We conducted a series of case studies, demonstrating the yield-enhancing effects of applying different toolbox elements of VALIDOGEN's Pichia pastoris protein expression system known as UNLOCK PICHIA
The application of a higher number of promoter variants from VALIDOGEN's UNLOCK PICHIA technology toolbox
as a pool typically leads to a significant productivity boost leveraging random genetic diversity and concomitant fine-tuning and optimization of gene dosage. The targeted application of VALIDOGEN's full toolbox by identification of optimum combinations with other elements such as strain background, helper factors, secretion signals or production regime further boosts productivity.
CASE STUDY 1 - UNLOCK PICHIA promoter variants
Yield optimization through application of a higher number of UNLOCK PICHIA promoter variants
- Bi-modular VHH
- Methanol-free and methanol-induced production
- Feasibility study (small screening, 90 transformants): 7 g/L (both conditions)
- Large screening (higher number of UNLOCK PICHIA promoter variants, 6,800 transformants): 15 g/L methanol-free - 18 g/L methanol-induced
CASE STUDY 2 - UNLOCK PICHIA helper factors
VHH product yield improvement through timely adjusted expression of helper proteins acting at multiple sites within the production host
- Bi-modular VHH
- Set of helper factors (HFs) acting at different sites in the cell
- Cytosolic chaperone - improvement factor: 2.0-fold
- Translocation engineering - improvement factor: 2.5-fold
- ER-resident helper - improvement factor: 2.7-fold
- All helper factors combined - improvement factor: 3.5-fold
CASE STUDY 3 - UNLOCK PICHIA toolbox "MeOH-free"
Yield optimization through application of the UNLOCK PICHIA toolbox and process optimization
- Single VHH
- Field of application required methanol-free production and ARMs-free cloning (antibiotic-resistance marker-free)
- Feasibility study (small screening, 90 transformants): 0.8 g/L
- Helper factor screening (on top of best clone from feasibility study): 1.3 g/L
- Production strain development (large screening with higher number of UNLOCK PICHIA promoter variants, including HF co-expression - 6,200 clones): 2.7 g/L
- Lean process optimization (4 runs in 5L-scale): 3.9 g/L
CASE STUDY 4 - UNLOCK PICHIA secretion signals
Yield optimization through application of a novel UNLOCK PICHIA secretion signal
- Bi-modular VHH
- Methanol-induced cultivation
- Classical secretion signal - small screening only (90 transformants): 8 g/L
- Novel UNLOCK PICHIA secretion signal - small screening only (90 transformants): 16.5 g/L
CASE STUDY 5 - UNLOCK PICHIA platform strains
Yield optimization through application of a novel UNLOCK PICHIA platform strain
- Tri-modular VHH
- Methanol-free and methanol-induced cultivation
- Small screening only (44 transformants)
- "UNLOCK PICHIA platform strain" 3.0 g/L versus "standard“ strain 1.8 g/L - methanol-induced
- "UNLOCK PICHIA platform strain" 1.8 g/L versus "standard“ strain 1.0 g/L - methanol-free
Applying the UNLOCK PICHIA toolbox elements
, we are able to generate a large number of different genetic constellations in thousands of strains for fine-tuning of expression thus unlocking Pichia´s potential for successful production of fully active, high quality proteins.
In our labs, expression constructs carrying UNLOCK PICHIA promoter variants, the gene of interest and potentially also genes encoding for different auxiliary (helper) proteins are transformed simultaneously in pools into selected Pichia platform strains, thereby generating a broad diversity of genetic arrangements in the host cells resulting in diverse expression profiles.
In this setting, protein production in Pichia pastoris is boosted significantly. Following specifically optimized protocols, thousands of clones can be screened in minimum time and most promising clones will be reliably identified already at microscale. Screening results are transferable into bioreactors making laborious intermediate steps for clone comparison obsolete.
UNLOCK PICHIA advantages
The case studies confirm the advantages of UNLOCK PICHIA as a very broad range Pichia-based platform incorporating numerous expression tools and different strategies for the enhancement of production yields and product quality.
- Boosted expression levels for many proteins (more than 20g/L) including monovalent- and multivalent VHH antibodies: up to 16.5 g/l
- Secreted products providing a beneficial starting point for protein purification
- Complete absence of methanol through VALIDOGEN´s methanol-free promoters
- Stable, robust and scalable processes for commercial manufacturing