Characterization of Therapeutic Antibodies and Related Products

Recent progress in development of siRNA delivery vehicles for cancer therapy

Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

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Expression of recombinant Antibodies

This Feature will introduce the strategies of therapeutic antibodies (mAbs) in-depth characterization by mass spectrometry (MS) and discuss analytical comparison of biosimilar to originator mAbs, with the cases of trastuzumab and cetuximab. In addition, the structural and functional insights gained both by state-of-the art and emerging MS methods used for biobetters and next generation antibodies design and optimization will also be highlighted.

http://pubs.acs.org/doi/pdf/10.1021/ac3002885

Biosimilar, Biobetter, and Next Generation Antibody Characterization by Mass Spectrometry

The state-of-play and future of antibody therapeutics ☆

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 fro...

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Unfortunately, in the original article, the name of the author Meehl Meehl was given incorrectly. The correct name is Michael Meehl for which we correctly presented above.

/pdf/erratum-to-improvement-of-n-glycan-site-occupancy-of-168yhjbuad.pdf

Rohan Patel

Papers

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Erratum to: Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris