By VALIDOGEN GmbH
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VALIDOGEN case studies demonstrate power of UNLOCK PICHIA toolbox in VHH antibody expression
Raaba-Grambach, Austria: Protein expression specialist VALIDOGEN has released a series of case studies that demonstrate the protein expression yield-enhancing effects of its UNLOCK PICHIA production toolbox across a range of different VHH antibody formats.
The case studies were generated in VALIDOGEN’s laboratories to show how expression of different VHH single domain antibody formats in Pichia pastoris could variously be boosted through application of different components of the company´s UNLOCK PICHIA toolbox.
UNLOCK PICHIA protein expression strategies
The exercise also aimed to show, among others, the benefits of applying a higher number of PAOX1 promoter variants from the toolbox in a ‘pool approach’ which typically leads to a significant productivity boost, at the same time enabling more rapid generation and identification of high-level production strains.
This approach leverages broad random diversity of genetic arrangements and expression profiles, with concomitant fine-tuning and optimization of gene dosage, applying promoter variants and helper factors simultaneously in pools of expression constructs.
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 regimes further boosts productivity.
The case studies applied a range of approaches, using different UNLOCK PICHIA toolbox elements and expression strategies:
Case Study 1: Applying more promoter variants
The first case study focused on bi-modular VHH yield optimization through application of a higher number of UNLOCK PICHIA promoter variants, analyzing 90 transformants in an initial small screening to reach 7g/L of culture supernatant in both methanol-free and methanol-induced production.
A subsequent large screening used a higher number of UNLOCK PICHIA promoter variants for 15 g/L yield methanol-free versus 18 g/L methanol-induced (analyzing 6800 transformants each).
The UNLOCK PICHIA promoter library includes methanol-inducible promoter variants for maximum output and methanol-free promoter variants for improved process safety and economy. The library allows closer fine-tuning of gene expression and significant productivity boost through an increase in promoter variants used in pool transformation (small screening vs. large screening).
Figure 1: Case study 1 – Yield optimization through application of a higher number of UNLOCK PICHIA promoter variants
Case Study 2: Helper factors with diverse functions
The second case study sought bi-modular VHH product yield improvement through timely adjusted expression of helper proteins acting at multiple sites within the production host.
Studying various helper factors (HFs) acting at different locations in the cell showed following specific improvements:
- 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.
UNLOCK PICHIA includes a broad range of exclusive helper factors that enhance protein production and improve product quality by timely adjusted co-expression, further boosting product yields.
Figure 2: Case Study 2 – VHH product yield improvement through timely adjusted expression of helper proteins acting at multiple sites within the production host
Case Study 3: “MeOH-free” process optimization
The third case study reviewed single VHH yield optimization through increasing the number of promoter variants combined with the application of HFs followed by process optimization for methanol-free production.
After ARMs-free cloning (antibiotic-resistance marker-free), the feasibility study used 90 transformants to achieve 0.8 g/L in a small screening and helper factor screening (on top of best clone from feasibility study) to increase yield to 1.3 g/L.
Production strain development (screening of 6,200 clones), combining higher number of UNLOCK PICHIA promoter variants and expression of the most promising HF reached 2.7 g/L while lean process optimization across four runs in 5L-scale achieved a further yield increase to 3.9 g/L.
Figure 3: Case Study 3 – Yield optimization through application of the UNLOCK PICHIA toolbox and process optimization
Case Study 4: Novel secretion signals
The fourth case study focused on bi-modular VHH yield improvement through application of a novel UNLOCK PICHIA secretion signal in methanol-induced cultivation.
While use of a standard secretion signal achieved 8 g/L, use of the novel UNLOCK PICHIA secretion signal under same conditions achieved more than double the yield at 16.5 g/L, in both cases in only a small screening with 90 transformants.
This demonstrated how novel UNLOCK PICHIA secretion signals can outperform standard leader sequences. The toolbox also includes hybrid versions of novel and standard secretion leader sequences.
Figure 4: Case Study 4 – Yield optimization through application of a novel UNLOCK PICHIA secretion signal
Case Study 5: Novel platform strains
The final case study looked at tri-modular VHH yield optimization through application of a novel engineered UNLOCK PICHIA platform strain in methanol-free and methanol-induced cultivation.
In small screening, using 44 transformants, use of the novel UNLOCK PICHIA platform strain achieved 3.0 g/L compared with 1.8 g/L for a “standard“ strain in methanol-induced cultivation.
In methanol-free cultivation, using the UNLOCK PICHIA platform strain achieved an even larger proportional increase at 1.8 g/L versus a “standard“ strain at 1.0 g/L.
UNLOCK PICHIA incorporates a variety of different platform strains including antibiotic resistance free host vector systems and strains with stably integrated helper factors.
Figure 5: Case Study 5 – Yield optimization through application of a novel UNLOCK PICHIA platform strain
The power of diversity
The case studies demonstrate how VALIDOGEN is able to apply the UNLOCK PICHIA toolbox elements to generate a large number of different genetic constellations in thousands of strains for fine-tuning of expression, proving the power of Pichia pastoris for successful production of fully active, high quality proteins.
In the VALIDOGEN laboratories, 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. Generating a broad diversity of genetic arrangements in the host cells results in diverse expression profiles and significantly boosting protein production in Pichia pastoris.
Following specifically optimized protocols, thousands of clones can be screened in minimum time with the most promising clones reliably identified at microscale. Screening results are transferable into bioreactors, rendering obsolete those laborious intermediate steps that have been conventionally used for clone comparison.
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.
About VALIDOGEN GmbH
VALIDOGEN GmbH (formerly VTU Technology), a leading contract research company, offers development services applying the broadest toolbox and most versatile technology platform available for Pichia pastoris recombinant protein expression and production.
VALIDOGEN´s yield-enhancing UNLOCK PICHIA expression platform is based on its proprietary AOX1 promoter libraries and set of expression enhancing helper factors, several platform strains with different genetic backgrounds and different secretion signals as well as elaborated cloning and transformation protocols, a high-throughput screening and cultivation regime combined with excellent know-how in fermentation and downstream process development for maximization of product yield and overall process performance. The platform enables highest productivities delivering up to 25 g/L of secreted protein (record productivities of 35 g/L) within a few months development time.
VALIDOGEN develops high performance expression strains and economically viable protein production processes for the manufacture of biopharmaceuticals, enzymes and various other recombinant proteins and provides tailor-made solutions for customers from different industry sectors – such as pharma, diagnostics, food & feed, chemical, agro and other industries.
Headquartered in Raaba-Grambach, near Graz, Austria, VALIDOGEN is a private company and independent subsidiary of the KonValue Group.
Further information at: https://www.validogen.com
Click on UNLOCK PICHIA for further information on Pichia pastoris production toolbox.
VALIDOGEN also used the recent BIO Digital event to show how different elements of the UNLOCK PICHIA production toolbox can be used to boost VHH antibody yields