, Volume 28, Issue 4, pp 393–402 | Cite as

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

  • Anne Pink
  • Aili Kallastu
  • Marina Turkina
  • Marianna Skolnaja
  • Priit Kogerman
  • Taavi Päll
  • Andres Valkna
Original Research Article


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.


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.


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.


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.



Area under curve


Initial plasma protein concentration


CD44 hyaluronan binding domain


Non-hyaluronan binding mutant of CD44HABD–CD44HABDR41AR78SY79S


Total body clearance


Column volumes


Flow through fraction


Gel filtration chromatography


CD44-3MUT GST-fusion protein




Inclusion bodies


Initial dose of injected protein


Ion exchange chromatography


Mouse lung endothelial cells


Mass-spectrometric analysis


Polyethylene glycol


Plasma half-life


Percent of total body weight


Volume of distribution

Supplementary material

40259_2014_89_MOESM1_ESM.pdf (1.2 mb)
Supplementary material 1 (PDF 1257 kb)


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Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Anne Pink
    • 1
    • 2
  • Aili Kallastu
    • 2
  • Marina Turkina
    • 2
  • Marianna Skolnaja
    • 1
    • 2
  • Priit Kogerman
    • 1
    • 2
  • Taavi Päll
    • 1
    • 2
  • Andres Valkna
    • 1
    • 2
  1. 1.Department of Gene TechnologyTallinn University of TechnologyTallinnEstonia
  2. 2.Competence Centre for Cancer ResearchTallinnEstonia

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