The Impact of Methanol Concentration on Recombinant Protein Glycosylation in Pichia pastoris SuperMan5

ABSTRACT The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii ) is a prominent platform for recombinant protein production, offering benefits such as thermo‐ and osmotolerance, high‐density growth, and efficient protein secretion. Its ability to metabolise methanol, an increasingly available carbon source, enhances its cost‐effectiveness and sustainability for industrial use. As a eukaryotic host, P. pastoris ensures proper protein folding and post‐translational modifications (PTMs), including glycosylation, which is essential for correct folding and endoplasmic reticulum (ER) quality control. While ER‐transferred glycans are critical for maturation, additional modification in the Golgi apparatus can yield larger glycans whose impact on stability, solubility, and bioactivity may be either beneficial or undesirable, depending on the application of the heterologous protein. The impact of induction conditions on glycosylation of proteins secreted by P. pastoris SuperMan5 was examined, using the DS‐1 (G2P[4]) and WA (G1P[8]) VP8* rotavirus capsid proteins as a model. An ELISA‐based screening system was employed for clone selection and media optimization, with results showing easy integration into automated workflows. Methanol concentration was found to impact both N ‐ and O‐ linked glycosylation complexity, shaping the glycosylation profile of the target protein as well as the P. pastoris secretome. This study underscores the importance of optimising cultivation conditions to enhance protein yield, refine glycosylation, and minimise impurities, all of which are crucial for large‐scale production and efficient downstream processing. It also suggests a method for easy modulation of glycosylation depending on the target application and the desired level of glycosylation.