Abstract
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.
Highlights
Pichia pastoris is frequently used as an expression system for the production of therapeutic proteins because it offers high growth rate and is able to grow on a relatively simple and inexpensive medium.A major advantage that Pichia offers over another popular microbial host, E. coli, is that Pichia is capable of inducing correct formation of disulfide bonds and to some extent glycosylation [1,2,3,4,5]
We have explored the possibility for clarification of the feed streams using microfiltration and depth filtration
It is seen that the particle size distribution of the fermentation broth includes a substantial population of particles greater than 200 nm and a significant portion of these particles would be expected to be retained by the membranes, most of which have the pore size ranging from 100–500 nm (Table 1)
Summary
Pichia pastoris is frequently used as an expression system for the production of therapeutic proteins because it offers high growth rate and is able to grow on a relatively simple and inexpensive medium.A major advantage that Pichia offers over another popular microbial host, E. coli, is that Pichia is capable of inducing correct formation of disulfide bonds ( reducing the need to refold protein) and to some extent glycosylation [1,2,3,4,5]. 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]. Membrane fouling can occur due to deposition of suspended particles on the external surfaces of the filter or within the filter’s pores This results in higher membrane resistance and affects quality of the permeate [11,12,13,14]. Cake filtration model assumes particle accumulation on the membrane surface in a permeable cake of increasing thickness
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