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Hollow fibre bioreactor delayed start-up, pH gradients and CHO cell product harvesting Lee, Yi-Ta

Abstract

Hollow fibre bioreactors (HFBRs) are widely used for monoclonal antibody production. Compared to spinners inoculated in parallel, ultrafiltration HFBR start-up lags are often significantly longer and HFBRs have a higher frequency of failure. A number of potential causes of these problems were identified, including inhibitory residual byproducts of the HFBR manufacturing process and the loss of the inoculum by cells settling into hypoxic manifold regions of the extracapillary space (ECS). Besides these primary effects, a number of secondary phenomena were also identified, e.g., factors released by the death of settled cells can decrease viable cell growth rates. Increased thermal degradation of L-glutamine to ammonium during the prolonged startup further contributed to decreasing cell growth. A combination of these effects can explain the higher frequency of HFBR startup failures. Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t-PA) were cultured to tissue-like density in a HFBR. A sharp decline in the product concentration was observed after 20 days. Another t-PA producing CHO cell line was cultured in a HFBR and a similar decline in the harvest product concentration was observed when the ECS was filled with cells. The harvested product concentrations were significantly lower than the ECS concentration measured at the end of the run. Large amounts of t-PA were recovered from inside the HFBR cartridge at the end of the runs, using a cell lysis buffer and a lysine analog. These results demonstrate that mass transport can become hindered in a packed HFBR to such an extent that the ability to harvest protein product is severely compromised. A mathematical model of HFBR mass transport was developed to describe the effects of CO₂ and lactate production on the ECS cell culture environment, in particular the pH. This mathematical model, using a set of previously reported diffusion coefficients along with some generalized assumptions for the specific lactic acid production rate (sLPR) and carbon dioxide evolution rate (sCER), predicted intracapillary space (ICS) pH levels similar to experimentally measured values. The variables that had the greatest impact on the ECS pH gradients were sLPR, the thickness of the ECS annulus and the ICS inlet pH. This model can therefore be a useful tool in determining the operating conditions needed to maintain a favourable pH environment in the ECS of an HFBR.

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