Development of an integrated continuous process for recombinant protein production using Pichia pastoris

This project will build on the successes of our two projects in phase 1 of the Bioprocessing Research Industry Club (BRIC1) to develop a system for the growth-associated production of commercially important protein products in a commercially-important production organism - the yeast Pichia pastoris.

Proteins, particularly antibodies, are now playing a major role in the treatment of human disease. They are usually produced using animal cells in culture, a procedure that is both costly and time-consuming. An attractive alternative is to engineer a yeast cell to produce human proteins of therapeutic value. Pichia pastoris is often used for this purpose since it may be grown at high cell densities and has an efficient secretion system to release the protein product from the cells. Current processes induce protein production by repeatedly adding methanol to the yeast cultures. Our studies in BRIC1 demonstrated that this is exactly the wrong way to go about this since the cells are repeatedly stressed and produced badly folded proteins that have low biological activity and are not secreted out of the cells. What is required is a continuous process for protein production and recovery and this is what our BRIC2 project aims to achieve.

The product proteins to be studied will be guided by our contacts within the BRIC member companies and include a number of proteins used in human therapies. Growth-associated production will enable continuous processes and thus increase commercial productivity. Computer models developed in BRIC1 will be improved, extended, and used to control the novel process. Moreover, the fast protein separation methods that we also developed in BRIC1, will permit the continuous retrieval of product, thus obviating the major barrier to the commercial adoption of continuous systems - the mismatch between continuous production and batchwise downstream processing.

We aim to improve both the quality and yield of proteins secreted from Pichia pastoris. This builds on the successes of our BRIC1 projects to develop a system for the growth-associated production of commercially important proteins. We have shown that the stresses of batch-wise production are sensed by the producer organism and inevitably reduce the quality and yield of secreted proteins. Growth-associated production will enable continuous processes that do not suffer these cell-associated stresses, thus increasing throughput. This will allow us to implement the modelling systems we developed in BRIC1 to effect process control. Moreover, the fast ion-exchange methods for protein separation that we also developed in BRIC1, will permit continuous retrieval of product, thus obviating the major barrier to the adoption of continuous systems - the mismatch between continuous production and batch-wise downstream processing.

We showed in BRIC1 that, in P.pastoris, secreted yields of r-proteins depend upon their native-state stabilities. Thus, we introduced the concept of engineering proteins for secreted yield, rather than just functionality a concept adopted successfully by industry. Understanding product yield's dependency on stability affords a basis for predicting secreted yield in system-wide models.
The production of r-proteins by P.pastoris exploits the powerful AOX1 promoter with expression repeatedly induced by methanol addition in a fed-batch process. In BRIC1, we demonstrated that such processes are doomed never to produce optimal yields of r-proteins since the cells are repeatedly subjected to a stress-inducing transient which both reduces yields and promotes protein misfolding. Thus, in BRIC2, we will develop an integrated continuous process for r-protein production and recovery in which product formation is growth-linked and in which medium formulation and process control are determined by a constraint-based model of P.pastoris metabolism.

The commercial production of recombinant proteins (r-proteins) of therapeutic value would be greatly facilitated, with enormous cost and efficiency benefits, if microbial processes were to supplant those based on cultured mammalian cells. Among the microbial 'cell factories' for r-protein production, the most commercially attractive is the yeast, Pichia pastoris, since it may be grown at high cell densities and an efficient secretion system that delivers the r-protein to the culture broth. However, it is our contention (based on our data from BRIC1) that currently used regimes for the production of r-proteins by P.pastoris, which exploit the powerful AOX1 promoter with expression repeatedly induced by methanol addition in a fed-batch process, are completely wrong-headed. This is because such regimes repeatedly expose the producer organism to stress-inducing transients that both reduce yields and promote protein misfolding. Thus both the quality and quantity of the r-protein product are compromised.

We propose to completely revolutionise r-protein production by this organism by developing an integrated continuous process in which product formation is growth-linked and in which medium formulation and process control are determined by a constraint-based model of P. pastoris metabolism. We will also intend to solve two problems that often militate against the adoption of continuous fermentation by the bioprocess industry - genetic instability of the producer organism and the mismatch between continuous production and batch-wise downstream processing. The first will be solved by the identification of medium constituents whose concentration may be varied without compromising product yield or quality - this removes the constant selection pressure that leads to genetic instability. The deployment of the fast ion-exchange methods for protein separation that we developed in BRIC1 will permit not only continuous retrieval of product, but also continuous verification of product quality - this will remove the second constraint on the commercial exploitation of continuous systems.

Although we intend to use Pichia pastoris as our chosen cell factory for this project, the lessons learned, and the approach to protein, organism, and process design that we shall develop, would be applicable to any eukaryotic expression system. Moreover, the systems that we shall put in place for the continuous retrieval of protein product from culture broth, and the design and operation of processes for continuous biomanufacture with continuous verification of product quality, will have wide applicability to the benefit of the Bioprocess Industry, particularly the members of BRIC.

The UK lags international competitors in the commercial supply of materials and equipment for biomanufacturing. The technologies described here are entirely novel and will be protected by patents, commercial manufacturing is to be tested and, upon achievement of the envisaged outcomes, lead times to launch of a continuous system could be short. BRIC is a good place to start with economic impact and to apply or test approaches with proteins of interest. Thus, we will engage with BRIC member companies through visits and workshops. Once we have patent protection, we can involve academics and companies beyond BRIC. Social impacts will accrue from our engagement with clinically informed companies within BRIC, which will lead to improved availability of pharmaceutical proteins at a lower price. Cambridge Enterprise (CE) will act, on behalf of both universities, to protect IP arising from the research. A Consortium Agreement will be put in place at the start of the Grant.