Dataset Information

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

ABSTRACT: CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this "difficult to-express" (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy's success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI-MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.

SUBMITTER: Billerhart M

PROVIDER: S-EPMC10341778 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

Billerhart Magdalena M Hunjadi Monika M Hawlin Vanessa V Grünwald-Gruber Clemens C Maresch Daniel D Mayrhofer Patrick P Kunert Renate R

International journal of molecular sciences 20230630 13

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this "difficult to-express" (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (A ...[more]

PMID: 37446069

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Project description:BackgroundTo ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.ResultsIn this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37 degrees C to 31 degrees C in comparison to standard batch culture maintained at 37 degrees C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-alpha), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2).ConclusionThese results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.

Project description:To function as intended in vivo, a majority of biopharmaceuticals require specific glycan distributions. However, achieving a precise glycan distribution during manufacturing can be challenging because glycosylation is a non-template driven cellular process, with the potential for significant uncontrolled variability in glycan distributions. As important as the glycan distribution is to the end-use performance of biopharmaceuticals, to date, no strategy exists for controlling glycosylation on-line. However, before expending the significant amount of effort and expense required to develop and implement on-line control strategies to address the problem of glycosylation heterogeneity, it is imperative to assess first the extent to which the very complex process of glycosylation is controllable, thereby establishing what is theoretically achievable prior to any experimental attempts. In this work, we present a novel methodology for assessing the output controllability of glycosylation, a prototypical example of an extremely high-dimensional and very non-linear system. We first discuss a method for obtaining the process gain matrix for glycosylation that involves performing model simulations and data analysis systematically and judiciously according to a statistical design of experiments (DOE) scheme and then employing Analysis of Variance (ANOVA) to determine the elements of process gain matrix from the resulting simulation data. We then discuss how to use the resulting high-dimensional gain matrix to assess controllability. The utility of this method is demonstrated with a practical example where we assess the controllability of various classes of glycans and of specific glycoforms that are typically found in recombinant biologics produced with Chinese Hamster Ovary (CHO) cells. In addition to providing useful insight into the extent to which on-line glycosylation control is achievable in actual manufacturing processes, the results also have important implications for genetically engineering cell lines design for enhanced glycosylation controllability.

Dataset Information

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

Publications

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

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