Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography

A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing

Longterm-habituation of the startle response in mice is stimulus modality, but not context specific

In mice, the specificity of longterm-habituation (LTH) of startle was tested in two experiments. In two strains of mice (C57Bl/6 and C3H) there was pronounced LTH over 10 days of acoustic stimulation in two different contexts of startle measurement. (We found LTH to be greater after stimulation with 14 kHz sine stimuli compared to noise or tactile stimuli). A change of context showed LTH to be independent of context, i.e., startle LTH in mice is a non-associative learning process. In the second experiment, 9 days of acoustic or tactile stimulation were given to C57B/6 mice. Both stimulus modalities produced LTH. When on the 10th day stimuli of the other modality were given, in both cases the long term habituated group showed no lower startle amplitude than a non-stimulated control group. This indicates LTH is stimulus-modality specific. Altogether, our results show that in mice—very similar to rats—LTH of startle is stimulus modality, but not context specific. In addition we found two indications that the LTH action site is on the sensory branch of the startle circuit

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography

A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing