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Elimination of β-mannose glycan structures in Pichia pastoris

Daniel Hopkins1, Sujatha Gomathinayagam, Alissa M Rittenhour

  • 1GlycoFi Inc., A wholly-Owned Subsidiary of Merck & Co. Inc., 21 Lafayette street, Suite 200, Lebanon, NH 03766, USA.

Glycobiology
|August 16, 2011
PubMed

Insights

Eliminating beta-mannose (β-Man) glycans in Pichia pastoris is crucial for therapeutic protein production. A quadruple knockout strain (bmt2,4,1,3) successfully removed these immunogenic glycans, enhancing the Pichia platform for glycoprotein manufacturing.

Area of Science:

  • Biotechnology
  • Glycobiology
  • Protein Engineering

Background:

  • Pichia pastoris is widely used for therapeutic protein production.
  • Fungal-like glycans, particularly those with beta-mannose (β-Man) linkages, can cause immune responses and reduce therapeutic efficacy.
  • The beta-mannosyl transferase (BMT) gene family in P. pastoris, especially Bmt2p, is responsible for most β-Man linkages.

Purpose of the Study:

  • To eliminate immunogenic β-Man glycans from therapeutic glycoproteins produced in Pichia pastoris.
  • To investigate the roles of the BMT gene family in glycan formation.
  • To develop a glycoengineered P. pastoris strain suitable for producing safer therapeutic proteins.

Main Methods:

  • Glycoengineering of Pichia pastoris by systematically knocking out genes from the BMT family.
  • Expression of recombinant human erythropoietin (rhEPO) in engineered strains.
  • Analysis of glycan profiles using techniques like PNGase F treatment and mass spectrometry.
  • Assessment of antigenicity associated with glycan structures.

Main Results:

  • A developmental strain lacking BMT2 showed cross-reactivity with an antibody, indicating glycan-associated antigenicity.
  • Complete elimination of α-mannosidase resistant glycoforms was achieved in a quadruple knockout strain (Δbmt2,4,1,3).
  • The additive elimination of Bmt2p, Bmt3p, and Bmt1p activities was necessary to abolish β-Man glycans and associated antigenicity.

Conclusions:

  • The quadruple knockout strain (Δbmt2,4,1,3) successfully removes β-Man containing glycoforms from therapeutic glycoproteins.
  • Genetic elimination of specific BMT family members is essential for producing non-immunogenic glycoproteins in P. pastoris.
  • This glycoengineering approach significantly advances the Pichia pastoris platform for safe and effective therapeutic glycoprotein production.

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