Process analytics for purification of monoclonal antibodies

Flatman, S.; Alam, Imtiaz; Gerard, Jeff; Mussa, Nesredin

doi:10.1016/j.jchromb.2006.11.018

Cited by 77 publications

(74 citation statements)

References 18 publications

(17 reference statements)

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“…A possible off-line alternative to GX, GX II, and MS for analyzing protein samples in a native state is analytical chromatography (Flatman et al, 2007;Maiser et al, 2014;Goetz et al, 2004). Major disadvantages are sample pre-treatment (e.g.…”

Section: Introductionmentioning

confidence: 99%

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Baumann

Bluthardt

Renner

et al. 2015

Journal of Biotechnology

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Section: Introductionmentioning

confidence: 99%

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Baumann

Bluthardt

Renner

et al. 2015

Journal of Biotechnology

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“…SDS-PAGE and Western blots have been used to characterize HCP species (Champion et al, 2005;Eaton, 1995;Flatman et al, 2007). A recent proteomic study has described the use of two-dimensional (2D)-gel electrophoresis on different CHO cell lines to support the use of multiproduct immunoassays to monitor HCP impurities (Krawitz et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Jin

Szapiel

Zhang

et al. 2009

Biotech & Bioengineering

151

165

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Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.

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“…Production of rmAb's from mammalian cells requires highly resolving analytical methods for the development and monitoring of the manufacturing processes as well as characterization of the final drug product [9]. Among those methods, electrophoretic techniques play an important role in the identity and purity testing of these large and complex molecules.…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype

et al. 2008

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Recombinant monoclonal antibodies of therapeutic interest were analyzed by a nonreduced CE-SDS (nrCE-SDS) method developed for the evaluation of size-based variants. We found that immunoglobulins analyzed by this technique exhibited different behavior depending on their subclasses. Under nrCE-SDS conditions, IgG1 molecules were separated in a well-resolved, single peak, whereas IgG2 molecules were consistently separated as a doublet. Investigation of these isoforms showed that they were structurally different, and that the difference was not caused by cell culture condition, glycosylation structure, or recombinant expression system. Commercially available IgG2 affinity-purified from human plasma also showed the presence of structural isoforms. The structural isoforms remained present under pH- and temperature-stressed conditions. Application of a mild cysteine/cystine redox potential converted the main peak doublet into a single peak, indicating that these isoforms were disulfide bond-related species. Bioactivity measured before and after application of a redox potential gave similar values, indicating that the structural isoforms have comparable potency. The nrCE-SDS technique described here demonstrated a unique capability to resolve IgGs, leading to the discovery of novel structural isoforms specific to the IgG2 isotype.

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Process analytics for purification of monoclonal antibodies

Cited by 77 publications

References 18 publications

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype

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