Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions

Valliere-Douglass, John; Wallace, Alison; Balland, Alain

doi:10.1016/j.chroma.2008.10.078

Cited by 77 publications

(78 citation statements)

References 53 publications

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“…4,21,22 Furthermore, because this approach is done under mild, non-denaturing conditions, the different variants have been successfully fractionated to near homogeneity while maintaining activity. 22 After application to a Dionex ProPac HIC-10 column, various mAb 001 lots were eluted with a gradient from 1 M ammonium sulfate in phosphate-buffered saline (PBS) to 0 M over one hour under ambient conditions.…”

Section: Resultsmentioning

confidence: 99%

Cysteinylation of a monoclonal antibody leads to its inactivation

McSherry

Ozaeta

et al. 2016

mAbs

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Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism.

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Section: Resultsmentioning

confidence: 99%

Cysteinylation of a monoclonal antibody leads to its inactivation

McSherry

Ozaeta

et al. 2016

mAbs

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“…An assortment of chromatographic techniques can be applied to resolve ADCs based on characteristics specific to conjugation, linker, and payload. Capillary electrophoresis (CE) and HIC are especially useful in resolving native ADCs (9,78,79). Although size exclusion chromatography is most useful as a means of ADC purification, it can be used to characterize variants in ADC samples, particularly when aggregates are suspected; however, the hydrophobicity of the payload may lead to nonspecific interactions with the stationary phase of columns.…”

Section: Uv/vismentioning

confidence: 99%

“…Disruption of the inter-chain disulfide bridges leads to instability under denaturing HPLC conditions due to incomplete covalent linkage between subunits; nevertheless, the heavy and light chains can be characterized separately (13). HIC in particular is advantageous for characterization of intact ADCs because of the pH-neutral and non-denaturing salt gradients used for separation (79). This method is most amenable for relatively homogeneous conjugations such as conjugates formed by inter-chain disulfide linkage and those formed by other site-specific conjugation methods such as glycoengineering (9,11,20,37).…”

Section: Uv/vismentioning

confidence: 99%

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

McCombs

Owen

2015

AAPS J

282

199

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Abstract. Antibody drug conjugates (ADCs) have emerged as an important pharmaceutical class of drugs designed to harness the specificity of antibodies with the potency of small molecule therapeutics. The three main components of ADCs are the antibody, the linker, and the payload; the majority of early work focused intensely on improving the functionality of these pieces. Recently, considerable attention has been focused on developing methods to control the site and number of linker/drug conjugated to the antibody, with the aim of producing more homogenous ADCs. In this article, we review popular conjugation methods and highlight recent approaches including "click" conjugation and enzymatic ligation. We discuss current linker technology, contrasting the characteristics of cleavable and noncleavable linkers, and summarize the essential properties of ADC payload, centering on chemotherapeutics. In addition, we report on the progress in characterizing to determine physicochemical properties and on advances in purifying to obtain homogenous products. Establishing a set of selection and analytical criteria will facilitate the translation of novel ADCs and ensure the production of effective biosimilars.

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“…Other methods such as hydrophobic-interaction HPLC 49,54 or cation-exchange HPLC 55 could detect peptide bond cleavage as well, but are mainly used to detect degradations of the amino acid side chains.…”

confidence: 99%