Cell culture process conditions including media components and bioreactor operation conditions have a profound impact on recombinant protein quality attributes. Considerable changes in the distribution of galactosylated glycoforms (G0F, G1F, and G2F) were observed across multiple CHO derived recombinant proteins in development at Eli Lilly and Company when switching to a new chemically defined (CD) media platform condition. In the new CD platform, significantly lower G0F percentages and higher G1F and G2F were observed. These changes were of interest as glycosylation heterogeneity can impact the effectiveness of a protein. A systematic investigation was done to understand the root cause of the change and control strategy for galactosylated glycoforms distribution. It was found that changes in asparagine concentration could result in a corresponding change in G0F, G1F, and G2F distribution. A follow-up study examined a wider range of asparagine concentration and it was found that G0F, G1F, and G2F percentage could be titrated by adjusting asparagine concentration. The observed changes in heterogeneity from changing asparagine concentration are due to resulting changes in ammonium metabolism. Further study ascertained that different integrated ammonium level during the cell culture process could control G0F, G1F, and G2F percentage distribution. A mechanism hypothesis is proposed that integrated ammonium level impacts intracellular pH, which further regulates β-1, 4 galactosyltransferase activity.

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG C 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.