XS™ Pichia Expression Systems

  • Introduction

    Although E. coli host systems remain the industry workhorse, there are times when an ideal expression outcome for your product is not achieved.  In these situations, we offer an alternative approach to microbial expression with a range of Pichia pastoris host systems.

    As a eukaryotic host organism, Pichia is able to effectively express small polypeptides, as well as large, complex proteins that require higher order post translational modifications. In addition, Pichia uses a signal sequence, which helps it to express soluble secreted product as opposed to undesirable intracellular inclusion bodies. Soluble secreted product, combined with use of minimal media makes recovery and downstream processing easier, more efficient, and improves overall process yields. Pichia also lacks endotoxin, therefore eliminating this typical purification challenge.

    Lonza has established a proprietary Pichia host which has been developed for excellent growth performance (see Figure 1 below). Our XS™ Pichia toolbox includes three different promoter systems: Methanol Inducible, Glucose Regulated and GAP Constitutive. This range of promoter options provides complementary mechanisms for the optimal expression of your product.  In addition, the toolbox also includes: novel signal peptides, zeocin selection marker, two chromosomal integration sites, and single or multi-copy insertion options.

    Lonza's XS™ Pichia Systems Include:    

    • Novel signal peptide that eliminates N-terminal variant formation
    • Soluble secreted expression simplifying downstream processing
    • Tunable copy number that increases the probability of creating the best production clone
    • Complementary Pichia systems increase the probability of expression success

    Figure 1.  Wet cell weight (WCW) and dry cell weight (DCW) were measured over time.  Similar growth and fermentation profiles were seen for both the Lonza host strain and an industry standard. The measured WCW and DCW concentrations were reproducible.

     

     

  • Methanol Inducible

    Pichia is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source via the AOX1 promoter. This expression mechanism has been the most widely-used Pichia system in the industry. For reasons described on the Glucose Regulated page (see tab on right), methanol is not an ideal inducer.  In some cases however, the advantages of higher titers may outweigh the issues related to methanol.

    Lonza has combined its proprietary XS™ Pichia host and signal sequences with this proven promoter system to offer customers a comprehensive Pichia expression toolbox. Multi-gram-per-liter titers have been achieved with this system (see Figure 2 below).

    This system is available for use in your own labs under our Research Evaluation Agreement.

     

    Methanol Inducible System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for all plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at 2 different chromosomal loci
    • Fed-batch fermentation platform protocols

     

    Figure 2. 10L fed-batch fermentation of a protein using the XS™ Pichia Methanol Inducible System showing titers in excess of 9 g/L. 

  • Glucose Regulated - XS™ Pichia 2.0

    For many years, the methanol inducible AOX1 promoter system has been the state-of-the-art system for Pichia as it generates high titers of heterologous product. Combining our Glucose Regulated System with G1-3, a new, proprietary inducible promoter, Lonza has developed the next generation Pichia system, Pichia 2.0.

    The novel G1-3 strong promoter avoids methanol and uses glucose as a trigger for induction of expression. Combining a glycerol-based biomass generation phase with a limiting glucose feed results in a drastically-reduced fermentation times that approximate those of E.coli. The Pichia 2.0. fermentation strategy has produced high, mutli-gram-per-liter titers with very short fermentation times of 2-3 days (see Figure 3).

     

    Figure 3. XS™ Pichia 2.0 fermentation for a single domain antibody-like molecule of human origin where 6.4 g/L of product was obtained in less than 60 hours of total fermentation time.

     

    In addition, Lonza offers tailor-made, model-based fermentation optimizations that allow tuning of the fermentation for the required outcome from maximum titer to maximum space-time yield. The model enables a predictable, product-specific and customizable fermentation set-up that can be tailored to the requirements of the full production process and the specifications of the production plants at any stage of process development.  (see Figure 4).

     

     

    Figure 4. Schematic representation of the process variability window where a tailor-made model is used to optimize the fermentation specifically to the needs and requirements of the production process and the plant.

     

    Our new Pichia 2.0 expression system provides additional benefits including straightforward and robust high-throughput screening using slow-releasing glucose polymers. The methanol-free induction results in highly-viable cells for improved product quality. Finally, the Pichia 2.0 system enables full-scalability from early screening to large scale production.

     

     XS™ Pichia 2.0 is available for use in your own labs under our Research Evaluation Agreement.

     

    Glucose Regulated System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for all plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at two different chromosomal loci
    • Fed-batch fermentation platform protocols

    To learn more about our XS™ Pichia 2.0 System from an industry expert, please download Christoph Kiziak's webinar entitled, “Achieving Speed and Scalability Through aValuable Alternative to Microbial Expression for Next-Gen Therapeutics"

     

  • GAP Constitutive

    Vectors containing a constitutive Pichia promoter derived from the glyceraldehyde-3-phosphate dehydrogenase gene (GAP), are widely known and used in the industry. For products that do not interfere with Pichia metabolism and cell viability, non-induced constitutive can be an attractive expression strategy.

    Lonza has combined this promoter with our novel XS™ Pichia host and signal sequences to offer customers another complementary Pichia option to screen.  This system has shown productivity levels in excess of 1 g/L for a novel therapeutic candidate (see Figure 5 below).  An additional benefit of this system is that it does not require the use of methanol.

    This system is available for use in your own labs under our Research Evaluation Agreement.

     

    GAP Constitutive System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for all plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at 2 different chromosomal loci
    • Fed-batch fermentation platform protocols

     

    Figure 5. 10L fed-batch fermentation of a novel therapeutic candidate using XS™ Pichia GAP Constitutive System showing titers in excess of 1 g/L.