Advancing Biologics Development Programs with Legacy Cell Lines: Advantages and Limitations of Genetic Testing for Addressing Clonality Concerns Prior to Availability of Late Stage Process and Product Consistency Data.

The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line "clonal origin" an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein referred to as "legacy cell lines," may not meet current regulatory expectations for demonstration of clonal derivation. In this article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of process consistency and product comparability of critical quality attributes throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications (BLA) submission.

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A.

Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells.

We have recently demonstrated an increase in recombinant factor VIII (rFVIII) secretion from BHK-21 cells (rBHK-21(host)) following an over-expression of the chaperone protein heat shock protein 70 (Hsp70) (rBHK-21(Hsp70)) due to an inhibition of apoptotic cell death and an increased cellular expression of rFVIII [Ishaque, A., Thrift, J., Murphy, J.E., Konstantin, K., 2007. Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol. Bioeng. Biotech. Bioeng. 97, 144-155]. In the present study we investigated the difference in adherence of rFVIII to the cell membrane surface by comparing changes in cell viability and extent of phosphatidylersine (PS) exposure in apoptosis between rBHK-21(host), rBHK-21(Hsp70), and parental BHK-21 cells devoid of rFVIII expression (BHK-21(native)) during batch cell culture experiments. The Zenon technique was used to double stain for cell surface and intracellular rFVIII using flow cytometric Guava PCA analysis. By this quantitative analysis intracellular rFVIII was shown to decrease in rBHK-21(host) cells as the cell viability declined while the rFVIII cell surface staining increased. Conversely, rBHK-21(Hsp70) cell cultures displayed higher cell viability and intracellular rFVIII with less cell surface rFVIII staining. Time dependent increases of rFVIII adherence to the surface of rBHK-21(host) cells and its reduction on the surface of rBHK-21(Hsp70) cells was also confirmed by fluorescence microscopy. Furthermore, greater rFVIII cell surface staining correlated with an increase in detectable PS exposure on the surface of BHK-21(native) batch cell cultures. However, PS exposure could not be identified to the same extent on rBHK-21(host) cells despite a similar decline in cell viability between rBHK-21(host) and BHK-21(native) batch cultures. Any exposed PS on rBHK-21(host) cells was most likely masked by secreted rFVIII, mimicking the effect on activated platelets where the externalization of PS also occurs, and serves as a ligand for FVIII activation in the blood coagulation cascade. Taken together we have identified that rFVIII sequestration on the membrane surface is another potential limitation to rFVIII productivity and one which can also be alleviated by reduction of apoptosis in a clone expressing human HSP70.

Anti-apoptotic genes Aven and E1B-19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture.

The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression, and improved product quality. In this study, a baby hamster kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In high cell density shake flask culture evaluation, 11 clonal cell lines expressing either E1B-19K or a combination of Aven and E1B-19K showed improved survival compared to both parental and blank vector cell line controls. These cell lines exhibited lower caspase-3 activation and reduced Annexin-V binding compared to the controls. Parental and blank vector cell lines were less than 50% viable after 48 h of exposure to thapsigargin while cell lines expressing E1B-19K with or without Aven maintained viabilities approaching 90%. Subsequently, the best Aven-E1B-19K candidate cell line was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue, so the bioreactors were operated at sequentially lower specific perfusion rates, while maintaining a cell density of 2 x 10(7) viable cells/mL. The viability of the parental cell line declined from nearly 100% at a perfusion rate of 0.5 nL/cell/day to below 80% viability, with caspase-3 activity exceeding 15%, at its lower perfusion limit of 0.15 nL/cell/day. In contrast, the Aven-E1B-19K cell line maintained an average viability of 94% and a maximum caspase-3 activity of 2.5% even when subjected to a lower perfusion minimum of 0.1 nL/cell/day. Factor VIII productivity, specific growth rate, and cell size decreased for both cell lines at lower perfusion rates, but the drop in all cases was larger for the parental cell line. Specific consumption of glucose and glutamine and production of lactate were consistently lower for the Aven-E1B-19K culture. Furthermore, the yield of ammonia from glutamine increased for the Aven-E1B-19K cell line relative to the parent to suggest altered metabolic pathways following anti-apoptosis engineering. These results demonstrate that expression of anti-apoptotic genes Aven and E1B-19K can increase the stability and robustness of an industrially relevant BHK-FVIII mammalian cell line over a wide range of perfusion rates.

Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion.

Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.

The "push-to-low" approach for optimization of high-density perfusion cultures of animal cells.

High product titer is considered a strategic advantage of fed-batch over perfusion cultivation mode. The titer difference has been experimentally demonstrated and reported in the literature. However, the related theoretical aspects and strategies for optimization of perfusion processes with respect to their fed-batch counterparts have not been thoroughly explored. The present paper introduces a unified framework for comparison of fed-batch and perfusion cultures, and proposes directions for improvement of the latter. The comparison is based on the concept of "equivalent specific perfusion rate", a variable that conveniently bridges various cultivation modes. The analysis shows that development of economically competitive perfusion processes for production of stable proteins depends on our ability to dramatically reduce the dilution rate while keeping high cell density, i.e., operating at low specific perfusion rates. Under these conditions, titer increases significantly, approaching the range of fed-batch titers. However, as dilution rate is decreased, a limit is reached below which performance declines due to poor growth and viability, specific productivity, or product instability. To overcome these limitations, a strategy referred to as "push-to-low" optimization has been developed. This approach involves an iterative stepwise decrease of the specific perfusion rate, and is most suitable for production of stable proteins where increased residence time does not compromise apparent specific productivity or product quality. The push-to-low approach was successfully applied to the production of monoclonal antibody against tumor necrosis factor (TNF). The experimental results followed closely the theoretical prediction, providing a multifold increase in titer. Despite the medium improvement, reduction of the specific growth rate along with increased apoptosis was observed at low specific perfusion rates. This phenomenon could not be explained with limitation or inhibition by the known nutrients and metabolites. Even further improvement would be possible if the cause of apoptosis were understood. In general, a strategic target in the optimization of perfusion processes should be the decrease of the cell-specific perfusion rate to below 0.05 nL/cell/day, resulting in high, batch-like titers. The potential for high titer, combined with high volumetric productivity, stable performance over many months, and superior product/harvest quality, make perfusion processes an attractive alternative to fed-batch production, even in the case of stable proteins.