Traditional production of therapeutic
glycoproteins relies on mammalian cell culture technology.
Glycoproteins produced by mammalian cells invariably display N-
glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic
proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked
glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked
glycans and the function of
glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human
lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-
glycan structures on in vitro secondary humoral immune responses.
Lactoferrin is considered an important first line defense
protein involved in protection against various microbial
infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical
protein and
glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the
biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (
sialic) acid as a terminal
sugar in propagation of proper immune responses.