Short cultivation time and high titer


Protein production in Pichia with maximized space-time yield

Protein production VALIDOGEN


Key facts summarizing case study results


  • 12 g/l of secreted bivalent VHH was produced in a 64 h methanol-free protein production process


  • No metabolic burden from switch of carbon source


  • By targeted application of VALIDOGEN´s UNLOCK PICHIA® toolbox final product titer was increased 5-fold



Key advantages


  • VALIDOGEN´s UNLOCK PICHIA® technology provides effective methanol-free protein production processes with maximized space-time yield


  • UNLOCK PICHIA® methanol-free processes are based on AOX1 promoter variants but use glycerol throughout the process, no methanol feed is required



VALIDOGEN's advanced strain development program - with manufacturability in mind


Targeted and time-saving generation of high-productivity strains is an integral part of effective bioprocess development. VALIDOGEN is addressing today's protein manufacturing needs by early consideration of production strain and process requirements.


The latest development & research programs (performed within Boehringer Ingelheim's Vienna and VALIDOGEN's joint technology program leveraging expertise from both companies) focus on the targeted development of bioreactor cultivation processes for protein production with Pichia pastoris* under methanol-free conditions with an emphasis on maximizing volumetric productivity (space-time yield).


Development of short, methanol-free protein production processes for maximization of volumetric productivity


In this case study, a VHH-expressing strain was used to demonstrate that MeOH-free cultivations based on our AOX1 promoter variants can deliver high product yields (>10 g/l of culture supernatant) with considerably shortened cultivation times (~60 h). Additionally, it shows how the targeted application of the UNLOCK PICHIA toolbox helps to incrementally boost productivity by identifying optimum combinations of different elements such as promoter variants, helper factors, secretion signals, and expression strategies. This technology is proofed to be ready for commercial use and large scale manufacturing at both parties.


Please contact us if you are interested to learn more about VALIDOGEN's strain development programs or any other services directly by speaking to our team.


Iskandar Dib, Principal Scientist DSP and BD manager, iskandar.dib@validogen.com

Rosie Maddock, BD manager, rosie.maddock@validogen.com


* Pichia pastoris was reclassified as Komagataella phaffii

Meet us at the following upcoming conferences


BIO-Europe Spring 2024, March 18-20, Barcelona, Spain


VALIDOGEN will be represented by Rosie Maddock, Business Development Manager. If you are interested in meeting Rosie, we would be very happy to arrange a meeting. Just contact us directly or via EBD’s Partnering System. We look forward to hearing from you.


Pichia 2024, March 24-27, San Diego, US


Join the Pichia community and our team, which will be represented by Roland Weis,  Head of Operations & CEO, Iskandar Dib, Prinicipal Scientist DSP & BD manager and Aid Atlic, Head of Fermentation Process Development, who will be presenting on



March 25, 2024

11 a.m. - 11.20 a.m. PST, 8 p.m. - 8.20 p.m. CET

Modification of the Pichia Secretory Mechanism for Enhanced Productivity


One of the major advantages of the Pichia expression host is that the target protein is mostly secreted into the cultivation medium. After cell separation, the product is found in the supernatant, and since no cell disruption is required and very low amounts of cell host proteins are natively secreted, this significantly simplifies the subsequent protein purification. However, the extent to which recombinant proteins are secreted is product-dependent, as not all proteins are produced in high amounts. By modifying the Pichia secretion mechanism using new elements of VALIDOGEN’s UNLOCK PICHIA toolbox, we are able to overcome some drawbacks and bottlenecks and significantly increase the productivity of many target proteins. Examples and case studies will be presented for methanol-induced and methanol-free protein production at various scales, including high-throughput microscale cultivations, microbioreactor system and lab-scale fermenter.


Aid Atlic, Head of Fermentation Process Development,  VALIDOGEN