title: Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response.
abstract: 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.
authors: GlycoFi, Inc., Lebanon, NH, USA.
authors: Byung-Kwon
authors: BK
authors: Choi
authors: Jeffrey K
authors: JK
authors: Actor
authors: Sandra
authors: S
authors: Rios
authors: Marc
authors: M
authors: d'Anjou
authors: Terrance A
authors: TA
authors: Stadheim
authors: Shannon
authors: S
authors: Warburton
authors: Erin
authors: E
authors: Giaccone
authors: Michael
authors: M
authors: Cukan
authors: Huijuan
authors: H
authors: Li
authors: Angela
authors: A
authors: Kull
authors: Nathan
authors: N
authors: Sharkey
authors: Paul
authors: P
authors: Gollnick
authors: Maja
authors: M
authors: Kocieba
authors: Jolanta
authors: J
authors: Artym
authors: Michal
authors: M
authors: Zimecki
authors: Marian L
authors: ML
authors: Kruzel
authors: Stefan
authors: S
authors: Wildt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D012756:Sheep; D012794:Sialic Acids / Q000737:chemistry / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D007781:Lactoferrin