Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Wang, Alice; Lewus, Rachael A.; Rathore, Anurag S.

doi:10.1002/bit.20816

Cited by 40 publications

(19 citation statements)

References 31 publications

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“…Additionally, the partially fermentative metabolism leads to a decreased biomass yield coefficient, so that less biomass is accumulated during the process, resulting in facilitated cell removal, which is a major challenge with high cell density cultures (Wang et al, 2006). As a low dissolved oxygen set point with limited oxygen concentration in the culture results in an increased oxygen transfer rate due to a higher concentration gradient, lower air-flow and stirrer speed are required to supply enough oxygen to the culture.…”

Section: Discussionmentioning

confidence: 99%

Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

Baumann

Maurer

Dragosits

et al. 2007

Biotech & Bioengineering

120

100

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High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.5-fold increase of the specific productivity q(P) at low oxygen supply was observed. At the same time the biomass decreased and ethanol was produced, indicating a shift from oxidative to oxidofermentative conditions. Based on these results we designed a feedback control for enhanced productivity in fed batch processes, where the concentration of ethanol in the culture was kept constant at approximately 1.0% (vv(-1)) by a regulated addition of feed medium. This strategy was tested successfully with three different protein producing strains, leading to a three- to sixfold increase of the q(P) and threefold reduced fed batch times. Taken together the volumetric productivity Q(P) increased 2.3-fold.

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Section: Discussionmentioning

confidence: 99%

Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

Baumann

Maurer

Dragosits

et al. 2007

Biotech & Bioengineering

120

100

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“…The fouling mechanism may include pore blockage, cake formation and/or pore constriction (39, 40). The high particulate load and high turbidity present in clarified cell culture supernatant adds challenges to the secondary clarification by depth filtration.…”

Section: Depth Filtrationmentioning

confidence: 99%

“…Small size depth filter disks are often used for the purpose of initial screening to compare different brands of depth filters to fit particular feed streams (51). Variations of up to 30% in filter performance of small filters are expected because of the wet‐laid filter manufacturing process.…”

Section: Scale‐down Models and Scale‐up Considerationsmentioning

confidence: 99%

Advances in Primary Recovery: Centrifugation and Membrane Technology

Roush

2008

Biotechnology Progress

119

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Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification ( 1). Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers ( 2, 3). Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead‐end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention ( 4). Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies ( 5). This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.

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“…The main purpose of clarification is to efficiently separate cells, cell debris, and other colloidal matter and deliver a particle-free feed to downstream process steps such as ion exchange and/or protein A chromatography. This is typically achieved by performing centrifugation or filtration based operations such as microfiltration or depth filtration or a combination of these [6][7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Modeling of Filtration Processes—Microfiltration and Depth Filtration for Harvest of a Therapeutic Protein Expressed in Pichia pastoris at Constant Pressure

2014

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Filtration steps are ubiquitous in biotech processes due to the simplicity of operation, ease of scalability and the myriad of operations that they can be used for. Microfiltration, depth filtration, ultrafiltration and diafiltration are some of the most commonly used biotech unit operations. For clean feed streams, when fouling is minimal, scaling of these unit operations is performed linearly based on the filter area per unit volume of feed stream. However, for cases when considerable fouling occurs, such as the case of harvesting a therapeutic product expressed in Pichia pastoris, linear scaling may not be possible and current industrial practices involve use of 20-30% excess filter area over and above the calculated filter area to account for the uncertainty in scaling. In view of the fact that filters used for harvest are likely to have a very limited lifetime, this oversizing of the filters can add considerable cost of goods for the manufacturer. Modeling offers a way out of this conundrum. In this paper, we examine feasibility of using the various proposed models for filtration of a therapeutic product expressed in Pichia pastoris at constant pressure. It is observed that none of the individual models yield a satisfactory fit of the data, thus indicating that more than one fouling mechanism is at work. Filters with smaller pores were found to undergo fouling via complete pore blocking followed by cake filtration. On the other hand, filters with larger pores were found to undergo fouling via intermediate pore blocking followed by cake filtration. The proposed approach can be used for more accurate sizing of microfilters and depth filters.

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Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Cited by 40 publications

References 31 publications

Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

Advances in Primary Recovery: Centrifugation and Membrane Technology

Modeling of Filtration Processes—Microfiltration and Depth Filtration for Harvest of a Therapeutic Protein Expressed in Pichia pastoris at Constant Pressure

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