Purification, Characterization and Plasma Half-Life of PEGylated Soluble Recombinant Non-HA-Binding CD44

Abstract

Background and Objectives

The aim of this study was to increase the serum half-life of recombinant CD44 hyaluronan (HA) binding domain by PEGylation. We have previously found that recombinant soluble CD44 HA binding domain (CD44HABD) and its non-HA-binding triple mutant CD44HABDR41AY78SY79S (CD44-3MUT) inhibits angiogenesis and subcutaneous tumor growth. However, this ~12 kDa recombinant protein displays a high serum clearance rate.

Methods

Here, we report the purification of monomeric CD44-3MUT from urea solubilized inclusion bodies using weak anion exchange chromatography and gel filtration. To increase the serum residence time of CD44-3MUT we PEGylated the resulting protein using 20 kDa methoxy-PEG-propionaldehyde.

Results

PEGylation of CD44-3MUT prolonged its in vivo serum half-life about 70-fold from 0.03 to 1.8 hours. Along with extended plasma residence time, PEGylation also increased the systemic exposure. By cell impedance assay we confirmed that PEGylated CD44-3MUT maintained its in vitro function. The results from the impedance assay additionally demonstrate that the CD44-3MUT effect on endothelial cells is mediated by vimentin.

Conclusions

In summary, we have developed a purification protocol for large-scale production of CD44-3MUT and generated a PEGylated form of CD44-3MUT. HA binding domain of CD44(CD44HABD) and its modified non-HA binding form (CD44-3MUT) inhibit angiogenesis and tumor growth in vivo without disturbing HA-binding functions. CD44-3MUT has been PEGylated for use as a new type of anti-angiogenic human drug. PEGylation of CD44-3MUT improved pharmacokinetic properties but retains its functional activity.

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Abbreviations

AUC:

Area under curve

C0 :

Initial plasma protein concentration

CD44HABD:

CD44 hyaluronan binding domain

CD44-3MUT:

Non-hyaluronan binding mutant of CD44HABD–CD44HABDR41AR78SY79S

CL:

Total body clearance

CV:

Column volumes

FT:

Flow through fraction

GF:

Gel filtration chromatography

GST-CD44-3MUT:

CD44-3MUT GST-fusion protein

HA:

Hyaluronan

IB:

Inclusion bodies

ID:

Initial dose of injected protein

IEC:

Ion exchange chromatography

MLEC:

Mouse lung endothelial cells

MS:

Mass-spectrometric analysis

PEG:

Polyethylene glycol

T 1/2 :

Plasma half-life

%TBW:

Percent of total body weight

V d :

Volume of distribution

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Acknowledgments and Disclosures

We are grateful to Anne Meikas for protein expression. This research was supported by the European Regional Development Fund via Enterprise Estonia grants (EU28138/EU28658, EU30013) to Competence Centre for Cancer Research and by Estonian Science Fund Grant 8116 to Priit Kogerman. The authors have no conflicts of interest that are directly relevant to the content of this article.

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Correspondence to Andres Valkna.

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Pink, A., Kallastu, A., Turkina, M. et al. Purification, Characterization and Plasma Half-Life of PEGylated Soluble Recombinant Non-HA-Binding CD44. BioDrugs 28, 393–402 (2014). https://doi.org/10.1007/s40259-014-0089-y

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Keywords

  • Nonreducing Condition
  • Disulphide Bridge
  • Autoinduction Medium
  • Subcutaneous Tumor Growth
  • Recombinant CD44