Biochemical and Functional Characterization of PEGylated rVWF.

Abstract

Covalent modification of therapeutic proteins by polyethylene glycol derivatives is an established method for improving pharmacokinetic properties of therapeutic proteins. Highly purified rVWF expressed in CHO cells, was chemically modified via PEGylation of lysine residues at mild alkaline pH with PEG succinimidyl succinate (linear 5 kDa PEG). Increasing the amount of PEG used in the coupling procedure, the molecular size of VWF increased, as demonstrated in SDS-PAGE and by agarose gel electrophoreses indicating an increase in size of the bands resembling the VWF multimers. PEGylation of rVWF reduced platelet-aggregating and collagen-binding functions by 60% (VWF:RCo activity) and 40% (VWF:CB activity). While FVIII-binding capacity, measured by a FVIII binding ELISA, was reduced and reduction correlated with the amount of PEG bound to VWF, there was almost no effect on FVIII binding affinity which remained in the same order of magnitude as measured with non-PEGylated VWF. Similar results were obtained when rVWF was PEGylated via carbohydrate moieties after oxidation and subsequent derivatization with monomethoxy-PEG hydrazide. PEGylated rVWF was applied to VWF-deficient mice at a dose of 40 VWF:Ag U/kg and plasma levels were monitored for up to 24 hours. As a control, non-modified rVWF was applied to the animals. PEGylated VWF had substantially prolonged survival in the circulation compared with non-modified rVWF with an increase of the AUC by a factor of >10. VWD mice substituted with human VWF show a secondary rise in FVIII bringing them into the FVIII levels measured in C57Bl/6 control mice. This secondary rise was sustained after treatment with PEGylated rVWF where FVIII levels above the starting level were measurable even 48 hours after injection while in the control group base line FVIII levels were reached already after 24 hours. rVWF is the largest protein ever PEGylated and PEGylation results in prolonged survival in the circulation while maintaining FVIII stabilizing functions of the VWF molecule in vivo.

MALDI linear TOF mass spectrometry of PEGylated (glyco)proteins

“…The rvWF dimer was purified from an rvWF preparation by using a combination of size‐exclusion and ion‐exchange chromatography. The rvWF preparation was PEGylated with high‐quality pharmaceutical 5‐kDa linear PEG and the dimer was isolated by size‐exclusion chromatography27 containing no starting materials. Formic acid (FA) and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany), and sinapinic acid (SA) was from Fluka (Buchs, Switzerland) in the highest MS quality available.…”

The biological efficacy profile of BAX 855, a PEGylated recombinant factor VIII molecule

“…The molecular size of FVIII is 280 kDa and is too large to be cleared by the kidney, thereby indicating an alternate mechanism of clearance. PEGylation may make FVIII less susceptible to proteolytic cleavage and degradation and/or alter its surface charge properties to interfere with low-density lipoprotein receptor (LRP)-mediated clearance processes [11][12][13].…”

“…In laboratory experiments, the covalent binding of PEG by amine coupling preferentially to lysine residues of full-length recombinant FVIII (rFVIII) slowed thrombin inactivation, but did not alter the biochemical or functional properties of the molecule [11].…”

“…The molecular size of FVIII is 280 kDa and is too large to be cleared by the kidney, thereby indicating an alternate mechanism of clearance. PEGylation may make FVIII less susceptible to proteolytic cleavage and degradation and/or alter its surface charge properties to interfere with low‐density lipoprotein receptor (LRP)‐mediated clearance processes . In laboratory experiments, the covalent binding of PEG by amine coupling preferentially to lysine residues of full‐length recombinant FVIII (rFVIII) slowed thrombin inactivation, but did not alter the biochemical or functional properties of the molecule .…”

Investigating the clearance of VWF A‐domains using site‐directed PEGylation and novel N‐linked glycosylation

“…Given the large number of free cysteines in full‐length VWF, this targeted cysteine‐mediated PEGylation approach is unlikely to be successful in developing an EHL VWF therapy. Moreover, previous work by Turecek et al 30 demonstrated that a lysine‐mediated PEGylation of full length VWF significantly prolonged survival compared to non‐modified VWF. However this non‐targeted and uncontrolled PEGylation approach also resulted in a significant reduction in VWF activity.…”