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Holmes, William J.; Smith, Rodney; Bill, Roslyn M.

doi:10.1186/1475-2859-5-s1-p30

Cited by 15 publications

(2 citation statements)

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“…Antifoams have previously been suggested to alter the growth of cells and influence protein yield in bioprocesses [2,11], but their addition to P. pastoris cultures has never been examined systematically. In this study, addition of the five antifoams tested increased the total yield of secreted recombinant GFP produced by 20 mL P. pastoris cultures.…”

Section: Discussionmentioning

confidence: 99%

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Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

et al. 2011

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BackgroundPichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.ResultsAddition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (kLa) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.ConclusionsWe show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.

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Section: Discussionmentioning

confidence: 99%

“…Foaming can lead to reduced yields since bursting bubbles can damage proteins [2] and can also result in a loss of sterility if the foam escapes [3]. In bioreactors, foaming can lead to over-pressure if a foam-out blocks an exit filter.…”

Section: Introductionmentioning

confidence: 99%

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

et al. 2011

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Identification of process conditions influencing protein aggregation in Chinese hamster ovary cell culture

Paul

Handrick

Ebert

et al. 2018

Biotech & Bioengineering

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Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells. Furthermore, rapid and efficient bioprocess optimization has been performed using design of experiments (DoE) strategies. In this work, we describe a DoE-based approach for the analysis of process parameters and cell culture additives influencing protein aggregation in Chinese hamster ovary (CHO) cell cultures. Important bioprocess variables influencing the aggregation of mAb and host cell proteins were identified in initial screening experiments. Response surface modeling was further applied in order to find optimal conditions for the reduction of protein aggregation during cell culture. It turned out that a temperature-shift to 31 °C, osmolality above 420 mOsm/kg, agitation at 100 rpm and 0.04% (w/v) antifoam significantly reduced the level of aggregates without substantial detrimental effects on cell culture performance in our model system. Finally, the aggregation reducing conditions were verified and applied to another production system using a different bioprocess medium and another CHO cell line producing another mAb. Our results show that protein aggregation can be controlled during cell culture and helps to improve bioprocessing of mAbs, by giving insights into the protein aggregation at its origin in mammalian cell culture.

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Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

Velugula‐Yellela

Williams

Trunfio

et al. 2017

Biotechnology Progress

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Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro-scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro-scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX-Cell Advanced and OptiCHO media, and 204, C, EX-Cell, SE-15 and Y-30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX-Cell Advanced and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25–35 × 106 cells-d/mL, while maintaining specific antibody production (Qp>2 pg/cell-d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX-Cell and SE-15 were capable of providing adequate control of foaming while antifoam 204 and Y-30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line.

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Cited by 15 publications

References 1 publication

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

Identification of process conditions influencing protein aggregation in Chinese hamster ovary cell culture

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

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