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

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. Themajority of theN-glycans of transgenic pigderived 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 detectableNeu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues.Additionally,wewere unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the publishedN-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

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

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. Themajority of theN-glycans of transgenic pigderived 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 detectableNeu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues.Additionally,wewere unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the publishedN-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

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

Analysis of the asparagine-linked oligosaccharides of recombinant human glycoproteins produced in the porcine mammary gland

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein, such as enzyme activity, protein stability, pharmacokinetics, and immunogenicity. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) and Protein C (tg-PC) produced in the transgenic pig mammary gland were determined. It has been found that the majority of N-glycans of tg-FIX and tg-PC are complex bi- and tri-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX and tg-PC produced in the porcine mammary epithelial cells differed with respect to N-glycans from endogenous glycoproteins produced in porcine thyroid, and B-cells. Tg-FIX and tg-PC contain no Neu5Gc moiety that is commonly found in porcine glycoproteins that are produced in porcine thyroid and B-cells. Additionally, tg-FIX and tg-PC do not contain any glycans that has a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. N-glycan structures of tg-FIX and tg-PC are also compared to the published N-glycan structures of recombinant human glycoproteins produced in different transgenic animals. While tg-FIX and tg-PC contain only complex structures, the transgenic animal derived antithrombin III, C1 inhibitor, and lactoferrin have both high mannose and complex structures. In addition, terminal Neu5Gc and Neu5Ac moieties are found in the transgenic animal derived antithrombin III, but only Neu5Ac in the other proteins. Collectively, these data indicate that there may be significant species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. We also have found that N-glycan profiles of tg-FIX are consistent during lactation with respect to the overall distribution of sialylated vs. neutral oligosaccharides. The results show that the porcine mammary gland can be a viable candidate bioreactor for production of recombinant human glycoproteins that require complex, sialylated N-linked glycans

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

Human protein C (hPC) is glycosylated at three Asn-X-Ser/Thr and one atypical Asn-X-Cys sequons. We have characterized the micro- and macro-heterogeneity of plasma-derived hPC and compared the glycosylation features with recombinant protein C (tg-PC) produced in a transgenic pig bioreactor from two animals having approximately tenfold different expression levels. The N-glycans of hPC are complex di- and tri-sialylated structures, and we measured 78% site occupancy at Asn-329 (the Asn-X-Cys sequon). The N-glycans of tg-PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn-X-Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. A distinct bias for particular structures was present at each of the four glycosylation sites for both hPC and tg-PC. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn-329 site. The N-glycan structures found for tg-PC are very similar to those reported for a recombinant Factor IX produced in transgenic pig milk, and similar to the endogenous milk protein lactoferrin, which may indicate that N-glycan processing in the porcine mammary epithelial cells is more uniform than in other tissues

Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography–tandem mass spectrometry

We demonstrate use of restricted access media with reversed phase functionality (RAM-RP) for analysis of low molecular weight proteins and peptides in mouse serum (75ml) using a custom designed modular automated processing system (MAPS). RAM-RP fractionation with simultaneous removal of high molecular weight and high abundance proteins is integrated with a follow-on buffer exchange module (BE) to ensure compatibility with subsequent processing steps (trypsin digestion and intact peptide separation prior to mass spectrometric analysis). The high sample capacity afforded by chromatographic methods generates enough sample to achieve comprehensive serum peptidome identification (357 proteins) through tandem mass spectrometric analysis of both intact and digested peptides. Sample losses during transfer between modules are minimized through precise fluidic control; no clogging occurred over several months of serum processing in our low back pressure system. Computer controlled operation of both modules and thorough optimization yield excellent run-to-run reproducibility and protein/peptide overlap in analytical repeats. The robustness of our results demonstrate that the RAM-RP-BE workflow executed on our MAPS platform shows tremendous potential for high throughput peptidome processing, particularly with regard to direct analysis of small-volume serum samples

Identification of alpha-Gal and non-Gal Epitopes in Pig Corneal Endothelial Cells and Keratocytes by Using Mass Spectrometry

Purpose: To investigate the expression of alpha-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry. Methods: The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immuno chemical staining was added to confirm the non-Gal antigen expression in pig corneal cells. Results: Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of alpha-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed. Conclusions: As well as alpha-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the alpha-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.Gil GC, 2008, ANAL BIOCHEM, V379, P45, DOI 10.1016/j.ab.2008.04.039Kim YG, 2008, PROTEOMICS, V8, P2596, DOI 10.1002/pmic.200700972Lee HI, 2007, XENOTRANSPLANTATION, V14, P612, DOI 10.1111/j.1399-3089.2007.00433.xAlvarez-Manilla G, 2007, GLYCOBIOLOGY, V17, P677, DOI 10.1093/glycob/cwm033ZHIQIANG P, 2007, XENOTRANSPLANTATION, V14, P603Hering BJ, 2006, NAT MED, V12, P301, DOI 10.1038/nm1369Kim YG, 2006, PROTEOMICS, V6, P1133Chen G, 2005, NAT MED, V11, P1295, DOI 10.1038/nm1330Lee CS, 2005, BIOTECHNOL BIOPROC E, V10, P212Kuwaki K, 2005, NAT MED, V11, P29, DOI 10.1038/nm1171Yamada K, 2005, NAT MED, V11, P32, DOI 10.1038/nm1172Kang P, 2005, RAPID COMMUN MASS SP, V19, P3421, DOI 10.1002/rcm.2210Kolber-Simonds D, 2004, P NATL ACAD SCI USA, V101, P7335Komoda H, 2004, XENOTRANSPLANTATION, V11, P237, DOI 10.1111/j.1399-3089.2004.00121.xMiwa Y, 2004, XENOTRANSPLANTATION, V11, P247, DOI 10.1111/j.1399-3089.2004.00126.xMorelle W, 2004, RAPID COMMUN MASS SP, V18, P2451, DOI 10.1002/rcm.1640Ciucanu I, 2003, J AM CHEM SOC, V125, P16213, DOI 10.1021/ja035660tAmano S, 2003, CURR EYE RES, V26, P313Phelps CJ, 2003, SCIENCE, V299, P411, DOI 10.1126/science.1078942Banerjee S, 2003, EXPERT OPIN INV DRUG, V12, P29Zhu A, 2002, XENOTRANSPLANTATION, V9, P376Gouw JW, 2002, RAPID COMMUN MASS SP, V16, P905, DOI 10.1002/rcm.654Zeng YJ, 2001, J BIOMECH, V34, P533QIAN Y, 2001, EXPERT REV MOL MED, P1Morozumi K, 1999, TRANSPLANT P, V31, P942Rocha G, 1998, CRIT REV IMMUNOL, V18, P305Viseux N, 1997, ANAL CHEM, V69, P3193Inohara H, 1996, CANCER RES, V56, P4530JAGER MJ, 1995, TRANSPL IMMUNOL, V3, P135COOPER DKC, 1994, IMMUNOL REV, V141, P31ORIOL R, 1993, TRANSPLANTATION, V56, P1433GALILI U, 1993, IMMUNOL TODAY, V14, P480COOPER DKC, 1993, TRANSPL IMMUNOL, V1, P198GOOD AH, 1992, TRANSPLANT P, V24, P559CORNIL I, 1990, J CELL BIOL, V111, P773HAZLETT LD, 1989, J HISTOCHEM CYTOCHEM, V37, P1215STREILEIN JW, 1979, NATURE, V282, P326

Qualitative and quantitative comparison of N-glycans between pig endothelial and islet cells by high-performance liquid chromatography and mass spectrometry-based strategy

N-glycan structures released from miniature pig endothelial and islet cells were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) negative ion electrospray ionization (ESI) MS/MS and normal-phase high performance liquid chromatography (NP-HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N-glycans including 129 sialylated and 18 alpha-galactosylated glycans from pig endothelial cells and 80 N-glycans including 41 sialylated and one alpha-galactosylated glycans from pig islet cells. The quantity of the alpha-galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc-terminated N-glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using alpha 1,3-galactosyltransferase gene-knockout (GaIT-KO) pig. Copyright (C) 2009 John Wiley & Sons, Ltd.Kim YG, 2008, PROTEOMICS, V8, P2596, DOI 10.1002/pmic.200700972Harvey DJ, 2008, ANAL BIOCHEM, V376, P44, DOI 10.1016/j.ab.2008.01.025Parry S, 2007, GLYCOBIOLOGY, V17, P646, DOI 10.1093/glycob/cwm024Kim JH, 2007, XENOTRANSPLANTATION, V14, P60, DOI 10.1111/j.1399-3089.2006.00364.xHering BJ, 2006, NAT MED, V12, P301, DOI 10.1038/nm1369Kim YG, 2006, PROTEOMICS, V6, P1133Rood PPM, 2006, CELL TRANSPLANT, V15, P89Jang-Lee J, 2006, METHOD ENZYMOL, V415, P59, DOI 10.1016/S0076-6879(06)15005-3Chen G, 2005, NAT MED, V11, P1295, DOI 10.1038/nm1330Kim D, 2005, CELL BIOL INT, V29, P638, DOI 10.1016/j.cellbi.2005.03.016Harvey DJ, 2005, J AM SOC MASS SPECTR, V16, P631, DOI 10.1016/j.jasms.2005.01.005Harvey DJ, 2005, J AM SOC MASS SPECTR, V16, P647, DOI 10.1016/j.jasms.2005.01.006Kuwaki K, 2005, NAT MED, V11, P29, DOI 10.1038/nm1171Kolber-Simonds D, 2004, P NATL ACAD SCI USA, V101, P7335Komoda H, 2004, XENOTRANSPLANTATION, V11, P237, DOI 10.1111/j.1399-3089.2004.00121.xCiucanu I, 2003, J AM CHEM SOC, V125, P16213, DOI 10.1021/ja035660tPhelps CJ, 2003, SCIENCE, V299, P411, DOI 10.1126/science.1078942Teranishi K, 2002, TRANSPLANTATION, V73, P129Buhler L, 2000, TRANSPLANTATION, V69, P2296Morozumi K, 1999, TRANSPLANT P, V31, P942Heald KA, 1999, J MOL MED-JMM, V77, P169Anumula KR, 1998, GLYCOBIOLOGY, V8, P685van der Burg MPM, 1998, TRANSPLANT P, V30, P362Dwek RA, 1996, CHEM REV, V96, P683COZZI E, 1995, NAT MED, V1, P964BORNSEN KO, 1995, RAPID COMMUN MASS SP, V9, P1031COOPER DKC, 1994, IMMUNOL REV, V141, P31MCKENZIE IFC, 1994, TRANSPL IMMUNOL, V2, P81ORIOL R, 1993, TRANSPLANTATION, V56, P1433VARKI A, 1993, GLYCOBIOLOGY, V3, P97GOOD AH, 1992, TRANSPLANT P, V24, P559GALILI U, 1984, J EXP MED, V160, P1519SETCAVAGE TM, 1976, INFECT IMMUN, V13, P600