Early Integration of Manufacturing Considerations for Pichia Pastoris Protein Production
In protein production, it is essential that strain and process design account for the requirements of large-scale manufacturing from the outset. Considering productivity, quality, safety, and consistency early helps prevent these factors from becoming obstacles later in development. Important aspects of process performance can already be assessed at the 1–10 L scale, enabling refinement before moving into costly pilot or production stages. At VALIDOGEN, the expression-enhancing power of our UNLOCK PICHIA® platform is combined with systematic process development and optimization to deliver Pichia pastoris strains and cultivation processes that are robust, scalable, and reliable across a wide range of applications.
VHH Case Study
One example of how we address large-scale manufacturing challenges is through process development tailored to different production requirements. To illustrate this, we carried out a three-part case study using a bivalent VHH (nanobody) expressed in Pichia pastoris. The scenarios include methanol-free processes, highly accelerated cultivation timelines, and titer-maximizing strategies.
The first two case studies addressed situations where methanol-free production is a prerequisite set by the manufacturing site. Accordingly, our methanol-free UNLOCK PICHIA® technology was applied to develop high-performance expression strains. In the first case, limited downstream processing (DSP) capacity was identified as a bottleneck. To address this, we implemented a short cultivation protocol (64 h), yielding 12 g/L of secreted bivalent VHH with a high space-time yield of 96 mg/L/h. By reducing batch size and increasing frequency, the DSP load per run was minimized - effectively eliminating the bottleneck without compromising overall throughput.
In the second case, no downstream processing (DSP) constraints were present, allowing the process to be optimized within VALIDOGEN’s standard methanol-free cultivation timeframe (~110 h) with the primary objective of maximizing product titer. This approach resulted in a VHH yield of 19 g/L in the culture supernatant and a high space-time yield of 95 mg/L/h - demonstrating the platform’s potential for volumetric productivity.
In the third case, methanol use could be facilitated and no other constraints were identified, and so the focus was simply to generate the highest titer possible. Utilizing our UNLOCK PICHIA® toolbox an evaluation of methanol-induced promoters, secretion signals and co-expression helper factors generated an initial high-performance strain. Under a generic protocol a productivity of 21 g/L was achieved in 1L bioreactors. Through targeted process optimization, and scaling to 10 L, specific productivity was increased by 80%, achieving a final titer of 31 g/L.
Proven scalability
From the earliest stages, we account for scale-up effects so that our lab-scale results translate reliably to pilot and production. This approach minimizes adaptation during technology transfer and enables smooth implementation at large scale. VALIDOGEN’s clients have successfully established Pichia pastoris processes developed in-house at volumes up to 100,000 L, confirming the robustness and scalability of the UNLOCK PICHIA® platform.