Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response.
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.
Choi
Byung-Kwon
BK
GlycoFi, Inc., Lebanon, NH, USA.
Actor
Jeffrey K
JK
Rios
Sandra
S
d'Anjou
Marc
M
Stadheim
Terrance A
TA
Warburton
Shannon
S
Giaccone
Erin
E
Cukan
Michael
M
Li
Huijuan
H
Kull
Angela
A
Sharkey
Nathan
N
Gollnick
Paul
P
Kocieba
Maja
M
Artym
Jolanta
J
Zimecki
Michal
M
Kruzel
Marian L
ML
Wildt
Stefan
S
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
D006023:Glycoproteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D007781:Lactoferrin