title: Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.
abstract: Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 μg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.
authors.affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA.
authors.forename: Jong Youn
authors.initials: JY
authors.lastname: Baik
authors.affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA.
authors.forename: Hussain
authors.initials: H
authors.lastname: Dahodwala
authors.affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA.
authors.forename: Eziafa
authors.initials: E
authors.lastname: Oduah
authors.affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA.
authors.forename: Lee
authors.initials: L
authors.lastname: Talman
authors.affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA.|Albany College of Pharmacy and Health Sciences, Albany, NY, USA.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Gemmill
authors.affiliation: Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Leyla
authors.initials: L
authors.lastname: Gasimli
authors.affiliation: Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Payel
authors.initials: P
authors.lastname: Datta
authors.affiliation: Department of Chemistry and Chemical Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.affiliation: Department of Chemistry and Chemical Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Guoyun
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Chemical Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.|Department of Chemistry and Chemical Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.|Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: Thermo Fisher Scientific Inc., Waltham, MA, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Campbell
authors.affiliation: Thermo Fisher Scientific Inc., Waltham, MA, USA.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Gorfien
authors.affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA. ssharfstein@sunycnse.com.
authors.forename: Susan T
authors.initials: ST
authors.lastname: Sharfstein
mesh_terms: D000818:Animals; D000925:Anticoagulants; D019149:Bioreactors; D053898:Biosynthetic Pathways; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006493:Heparin / Q000096:biosynthesis* / Q000378:metabolism; D060847:Metabolic Engineering
chemical_list: D000925:Anticoagulants; D006493:Heparin
keywords: CHO cells; Disaccharide analysis; Fed-batch cultures; Glycosaminoglycans; Metabolic engineering