Combinatorial Fusion TAG yields powerful platform process for the production of pharmaceutically relevant proteins

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Antibody charge heterogeneity formation in a mammalian cell culture fed-batch process

The charge heterogeneity of a monoclonal antibody (mAb) is as a sum factor of several post translational modifications and most of them are of high importance regarding product quality and efficacy. For this reason monitoring and controlling of this sum factor can be beneficial. The work presented here builds the basis for on-line monitoring and will help to achieve Quality by Control (Sommeregger et al., 2017). The aim of this work was to develop a method that allows fast and accurate determination of the charge profile of monoclonal antibodies directly from cell culture supernatants. We were able to circumvent a pre-purification step by adapting a cation exchange method (CEX) using a highly linear pH gradient (Lingg et al, 2013). The established method was then used to gain information about the formation of charge variants during a fed-batch process of an industrial relevant mAb produced by a Chinese Hamster Ovary (CHO) cell line. Please click Additional Files below to see the full abstract

Change of charge variant composition of trastuzumab upon stressing at physiological conditions

Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab.</p

Chromatographic characterization of immunoglobulin G affinity

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Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range

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Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range: Validation of the method parameters

International audienceCation exchange chromatography has been routinely used for the quantification of monoclonal antibody (mAb) charge heterogeneity. A previously developed method utilizing pH gradients for the elution instead of salt gradients was validated according to current guidelines proposed by the ICH. The linearity, stability, accuracy, precision and the lower limit of quantification have been determined, using pure charge variant standards. The method is valid for the quantification of mAb samples with a charge heterogeneity between 1% and 50%. Three different approaches to obtaining pure standard material for the validation of bio-analytical methods for the quantification of charge heterogeneity of IgG are presented. These methods are based on salt gradient elution, pH gradient elution and displacement in cation exchange chromatography

Change of charge variant composition of trastuzumab upon stressing at physiological conditions

Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab