title: Alternative antibody Fab' fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering.
abstract: Antigen-binding fragments (Fab') of antibodies can be site specifically PEGylated at thiols using cysteine reactive PEG-maleimide conjugates. For therapeutic Fab'-PEG, conjugation with 40 kDa of PEG at a single hinge cysteine has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. Previous methods have activated the hinge cysteine using mildly reducing conditions in order to retain an intact interchain disulphide. We demonstrate that the final Fab-PEG product does not need to retain the interchain disulphide and also therefore that strongly reducing conditions can be used. This alternative approach results in PEGylation efficiencies of 88 and 94% for human and murine Fab, respectively. It also enables accurate and efficient site-specific multi-PEGylation. The use of the non-thiol reductant tris(2-carboxyethyl) phosphine combined with protein engineering enables us to demonstrate the mono-, di- and tri-PEGylation of Fab fragments with a range of PEG size. We present evidence that PEGylated and unPEGylated Fab' molecules that lack an interchain disulphide bond retain very high levels of chemical and thermal stability and normal performance in PK and efficacy models.
authors: UCB-Celltech, 216 Bath Road, Slough, SL1 4EN, UK. david.humphreys@celltech.ucb-group.com
authors: David P
authors: DP
authors: Humphreys
authors: Sam P
authors: SP
authors: Heywood
authors: Alistair
authors: A
authors: Henry
authors: Layla
authors: L
authors: Ait-Lhadj
authors: Pari
authors: P
authors: Antoniw
authors: Roger
authors: R
authors: Palframan
authors: Kevin J
authors: KJ
authors: Greenslade
authors: Bruce
authors: B
authors: Carrington
authors: Dominc G
authors: DG
authors: Reeks
authors: Leigh C
authors: LC
authors: Bowering
authors: Shauna
authors: S
authors: West
authors: Helen A
authors: HA
authors: Brand
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019163:Reducing Agents / Q000737:chemistry*
chemical_list: D003545:Cysteine; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D010720:Phosphines; D011092:Polyethylene Glycols; D019163:Reducing Agents; C080938:tris(2-carboxyethyl)phosphine