Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells.
CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.
Borys
Michael C
MC
Process Sciences, Worldwide Medicines Group, Bristol-Myers Squibb, Syracuse, NY 13221, USA.
Dalal
Nimish G
NG
Abu-Absi
Nicholas R
NR
Khattak
Sarwat F
SF
Jing
Ying
Y
Xing
Zizhuo
Z
Li
Zheng Jian
ZJ
D000818:Animals; D019149:Bioreactors; D002087:Butyrates / Q000737:chemistry; D016466:CHO Cells / Q000378:metabolism*; D002245:Carbon Dioxide / Q000737:chemistry; D002254:Carbonates / Q000737:chemistry; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D009438:Neuraminic Acids / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012972:Sodium Hydroxide / Q000737:chemistry; D013696:Temperature
D002087:Butyrates; D002254:Carbonates; D003470:Culture Media; D006023:Glycoproteins; D009438:Neuraminic Acids; D011993:Recombinant Fusion Proteins; C032592:N-glycolylneuraminic acid; D002245:Carbon Dioxide; C005686:sodium carbonate; D012972:Sodium Hydroxide; D019158:N-Acetylneuraminic Acid