A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant.
Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.
Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark; Lund University, Department of Chemistry, Division of Biotechnology, Getingevägen 60, SE-221 00 Lund, Sweden.
Gry Ravn
GR
Jespersen
Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark.
Finn
F
Matthiesen
Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark.
Anja Kallesøe
AK
Pedersen
Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark.
Henrik Sune
HS
Andersen
Lund University, Department of Chemistry, Division of Biotechnology, Getingevägen 60, SE-221 00 Lund, Sweden.
Harald
H
Kirsebom
Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark. Electronic address: AeLN@novonordisk.com.
Anders Lærke
AL
Nielsen
D059985:Cryogels / Q000737:chemistry*; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D006207:Half-Life; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D059985:Cryogels; D004220:Disulfides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D019382:Human Growth Hormone; D003545:Cysteine
Human growth hormone; Macroporous monolith; Peptide disulfide; Solid phase reducing agent