PEDS Advance Access published online on April 23, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm015
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Alternative antibody Fab' fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering
1 UCB-Celltech, 216 Bath Road, Slough, SL1 4EN, UK
2 To whom correspondence should be addressed. E-mail: david.humphreys{at}celltech.ucb-group.com
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.
Keywords: disulphide engineering/Fab'/PEGylation/periplasm
Received October 16, 2006; revised January 9, 2007; accepted February 14, 2007.
Abbreviations: BSA, bovine serum albumin; ß-ME, ß-mercaptoethanol; ß-MA, ß-mercaptoethylamine; DTT, dithiothreitol; GuHCL, guanidine hydrochloride; GSH, glutathione (reduced); TBP, tris(butyl) phosphine; TCEP, tris(2-carboxyethyl) phosphine; Fab', Fragment antigen binding; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; AUC, area under the curve; PEG, polyethylene glycol; PK, pharmacokinetics; LC, light chain; HC, heavy chain; IPTG, isopropyl ß-D-thiogalactopyranoside; EDTA, ethylenediaminetetraacetic.