781-597-9197

Technologies

Contact us

Glycoengineered yeast Pichia pastoris Expression System

Yeast expression system, especially Pichia pastoris (P. pastoris), has a number of advantages over other expression systems for the production of recombinant protein and antibody. It is ease of genetic manipulation, has low cost for high protein production, and can perform higher eukaryotic protein modifications, such as glycosylation, disulfide bond formation and proteolytic processing. Unlike mammalian cells, yeast P. pastoris does not require serum in culture medium, and can be grown to very high cell densities using minimal media. The fermentation processes can be readily scaled up to meet greater demands, and the integrated vectors have genetic stability in continuous and large-scale fermentation. Although yeast P. pastoris can perform N-glycosylation modification, it is also a serious disadvantage of yeast P. pastoris to express glycoprotein. Protein N-glycosylation in yeast P. pastoris has glycans of the high mannose type with up to 100 or more mannose residues (hypermannosylation), which differ significantly from those of mammalian cells, and alter protein structure and function.
Genekine has genetically re-engineered N-glycosylation modification in yeast P. pastoris to mimic N-glycosylation modification in mammalian cells. By deleting the intrinsic N-glycosylation genes and integrating mammalian N-glycosylation genes in the genome of P. pastoris, we have constructed glycoengineered yeast P. pastoris strains, which yield human-like N-glycans on protein and antibody. Since the hypermannosylation of N-glycosylation is eliminated, our glycoengineered P. pastoris offer substantial advantages over P. pastoris and other expression systems to produce high level of protein and antibody for both basic research and industrial manufacture.

Full-length IgG Yeast Surface Display

Surface display technologies are powerful tools for high-throughput screening in antibody discovery and optimization. Phage display and yeast display are commonly used for displaying and screening the library of antibody fragment such as single-chain variable fragment (ScFv). However, for pharmaceutical development, scFv requires format conversion into full-length IgG (two heavy chain and two light chain) for further characterization, namely IgG reformatting. The converted full-length IgG may lose activity and often require further affinity maturation. The selected scFv fragment may be difficult to re-engineer and to generate full-length IgG which has good expression and biophysical properties required for pharmaceutical development. Mammalian cells are idea system to display full-length IgG in native form and allow selection of antibody for functional properties. However, mammalian cell display system has some disadvantages, such as small library size, difficulty in genetic manipulation, slow generation time, and high cost. Mammalian cell display system is not an efficient system for antibody maturation.
Like mammalian cell expression system, glycoengineered yeast P. pastoris cells contain eukaryotic internal quality control apparatus for antibody correct folding and post-translational modification (such as human-like N-glycosylation). We have applied glycoengineered yeast P. pastoris expression system to develop a platform of full-length IgG yeast surface display. The platform uses gene editing technology to efficiently direct site-specific integration of antibody genes into a single locus within the cell genome. This enables construction of libraries of many millions of monoclonal stable cell lines displaying full-length IgG antibodies on their surface. Many millions of clones can be screened directly for high affinity and/or improved developability profiles (“Well behaved” Antibody) by using fluorescence-activated cell sorting (FACS). This advanced platform combines the throughput of yeast display with mammalian-cell quality control in one platform. Full-length IgG antibody is displayed as close to native form in molecular structure, biophysical property, and biological function. This display system is well applicative in real-time selecting full-length antibodies and antibody fragments (e.g scFv, Fab), which cannot be done by other display systems like phage display, ribosome display, and bacterial display. It can be employed for antibody discovery, humanization, and optimization in native IgG format. The selected antibody can be used directly for animal study and clinical trial. Based on our proprietary platform of full-length IgG yeast surface display, Genekine can provide comprehensive solutions for antibody humanization, affinity maturation, and developability improvement. Clients will maximize their chances of identifying the best therapeutic or diagnostic antibody leads for the toughest targets.

Schematic illustration of IgG yeast surface display

The yeast surface display of IgG antibody libraries can be generated by transformation of yeast P. pastoris with plasmids encoding for anchor protein, heavy chains and light chains. The use of genetic engineering tools ensures plasmid integration into predefined chromosome sites and hence, full-length IgG antibody libraries are expressed and displayed on the surface of yeast cells. IgG-displaying yeast cells are incubated with the fluorophore-labeled antigen and fluorophore-labeled detection antibody (i.e. fluorophore-labeled anti-kappa-antibody) for sorting by FACS. FACS sorting of IgG display libraries is based on both antibody affinity and display level. It can eliminate the deviation caused by expression levels and distinguish clones with small affinity.

Full-length IgG library sorting by FACS

In yeast surface display of IgG antibody library, each individual yeast cell is double labeled and examined simultaneously for its antigen binding and IgG display level by two-dimensional FACS analysis. In bi-variate dot plot, each dot represents two fluorescent signals of a separate yeast cell in the display library. The x-axis is a measure of the amount of the fuorescent antigen that is bound (i.e. APC fuorescence) whereas the y-axis gives an indication of the IgG display level (i.e. FITC fuorescence). Yeast cell subpopulation in Q4 gate does not express and display IgGs on the cell surface (Unstained control). Subpopulation in Q1 gate expresses no affinity IgGs on the cell surface. Subpopulation in gate Q2 expresses high affinity IgGs on the cell surface.

Advantages of Full-length IgG Yeast Surface Display

  • In glycoengineered yeast P. pastoris, full-length IgG is displayed as close to native form in molecular structure, biophysical property, and biological function. However, ScFv in phage or yeast display requires format conversion into full-length IgG, which may lose activity.
  • Comparing to the mammalian cell display, full-length IgG yeast surface display has many advantages, such as large library size, easy genetic manipulation, fast generation time, and low cost screening.
  • FACS sorting of full-length IgG in yeast surface display system is based on both IgG antibody affinity and display level. It can eliminate the deviation caused by expression levels and distinguish clones with small affinity. However, phage display screen is usually adversely affected not only by affinity, but also by the expression level.
  • FACS sorting technology can directly select high, medium, and low affinity clones at a time.

IgG-like Bispecific Antibody

A full-length IgG contains two identical heavy chains and light chains. Each heavy chain associates with a light chain through a disulfide bond and non-covalent interactions to form a heterodimer, and both heterodimers associate to forming a complex Y-shaped antibody. Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously bind to two different antigens or epitopes. Bispecific antibodies are emerging as the next wave of antibody-based therapies. Advances in genetic engineering technology has resulted in a range of recombinant bispecific antibody formats. Generally, bispecific antibody can be divided into two major categories, those with an Fc region and those without an Fc region. Nevertheless, most of these formats have been limited by some of their liabilities, such as instability, short half-life, poor manufacturability, and immunogenicity. Heterodimeric IgG-like bispecific antibody, which is based on the heterodimerization of two different IgG molecules, is a promising format because it maintains the overall size and natural structure of human IgG with good stability, half-life, and pharmacokinetics profile. To generate heterodimeric IgG-like bispecific antibody, two challenges have to overcome. One is to facilitate heterodimerization of two distinct heavy chains and prevent homodimerization of two identical heavy chains. The second is to have correct pairing of cognate heavy and light chain. An efficient strategy to overcome these challenges is to use knobs-into-holes (KIH) technology to prioritize heterodimerization of two different heavy chains and combine with a common light chain, based on generally accepted fact that the affinity and specificity of an antibody is predominantly defined by the heavy chain, and it can be maintained when an antibody has a non-cognate light chain.
Genekine applys a versatile approach to directly generate common light chain bispecific antibodies by using yeast surface display of heterodimeric IgG-like bispecific antibody. It can provide service to efficiently isolate common light chains for various combinations of two existing heavy chains.
Copyright © 2022 Network Solutions, LLC. All rights reserved.
Contacts
Mail Address
142 Lowell Road Ste 17-216 Hudson, NH 03051