon lyophilization

    J Mol Biol

    (2001)
  • S.J. Shire et al.

    Challenges in the development of high protein concentration formulations

    J Pharm Sci

    (2004)
  • G. Schreiber

    Kinetic studies of protein-protein interactions

    Curr Opin Struct Biol

    (2002)
  • J.L. Cleland et al.

    A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

    J Pharm Sci

    (2001)
  • Y.F. Maa et al.

    Spray-drying of air-liquid interface sensitive recombinant human growth hormone

    J Pharm Sci

    (1998)
  • W.K. Hartmann et al.

    Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

    Anal Biochem

    (2004)
  • P.M. Doran

    Loss of secreted antibody from transgenic plant tissue cultures due to surface adsorption

    J Biotechnol

    (2006)
  • A.A. Morales et al.

    Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: Additives, biodistribution, and stability

    Nucl Med Biol

    (1999)
  • A. Usami et al.

    Effect of pH, hydrogen peroxide and temperature on the stability of human monoclonal antibody

    J Pharm Biomed Anal

    (1996)
  • M. Xie et al.

    Secondary structure and protein deamidation

    J Pharm Sci

    (1999)
  • R. Tyler-Cross et al.

    Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

    J Biol Chem

    (1991)
  • X.M. Lam et al.

    Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2

    J Pharm Sci

    (1997)
  • R.J. Harris

    Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture

    J Chromatogr A

    (1995)
  • J. Liu et al.

    Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution

    J Pharm Sci

    (2005)
  • H.C. Mahler et al.

    Induction and analysis of aggregates in a liquid IgG1-antibody formulation

    Eur J Pharm Biopharm

    (2005)
  • E. Ha et al.

    Peroxide formation in polysorbate 80 and protein stability

    J Pharm Sci

    (2002)
  • W. Wang

    Lyophilization and development of solid protein pharmaceuticals

    Int J Pharm

    (2000)
  • E.D. Breen et al.

    Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation

    Pharm Res

    (2001)
  • A.C.A. Roque et al.

    Antibodies and genetically engineered related molecules: Production and purification

    Biotechnol Prog

    (2004)
  • S.P. Martsev et al.

    Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability

    Protein Eng Des Sel

    (2004)
  • R.J. Harris et al.

    Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies

    Drug Dev Res

    (2004)
  • C.A. Janeway et al.

    Immunobiology

    (2001)
  • W. Zhang et al.

    Free sulfhydryl in recombinant monoclonal antibodies

    Biotechnol Prog

    (2002)
  • R. Glockshuber et al.

    The disulfide bonds in antibody variable domains: Effects on stability, folding in vitro, and functional expression in Escherichia coli

    Biochemistry

    (1992)
  • D.J. Kroon et al.

    Identification of sites of degradation in a therapeutic monoclonal antibody by peptide mapping

    Pharm Res

    (1992)
  • M.H. Tao et al.

    Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region

    J Immunol

    (1989)
  • H. Leibiger et al.

    Variable domain-linked oligosaccharides of a human monoclonal IgG: Structure and influence on antigen binding

    Biochem J

    (1999)
  • S. Hermeling et al.

    Structure-immunogenicity relationships of therapeutic proteins

    Pharm Res

    (2004)
  • W. Jiskoot et al.

    Analytical approaches to the study of monoclonal antibody stability

    Pharm Res

    (1990)
  • L. Wang et al.

    Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry

    Pharm Res

    (2005)
  • M. Wan et al.

    Variant antibody identification by peptide mapping

    Biotechnol Bioeng

    (1999)
  • P.A. Ramsland et al.

    Crystal structures of human antibodies: A detailed and unfinished tapestry of immunoglobulin gene products

    J Mol Recognit

    (2002)
  • B. Chen et al.

    Influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms

    Pharm Res

    (2003)
  • A. Worn et al.

    Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability

    Biochemistry

    (1998)
  • Cited by (773)

    View all citing articles on Scopus

    Published online in Wiley InterScience (www.interscience.wiley.com).

    View full text