Limits...
Impact of large aggregated uricases and PEG diol on accelerated blood clearance of PEGylated canine uricase.

Zhang C, Fan K, Ma X, Wei D - PLoS ONE (2012)

Bottom Line: In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase.Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC.Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.

ABSTRACT

Background: Uricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated.

Methods and findings: Recombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC.

Conclusions: The size of conjugates is important for triggering such phenomena and we speculate that 40-60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

Show MeSH

Related in: MedlinePlus

Representative chromatogram of mPEG-rCU purificationThe three different chromatograms correspond to mPEG-rCU protein (A), unconjugated mPEG (B) and N-hydroxysuccinimide acid (C), respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383732&req=5

pone-0039659-g003: Representative chromatogram of mPEG-rCU purificationThe three different chromatograms correspond to mPEG-rCU protein (A), unconjugated mPEG (B) and N-hydroxysuccinimide acid (C), respectively.

Mentions: Based on the differences in their electostatic charge, anion-exchange chromatography was used to remove the Di-acid PEG from the mono-acid mPEG. Di-acid PEG bound more tightly to the anion-exchange column than the mono-acid form. As shown in Figure 2, the content of Di-acid PEG in purified mPEG-PA was lower than 0.2%, as measured by SE-HPLC, whereas the amount in the initial, unfractionated 5 kDa mPEG-PA was about 2.7%. Both purified and unfractionated mPEG-PA were then converted to 5 kDa mPEG-SPA. Following modification, tetrameric and aggregated rCU were separately modified with the purified mPEG-SPA. A further sample of tetrameric rCU was also modified by unfractionated mPEG-SPA, resulting in three types of mPEG-rCU, as shown in Table 1. The three conjugates were further purified by size exclusion chromatography (Figure 3). By-products of PEGylation reactions (e.g. unconjugated PEG and N-hydroxysuccinimide acid) were efficiently removed (for detailed information see material S1). RP-HPLC and SE-HPLC showed that only a single peak existed after the above purification.


Impact of large aggregated uricases and PEG diol on accelerated blood clearance of PEGylated canine uricase.

Zhang C, Fan K, Ma X, Wei D - PLoS ONE (2012)

Representative chromatogram of mPEG-rCU purificationThe three different chromatograms correspond to mPEG-rCU protein (A), unconjugated mPEG (B) and N-hydroxysuccinimide acid (C), respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383732&req=5

pone-0039659-g003: Representative chromatogram of mPEG-rCU purificationThe three different chromatograms correspond to mPEG-rCU protein (A), unconjugated mPEG (B) and N-hydroxysuccinimide acid (C), respectively.
Mentions: Based on the differences in their electostatic charge, anion-exchange chromatography was used to remove the Di-acid PEG from the mono-acid mPEG. Di-acid PEG bound more tightly to the anion-exchange column than the mono-acid form. As shown in Figure 2, the content of Di-acid PEG in purified mPEG-PA was lower than 0.2%, as measured by SE-HPLC, whereas the amount in the initial, unfractionated 5 kDa mPEG-PA was about 2.7%. Both purified and unfractionated mPEG-PA were then converted to 5 kDa mPEG-SPA. Following modification, tetrameric and aggregated rCU were separately modified with the purified mPEG-SPA. A further sample of tetrameric rCU was also modified by unfractionated mPEG-SPA, resulting in three types of mPEG-rCU, as shown in Table 1. The three conjugates were further purified by size exclusion chromatography (Figure 3). By-products of PEGylation reactions (e.g. unconjugated PEG and N-hydroxysuccinimide acid) were efficiently removed (for detailed information see material S1). RP-HPLC and SE-HPLC showed that only a single peak existed after the above purification.

Bottom Line: In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase.Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC.Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.

ABSTRACT

Background: Uricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated.

Methods and findings: Recombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC.

Conclusions: The size of conjugates is important for triggering such phenomena and we speculate that 40-60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

Show MeSH
Related in: MedlinePlus