Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function

Harris, Reed J.; Chin, Edward T.; Macchi, Frank; Keck, Rodney G.; Shyong, Bao-Jen; Ling, Victor; Cordoba, Armando J.; Marian, Melinda; Sinclair, Don; Battersby, John E.; Jones, Andy J. S.

doi:10.1007/978-0-387-76643-0_12

Cited by 69 publications

(95 citation statements)

References 48 publications

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“…This finding was confirmed by Western blotting analyses: (1) protein A-purified samples from cell line A, but not from cell line B, exhibit a HMW band of M r extrapolated as 1.1×10 6 under non-reducing conditions (Fig. 3A); (2) protein A-purified samples from cell line A exhibit a stronger band, than from cell line B, at an M r of approximately 8.1×10 4 observed by SDS-PAGE under reducing conditions (Fig. 3F), indicating the presence of a covalently bonded species that is not linked by disulfide bonds.…”

Section: Characterization Of Antibody Aggregatessupporting

confidence: 52%

“…The quality of therapeutic mAbs expressed is also critical because mAbs produced from Chinese hamster ovary (CHO) cells are heterogeneous, owing to aggregation 2,3) and various post-translational modifications. 4,5) This aggregation and modification of antibodies may lead to a reduction in biological activity and/or an increase in undesirable side effects under certain circumstances. The content of aggregated species is one of the most critical sources of heterogeneity because protein aggregates may induce immunogenic responses and result in adverse events following administration.…”

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confidence: 99%

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Comparison of Antibody Molecules Produced from Two Cell Lines with Contrasting Productivities and Aggregate Contents

Ishii

Imamoto

Yamamoto

et al. 2015

Biological & Pharmaceutical Bulletin

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Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic C H 3 domain, which may serve as nuclei for aggregation.Key words antibody aggregate; light chain production; cell line; antibody fragment; covalent aggregate; noncovalent aggregate Therapeutic monoclonal antibodies (mAbs) have been widely used because of their high antigenic specificity, long serum half-life, and low incidence of undesirable side effects. The global market for therapeutic mAbs is growing annually. 1)Because large doses of therapeutic mAbs are usually required to achieve clinical success, mAb production processes with high productivity (titer) are required to reduce the expense to patients. The quality of therapeutic mAbs expressed is also critical because mAbs produced from Chinese hamster ovary (CHO) cells are heterogeneous, owing to aggregation 2,3) and various post-translational modifications. 4,5) This aggregation and modification of antibodies may lead to a reduction in biological activity and/or an increase in undesirable side effects under certain circumstances. The content of aggregated species is one of the most critical sources of heterogeneity because protein aggregates may induce immunogenic responses and result in adverse events following administration. 6,7)Therefore, the aggregate content of mAbs must be precisely controlled. Because protein aggregates are heterogeneous, they are classified on the basis of characteristics such as size, solubility, covalence, reversibility, structure (native/denatured), and formation mechanisms.2,8-11) Aggregate formation varies depending on cell line characteristics, physical properties of mAbs, medium compositions, and culture conditions. 2,[12][13][14] Because a serious concern has recently been raised that visible and subvisible particles, in addition to soluble aggregates, may potentially induce immunogenicity [15][16][17] elucidating the mechanism of aggre...

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Section: Characterization Of Antibody Aggregatessupporting

confidence: 52%

mentioning

confidence: 99%

Comparison of Antibody Molecules Produced from Two Cell Lines with Contrasting Productivities and Aggregate Contents

Ishii

Imamoto

Yamamoto

et al. 2015

Biological & Pharmaceutical Bulletin

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“…In a more recent study, high mannose (Man5) showed increased clearance in humans (15). However, earlier studies with omalizumab (IgG1) showed that Fc glycan structure, including high mannose, did not alter the antibody's clearance in mice, but this could be due to the fact that only a small percentage of the antibody contained high mannose (14). Furthermore, the degree of Fv sialylation did not appear to impact antibody PK (35) nor did complete desialylation (36).…”

Section: Glycosylationmentioning

confidence: 95%

“…Research on how manipulating antibody charge, size, valence, FcRn-binding affinity, and degree and type of glycosylation can influence their metabolism, clearance, penetration, and lymphatic absorption has been conducted (7)(8)(9)(10)(11)(12)(13)(14)(15). Furthermore, a need exists to further optimize drug delivery methods through formulation, dose, injection volume, and injection site (16)(17)(18)(19).…”

Section: Introductionmentioning

confidence: 99%

Physiochemical and Biochemical Factors Influencing the Pharmacokinetics of Antibody Therapeutics

Bumbaca¹,

Boswell²,

Fielder³

et al. 2012

AAPS J

110

107

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Abstract. Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncology, immunology, neurology, and ophthalmology. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biology, immunoglobulin G (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromolecular therapeutics, which in turn could have ramifications on their efficacy and safety. This minireview examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was observed that a net charge modification of at least a 1-unit shift in isoelectric point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to determine if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.

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“…These variants are commonly the result of modifications that occur during cell culture production due to enzymatic processes or spontaneous degradation, and can accumulate during production, purification, formulation and storage. [1][2][3][4][5][6][7][8] Heterogeneity in mAbs is represented by charge variants, typically caused by deamidation, isomerization, succinimide formation, oxidation, sialylation, N-terminal pyroglutamic acid or C-terminal lysine (Lys) clipping. [9][10][11][12] In addition to these variants, 'species' of unknown origin may also exist, and these species must be characterized to ensure the safety and efficacy of the products.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the basic charge variants of a human IgG1

Kaschak

Boyd

et al. 2011

mAbs

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Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function

Cited by 69 publications

References 48 publications

Comparison of Antibody Molecules Produced from Two Cell Lines with Contrasting Productivities and Aggregate Contents

Comparison of Antibody Molecules Produced from Two Cell Lines with Contrasting Productivities and Aggregate Contents

Physiochemical and Biochemical Factors Influencing the Pharmacokinetics of Antibody Therapeutics

Characterization of the basic charge variants of a human IgG1

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