Kripa Ram

Development and scale‐up of a commercial fed batch refolding process for an anti‐CD22 two chain immunotoxin

We describe the development and scale‐up of a novel two chain immunotoxin refolding process. This work provides a case study comparing a clinical manufacturing process and the commercial process developed to replace it. While the clinical process produced high quality material, it suffered from low yield and high yield variability. A systematic approach to process development and understanding led to a number of improvements that were implemented in the commercial process. These include a shorter inclusion body recovery process, limiting the formation of an undesired deamidated species and the implementation of fed batch dilution refolding for increased refold titers. The use of a combination of urea, arginine and DTT for capture column cleaning restored the binding capacity of the capture step column and resulted in consistent capture step yields compared to the clinical process. Scalability is shown with data from 250 L and 950 L scale refolding processes. Compared to the clinical process it replaces, the commercial process demonstrated a greater than fivefold improvement in volumetric productivity at the 950 L refolding scale.

Application of multivariate analysis and mass transfer principles for refinement of a 3-L bioreactor scale-down model—when shake flasks mimic 15,000-L bioreactors better

Characterization of manufacturing processes is key to understanding the effects of process parameters on process performance and product quality. These studies are generally conducted using small‐scale model systems. Because of the importance of the results derived from these studies, the small‐scale model should be predictive of large scale. Typically, small‐scale bioreactors, which are considered superior to shake flasks in simulating large‐scale bioreactors, are used as the scale‐down models for characterizing mammalian cell culture processes. In this article, we describe a case study where a cell culture unit operation in bioreactors using one‐sided pH control and their satellites (small‐scale runs conducted using the same post‐inoculation cultures and nutrient feeds) in 3‐L bioreactors and shake flasks indicated that shake flasks mimicked the large‐scale performance better than 3‐L bioreactors. We detail here how multivariate analysis was used to make the pertinent assessment and to generate the hypothesis for refining the existing 3‐L scale‐down model. Relevant statistical techniques such as principal component analysis, partial least square, orthogonal partial least square, and discriminant analysis were used to identify the outliers and to determine the discriminatory variables responsible for performance differences at different scales. The resulting analysis, in combination with mass transfer principles, led to the hypothesis that observed similarities between 15,000‐L and shake flask runs, and differences between 15,000‐L and 3‐L runs, were due to pCO2 and pH values. This hypothesis was confirmed by changing the aeration strategy at 3‐L scale. By reducing the initial sparge rate in 3‐L bioreactor, process performance and product quality data moved closer to that of large scale.

Protein Production in Eukaryotic Cells

The scientific and engineering aspects of design, development, scale-up, and manufacture of monoclonal antibodies are summarized in this chapter by outlining the key elements in the development of the expression cell line, cell culture, cell harvest, and protein purification process and exploring the effect of process technologies on production economics.

Functional Analysis of ER Stress Pathway Genes for Apoptosis of NS/0 Cell Line Using RNAi Methods

We characterized the expression of three ER stress pathway genes: IRE1, ATF4 and ATF6, in a GS-NS/0 antibody production cell line that was grown in animal protein free (APF) medium and serum containing medium. Differential expression was analyzed by microarray and qPCR techniques. Functions of the three genes were characterized using RNAi technology. Results show that the knocking- down of the ATF6 gene can cause increase in percentage of apoptotic cells, and also silencing of ATF6 had negative impact on cell growth, during late stage culture. However, silencing ATF4 and IRE1 only can induce more apoptotic cells at day 9 cultures while no impacts on cell growth. These results suggest that IRE1, ATF4 and ATF6 may play roles in apoptotic pathway in GS-NS/0 cell lines.

Characterization of Cholesterol-Independent GS-NS/0 Recombinant Antibody Cell Lines

GS-NS/0 recombinant antibody production cell line was adapted to cholesterol-free and animal protein free (APF) medium. Lower peak viable cell density (VCD) and higher productivities were seen in the cholesterol-independent cells than in parental cell line while the quality of the antibodies was similar. The cholesterol-independent cell line were then selected and characterized.