LC/MS-based Intact IgG and Released Glycan Analysis for Bioprocessing Applications.

Robust plate based antibody glycan analysis platforms are urgently needed for biopharmaceutical development and manufacturing as well as for clinical biomarker research. A 96-well plate based workflow has been developed to analyze both intact IgG antibodies and released N-glycans using an Orbitrap Fusion Mass Spectrometer and an LC/MS method on the Waters UNIFI platform. Here, such a workflow including protein A purification, PNGaseF digestion, 2-AB labeling, and SPE clean-up is described. The measured IgG glycan profile is consistent with that obtained from non-plate based method and commercial kit and has the advantage of less hands-on time. Also the application of the workflow in cell culture monitoring and clonal selection work is demonstrated. Apart from checking the major glycan structure changes among clones, post translational modifications (PTMs) such as C-terminal lysine residue clipping and N-terminal pyroglutamic acid formation can also be deduced from the workflow.

Optimized Selection Marker and CHO Host Cell Combinations for Generating High Monoclonal Antibody Producing Cell Lines.

There are several selection markers which are suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells for obtaining high productivity. Using an internal ribosome entry site (IRES)-mediated tricistronic vector and a set of IRES variants with different strengths, the expression of five antibiotics resistance genes (ARGs) in CHO-K1 cells and dihydrofolate reductase (DHFR) in CHO DG44 cells is optimized to enhance the stringency of selection for high producing cells. There is an obvious optimal expression level for every selection marker, below or above which, the productivity is significantly lower. The enhanced productivity in ARG generated CHO K1 cells is due to selective integration of active site while the enhanced productivity in the amplified CHO DG44 cells results from increased gene copies. The high producing CHO K1 pools and clones generated using ARG exhibit better production stability than the amplified high producing CHO DG44 pools and clones. Loss of expression for the CHO K1 cell lines is due to loss of gene copies while for CHO DG44 is due to transcriptional silencing. mAb glycan profile also differed significantly between CHO K1 and CHO DG44 cell lines. These results would be helpful when developing optimized vectors for generating high mAb producing CHO cell lines.

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors.

We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials. 1,2,3 We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted.

Impact of hydrolysates on monoclonal antibody productivity, purification and quality in Chinese hamster ovary cells.

Plant and yeast derived hydrolysates are economical and efficient alternative medium supplements to improve mammalian cell culture performance. We supplemented two commercial Chinese hamster ovary (CHO) culture media with hydrolysates from four different sources, yeast, soybean, Ex-Cell CD (a chemically defined hydrolysate replacement) and wheat to improve the productivity of two cell lines expressing different monoclonal antibodies (mAbs). Yeast, soybean and Ex-Cell CD improved the final mAb titer by increasing the specific productivity (qP) and/or extension of the culture period. Wheat hydrolysates increased peak viable cell density but did not improve productivity. IgG recovery from protein A purification was not compromised for all cultures by adding yeast, soybean and Ex-Cell CD hydrolysates except for one sample from soybean supplemented culture. Adding these three hydrolysates neither increased the amount of host cell protein, DNA or aggregate impurity amounts nor affect their clearance after purification. Profiling of the glycan types revealed that yeast and soybean hydrolysates could affect the distribution of galactosylated glycans. Ex-Cell CD performed the best at maintaining glycan profile compared to the non-supplemented cultures. Overall, yeast performed the best at improving CHO culture growth and productivity without being detrimental to downstream protein A processes but could affect mAb product glycan distribution while Ex-Cell CD yielded lower titers but has less effect on glycosylation. The hydrolysate to use would thus depend on the requirements of each process and our results would provide a good reference for improving culture performance with hydrolysates or related studies.

Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs.

Glycosylation of therapeutic proteins has a profound impact on their safety and efficacy. Many factors shape the glycosylation of biotherapeutics, ranging from expression systems and cell culture processes to downstream purification strategies. Various analytical technologies have been developed to address questions concerning different aspects of glycosylation. Informatics tools are also crucial for a systematic understanding of the glycosylation processes. Hence, an integrated approach is required to harness glycosylation for the production of optimal and consistent glycoprotein-based therapeutic drugs. Here, we review the latest developments and challenges in glycosylation analysis and control in the context of bioprocessing monoclonal antibodies.

Encapsulation of conventional and unconventional water dimers by water-binding foldamers.

Water-binding foldamers have been rarely studied. By orienting both H-bond donors and acceptors toward their interior, two pyridine-derived crescent-shaped folding oligoamides were found to be capable of trapping both conventional and unconventional water dimer clusters in their cavity (∼2.5 Å radius). In the unconventional water dimer cluster, the two water molecules stay in contact via an unusual H-H interaction (2.25 Å) rather than the typical H-bond.