title: Chemically defined media modifications to lower tryptophan oxidation of biopharmaceuticals.
abstract: Oxidation of biopharmaceuticals is a major product quality issue with potential impacts on activity and immunogenicity. At Eli Lilly and Company, high tryptophan oxidation was observed for two biopharmaceuticals in development produced in Chinese hamster ovary cells. A switch from historical hydrolysate-containing media to chemically defined media with a reformulated basal powder was thought to be responsible, so mitigation efforts focused on media modification. Shake flask studies identified that increasing tryptophan, copper, and manganese and decreasing cysteine concentrations were individual approaches to lower tryptophan oxidation. When amino acid and metal changes were combined, the modified formulation had a synergistic impact that led to substantially less tryptophan oxidation for both biopharmaceuticals. Similar results were achieved in shake flasks and benchtop bioreactors, demonstrating the potential to implement these modifications at manufacturing scale. The modified formulation did not negatively impact cell growth and viability, product titer, purity, charge variants, or glycan profile. A potential mechanism of action is presented for each amino acid or metal factor based on its role in oxidation chemistry. This work served not only to mitigate the tryptophan oxidation issue in two Lilly biopharmaceuticals in development, but also to increase our knowledge and appreciation for the impact of media components on product quality.
authors.affiliation: Bioproduct Research and Development, Eli Lilly and Company, Indianapolis, IN, 46285.
authors.forename: Laurie B
authors.initials: LB
authors.lastname: Hazeltine
authors.affiliation: Bioproduct Research and Development, Eli Lilly and Company, Indianapolis, IN, 46285.
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Knueven
authors.affiliation: Bioproduct Research and Development, Eli Lilly and Company, Indianapolis, IN, 46285.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Bioproduct Research and Development, Eli Lilly and Company, Indianapolis, IN, 46285.
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Lian
authors.affiliation: Bioproduct Research and Development, Eli Lilly and Company, Indianapolis, IN, 46285.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Olson
authors.affiliation: Bioproduct Research and Development, Eli Lilly and Company, Indianapolis, IN, 46285.
authors.forename: Anli
authors.initials: A
authors.lastname: Ouyang
mesh_terms: D000818:Animals; D001702:Biopharmaceutics; D019149:Bioreactors; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry*; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D003470:Culture Media; D014364:Tryptophan
keywords: Chinese hamster ovary; cell culture; chemically defined media; tryptophan oxidation