Nicholas R. Abu-Absi

Real time monitoring of multiple parameters in mammalian cell culture bioreactors using an in‐line Raman spectroscopy probe

The FDAs process analytical technology initiative encourages drug manufacturers to apply innovative ideas to better understand their processes. There are many challenges to applying these techniques to monitor mammalian cell culture bioreactors for biologics manufacturing. These include the ability to monitor multiple components in complex medium formulations non-invasively and in-line. We report results that demonstrate, for the first time, the technical feasibility of the in-line application of Raman spectroscopy for monitoring a mammalian cell culture bioreactor. A Raman probe was used for the simultaneous prediction of culture parameters including glutamine, glutamate, glucose, lactate, ammonium, viable cell density, and total cell density.

Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells.

CHO cells express glycoproteins containing both the N‐acetylneuraminic acid (Neu5Ac) and minor amounts of the N‐glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well‐known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50–62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early‐temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO2 with this product showed that the Neu5Gc levels were 46% lower at high pCO2 conditions (140 mmHg) compared to moderate pCO2 levels (20–80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics. Biotechnol. Bioeng. 2010;105: 1048–1057.

The use of ‘Omics technology to rationally improve industrial mammalian cell line performance

Biologics represent an increasingly important class of therapeutics, with 7 of the 10 top selling drugs from 2013 being in this class. Furthermore, health authority approval of biologics in the immuno‐oncology space is expected to transform treatment of patients with debilitating and deadly diseases. The growing importance of biologics in the healthcare field has also resulted in the recent approvals of several biosimilars. These recent developments, combined with pressure to provide treatments at lower costs to payers, are resulting in increasing need for the industry to quickly and efficiently develop high yielding, robust processes for the manufacture of biologics with the ability to control quality attributes within narrow distributions. Achieving this level of manufacturing efficiency and the ability to design processes capable of regulating growth, death and other cellular pathways through manipulation of media, feeding strategies, and other process parameters will undoubtedly be facilitated through systems biology tools generated in academic and public research communities. Here we discuss the intersection of systems biology, ‘Omics technologies, and mammalian bioprocess sciences. Specifically, we address how these methods in conjunction with traditional monitoring techniques represent a unique opportunity to better characterize and understand host cell culture state, shift from an empirical to rational approach to process development and optimization of bioreactor cultivation processes. We summarize the following six key areas: (i) research applied to parental, non‐recombinant cell lines; (ii) systems level datasets generated with recombinant cell lines; (iii) datasets linking phenotypic traits to relevant biomarkers; (iv) data depositories and bioinformatics tools; (v) in silico model development, and (vi) examples where these approaches have been used to rationally improve cellular processes. We critically assess relevant and state of the art research being conducted in academic, government and industrial laboratories. Furthermore, we apply our expertise in bioprocess to define a potential model for integration of these systems biology approaches into biologics development. Biotechnol. Bioeng. 2016;113: 26–38.

Understanding and Controlling Sialylation in a CHO Fc-Fusion Process

A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability.