Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

Abstract

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

“…An additional 14% of the glycans are triantennary with a mixture of α2,3/6-linked terminal sialic acid residues (21). Factor IX is a coagulation factor used to treat hemophilia B, a congenital disease caused by a factor IX deficiency (22,23). The protein contains two N-linked glycans, of which approximately 65% are tetraanntennary and 20% are triantennary glycans (24), with a mixture of α2,3/6-linked terminal sialic acid residues.…”

N‐glycosylation microheterogeneity and site occupancy of an Asn‐X‐Cys sequon in plasma‐derived and recombinant protein C

“…Additionally, we investigate if there is evidence for rate limitations in the N-glycan attachment and processing in the porcine mammary gland by comparing two transgenic lineages having up to ten-fold different expression levels. We also compare N-glycosylation of tg-PC with another recombinant vitamin K-dependent glycoprotein that has been produced in the transgenic pig mammary gland, Factor IX (tg-FIX) [23], and discuss the features of N-glycan processing of recombinant proteins that are evident in the transgenic pig bioreactor.…”

Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics - recent achievements

“…Their synthesis in the secretory epithelial cells of udder lactogenic vesicles can be induced through transfection of nuclear donor somatic cells with structure transgenes under the control of promoters, including the regulatory/intron sequences of either genes encoding protein variants/isoforms of casein (α S1 -, α S2 -, β-and κ-casein) or the genes encoding milk whey proteins (i.e., β-lactoglobulin, α-lactalbumin, whey acidic protein/WAP). Such biopharmaceuticals as human recombinant α-1-antitrypsin (McCreath et al 2000), antithrombin III-α (Baguisi et al 1999), human coagulation/clotting factors VIII and IX (Schnieke et al 1997, Chen et al 2002a, Lee et al 2003, Van Cott et al 2004, Gil et al 2008) and erythropoietin (Cheng et al 2002) have so far been excreted in the milk of transgenic cloned sheep, goats and pigs (Table 1). The transgenic approach can also provide a practical means of humanizing the milk of cloned ruminants via onset of expression in their mammary glands of foreign (xenogeneic) genes encoding the bionutriceuticals/bionutraceuticals, which include different recombinant human milk proteins.…”