One of the most important goals of genetic engineering, especially within the biopharmaceutical industry space, is the ability to produce therapeutic proteins.
Monoclonal antibodies (mAbs) continue to prevail in the biopharmaceutical therapeutic market however, new molecule formats such as novel scaffolds, fusion proteins, recombinant proteins, enzymes and plasmid DNA are also gaining traction. The increasing demand for these new molecule formats is also reflected in Lonza’s current and projected program portfolio, which is increasingly diverse and requires the utilization of increasingly sophisticated expression technologies.
For recombinant protein, several expression systems are available to produce them that utilize bacteria, mammalian cells, plant cells, yeast, mold, and insect cells. Within the yeast expression system space, Pichia pastoris (P. pastoris) represents a popular choice for both laboratory and industrial scale production due to the appropriate folding and secretion of recombinant proteins expressed in this system and their ease of purification.
In my experience working on the production of recombinant proteins and chemical compounds, P. pastoris is my favored expression system. Our vision at Lonza is to bring any therapy to life. For me, P. pastoris—a multifaceted cell factory that can secrete therapeutic proteins, chemicals, and natural products alike—is a great representation of this vision.
Market-leading Pichia expression system
For years, P. pastoris has been utilized as a versatile cell factory for the recombinant production of biomolecules at an industrial scale. For example, Eptinezumab, an FDA-approved mAb drug substance for episodic migraine, is produced on a large scale in a P. pastoris (Komagataella phaffii) strain[1]. Moreover, Nanobody® ALX0061 (recombinant anti-hIL6 receptor single domain antibody fragment) and Nanobody®ALX00171 (recombinant anti-RSV single domain antibody fragment) produced in P. pastoris strains have reached the clinical stage[2,3].
P. pastoris fills a gap between bacterial and mammalian expression systems and brings the best of both worlds. Development or expression constructs in P. pastoris is rapid and the expression system is capable of producing high yields of recombinant proteins. Additionally, the downstream processing is simplified by avoidance of a cell lysis step typically required in other expression systems as the recombinant protein is excreted.
Lonza has developed the XS® Pichia Expression System to help accommodate industry trends, eliminate production bottlenecks, align downstream processing, and drive breakthroughs into the clinic[4]. This powerful toolbox for Pichia strain development is comprised of more than twenty optimized strain variants, six plasmids, over ten methanol-free promoters, six novel signal sequences, and more than twenty helper factors. Additionally, a combinatorial screening workflow is available to identify the ideal combination of expression components for each protein of interest. The XS® Pichia Expression System offers a tailored strain development solution for each product and ensures high productivity, scalability, and reliability for manufacturing.
A key advantage of the XS® Pichia Expression System is the availability of alternative promoters to the methanol-inducible promoter AOX1. Switching to a methanol-free promoter, e.g. G1-3, has great benefits related to sustainability and process safety. Additionally, methanol induction requires long fermentation times while offering low space-time yields. From an operational perspective, avoiding the use of methanol during production mitigates the need for expensive explosion-proof production facilities.
XS® Pichia system delivers greater yields in the short fermentation times of bacteria-like expression systems.
The superior methanol-free promoter system uses glucose as the sole carbon and energy source. By eliminating potential methanol toxicity, the glucose-feed primary screening allows the selection of more reproducible and scalable clones. A glucose-based induction and feeding strategy dramatically simplifies the fermentation regime on the manufacturing scale. A favorable correlation of the promoter activity between growth rate and biomass-specific product secretion rate brings flexibility to optimize the feed profile between either high titer with prolonged fermentation times or maximum space-time yield with short, bacterial-like fermentation times.
Continued development to improve performance
Over the last few years, our team has established a model-based, product-specific process development approach exclusively for the XS® Pichia Expression System. For each protein of interest, only a minimal set of cultivation parameters needs to be screened. Once the optimal conditions are determined, we generate data for model training, which is used to explore the process-operation space and to generate an optimal feed profile considering manufacturing-constraints such as time, dry cell weight, or oxygen transfer.
The resulting tailored speed fermentation strategies, with bacterial-like fermentation time, ensure maximum titer for the production of several novel scaffolds and enzymes. The combination of a methanol-free promoter and model-based process development approach also ensures flexibility to adjust a methanol-free fed-batch fermentation strategy to the limitations of the selected production facility, further de-risking large-scale manufacturing.
As the recombinant protein landscape continues to evolve, we are continuously developing and optimizing our Pichia expression system by expanding our toolbox of components to help meet expected market demand. This includes additional promoter variants, improved host strains and newly identified helper factors.
References:
[1] Berger, Amnon A et al. “Eptinezumab-jjmr, a humanized monoclonal specific to Calcitonin Gene Related Peptide, for the preventive treatment of migraine in adults.” Health psychology research vol. 10,5 38439. 12 Nov 2022, doi:10.52965/001c.38439
[2] Detalle, Laurent et al. “Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection.” Antimicrobial agents and chemotherapy vol. 60,1 6-13. 5 Oct 2015, doi:10.1128/AAC.01802-15
[3] Van Roy, Maarten et al. “The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis.” Arthritis research & therapy vol. 17,1 135. 20 May 2015, doi:10.1186/s13075-015-0651-0
[4] Lonza | XS Technologies® Microbial Platform for Protein Expression Brochure