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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.
Item Metadata
Title |
Hollow fibre bioreactor delayed start-up, pH gradients and CHO cell product harvesting
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2004
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Description |
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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Genre | |
Type | |
Language |
eng
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Date Available |
2009-12-23
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0058860
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2005-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.