As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.

CASTing for success: The traditional problem of expanding the range of substrate acceptance of enzymes can be solved by creating focused libraries of mutants resulting from randomization of pairs of properly chosen amino acids around the active site (see example with the lipase from Pseudomonas aeruginosa, amino acid pairs are shown in the same color). CAST=combinatorial active‐site saturation test.

Mit CASTing zum Erfolg: Das bekannte Problem, die Substratakzeptanz von Enzymen zu erweitern, lässt sich dadurch lösen, dass fokussierte Mutantenbibliotheken durch Randomisierung von geeignet gewählten Aminosäurepaaren um das aktive Zentrum erzeugt werden (siehe das Beispiel mit der Lipase aus Pseudomonas aeruginosa; die Aminosäurepaare haben jeweils die gleiche Farbe). CAST=combinatorial active‐site saturation test.

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Recombinant methods work exceedingly well in the directed evolution of an enantioselective enzyme. For the kinetic resolution of the ester rac‐1 by a lipase [Eq. (a)] three steps were applied: 1) Generation of mutants by the error‐prone polymerase chain reaction (epPCR), 2) identification of “hot spots” in the enzyme by epPCR and simplified combinatorial multiple‐cassette mutagenesis (CMCM), and 3) extension of the process of CMCM to cover a defined region of protein sequence space. From lass then 40 000 enzyme varients generated, one was found which catalyzes the reaction with almost 50‐times higher enantioselectivity than the wild‐type enzyme.

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