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      Suppression of IgM Proteolysis by Conformational Stabilization Through Excipients

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          Abstract

          Protease activity from host cell lines may cause product loss or affect the quality of recombinant proteins. In this study, we showed that excipients like glycine and sorbitol reduce the proteolysis of an immunoglobulin M (IgM) in the presence of added proteases like α-chymotrypsin, papain, and pepsin. The activity of the proteases in the IgM-protective environments was conserved or even enhanced as tested using low molecular weight substrates. Thus, a higher resistance against proteolytic degradation appears to be caused by the conformational stabilization of the IgM due to preferential exclusion of sorbitol and glycine.

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          Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1.

          Miranda Yap, Angela Chin, Jason W Fong … (2008)
          Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.
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            Preferential interactions of proteins with solvent components in aqueous amino acid solutions.

            S Timasheff, T. Arakawa (1983)
            The preferential interactions of proteins with solvent components in concentrated amino acid solutions were measured by high-precision densimetry. Bovine serum albumin and lysozyme were preferentially hydrated in all of the amino acids examined, glycine, alpha- and beta-alanine, and betaine, i.e., addition of these amino acids resulted in an unfavorable free energy change. It was shown that, for the former three amino acids, known to have a positive surface tension increment, their perturbation of the surface free energy of water is consistent with their preferential exclusion from the protein surface. In the case of betaine, which does not increase the surface tension of water, preferential exclusion from protein surface must reflect the chemical structure of this cosolvent, which is considerably more hydrophobic than that of the other three amino acids.
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              Degradation of an Fc-fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease.

              Flavie Robert, Horst Bierau, Mara Rossi … (2009)
              A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Sci Pharm
                Journal ID (iso-abbrev): Sci Pharm
                Journal ID (publisher-id): SciPharm
                Title: Scientia Pharmaceutica
                Publisher: The Austrian Journal of Pharmaceutical Sciences (Austria )
                ISSN (Print): 0036-8709
                ISSN (Electronic): 2218-0532
                Publication date Collection: 2015
                Publication date (Electronic): 04 March 2015
                Volume: 83
                Issue: 2
                Pages: 401-410
                Affiliations
                [1 ]Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
                [2 ]Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore
                Author notes
                [* ]Corresponding author. E-mail: monika.mueller@ 123456univie.ac.at (M. Mueller)
                Article
                Publisher ID: SciPharm-83-401
                DOI: 10.3797/scipharm.1501-12
                PMC ID: 4727821
                PubMed ID: 26839826
                SO-VID: 83be773b-0726-44ad-86af-fac18a8c8f7d
                Copyright © Copyright: © Mueller et al.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 21 January 2015
                Date accepted : 04 March 2015
                Categories
                Subject: Research Article

                ScienceOpen disciplines: Pharmacology & Pharmaceutical medicine
                Keywords: igm,proteolysis,conformational stability,sorbitol,glycine
                Data availability:
                ScienceOpen disciplines: Pharmacology & Pharmaceutical medicine
                Keywords: igm, proteolysis, conformational stability, sorbitol, glycine

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