Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.
This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product.
NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland. Electronic address: radka.fahey@nibrt.ie.
Radka
R
Saldova
Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland; Microbiology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland. Electronic address: Michelle.Kilcoyne@nuigalway.ie.
Michelle
M
Kilcoyne
NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland. Electronic address: henning.stoeckmann@cantab.net.
Henning
H
Stöckmann
NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland. Electronic address: silvia.millanmartin@nibrt.ie.
Silvia
S
Millán Martín
Bristol-Myers Squibb, BMS, Biologics Development, 38 Jackson Road, Devens, MA 01434, USA. Electronic address: amanda.lewis@bms.com.
Amanda M
AM
Lewis
Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland. Electronic address: cath.tuite@gmail.com.
Catherine M E
CM
Tuite
Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland; Regenerative Medicine Institute, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland. Electronic address: jared.gerlach@nuigalway.ie.
Jared Q
JQ
Gerlach
Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland. Electronic address: marie.leberre@nuigalway.ie.
Marie
M
Le Berre
Bristol-Myers Squibb, BMS, Biologics Development, 38 Jackson Road, Devens, MA 01434, USA. Electronic address: michael.borys@bms.com.
Michael C
MC
Borys
Bristol-Myers Squibb, BMS, Biologics Development, 38 Jackson Road, Devens, MA 01434, USA. Electronic address: zhengjian.li@bms.com.
Zheng Jian
ZJ
Li
Bristol-Myers Squibb, BMS, Biologics Development, 38 Jackson Road, Devens, MA 01434, USA. Electronic address: nicholas.abuabsi@abbvie.com.
Nicholas R
NR
Abu-Absi
Bristol-Myers Squibb, BMS, Biologics Development, 38 Jackson Road, Devens, MA 01434, USA. Electronic address: leistk222@gmail.com.
Kirk
K
Leister
Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland. Electronic address: lokesh.joshi@nuigalway.ie.
Lokesh
L
Joshi
NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland. Electronic address: pauline.rudd@nibrt.ie.
Pauline M
PM
Rudd
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics*; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003412:Cricetulus; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D040081:Protein Array Analysis / Q000295:instrumentation*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry* / Q000302:isolation & purification
D000906:Antibodies; D037102:Lectins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D012794:Sialic Acids
Bioprocess engineering; CHO cells; Glycosylation; HILIC; Lectin microarrays; UPLC