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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.

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Related in: MedlinePlus

ELISA analysis of IgG antibody against mPEG-rCU.Serum samples were collected 24 hours before each of four weekly injections of mPEG-rCU. Microtiter plates were coated with 50 µg/ml of mPEG-rCU-1.
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pone-0039659-g008: ELISA analysis of IgG antibody against mPEG-rCU.Serum samples were collected 24 hours before each of four weekly injections of mPEG-rCU. Microtiter plates were coated with 50 µg/ml of mPEG-rCU-1.

Mentions: The serum levels of IgG and IgM antibodies reactive to mPEG-rCU after every repeat injection were measured by ELISA. A screening ELISA performed on 1∶50 and 1∶100 dilutions failed to detect IgM and IgG antibodies against mPEG-rCU-1. The next screening was performed at 1∶10 dilutions of plasma. As shown in Figure 8, no obvious IgG antibodies were observed in either the normal rats or those that displayed RLEA during the four injections, suggesting that the RLEA was not mediated by anti-mPEG-rCU IgG antibodies. However, there were significant differences in the amounts of anti-mPEG-rCU IgM antibodies measured between the normal rats in group one and the rats in group two, which displayed RLEA (Figure 9A). The amount of IgM antibody reactive to mPEG-rCU in all cases reached its maximum level before the second injection (at day 7) and decreased in later injections. Only the rats in group two that displayed RLEA gave a positive response during the injections (where anti-mPEG-rCU IgM was regarded as positive if the optical density at 490 nm wavelength (OD490) was more than 2.1 times that of the same rats before injection). The amounts of IgM antibody in group one rats showed a slight increase but they nevertheless showed a negative response after the first injection. Similarly positive IgM antibodies were observed in one rat of group three, which also showed RLEA (data not shown).


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)

ELISA analysis of IgG antibody against mPEG-rCU.Serum samples were collected 24 hours before each of four weekly injections of mPEG-rCU. Microtiter plates were coated with 50 µg/ml of mPEG-rCU-1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039659-g008: ELISA analysis of IgG antibody against mPEG-rCU.Serum samples were collected 24 hours before each of four weekly injections of mPEG-rCU. Microtiter plates were coated with 50 µg/ml of mPEG-rCU-1.
Mentions: The serum levels of IgG and IgM antibodies reactive to mPEG-rCU after every repeat injection were measured by ELISA. A screening ELISA performed on 1∶50 and 1∶100 dilutions failed to detect IgM and IgG antibodies against mPEG-rCU-1. The next screening was performed at 1∶10 dilutions of plasma. As shown in Figure 8, no obvious IgG antibodies were observed in either the normal rats or those that displayed RLEA during the four injections, suggesting that the RLEA was not mediated by anti-mPEG-rCU IgG antibodies. However, there were significant differences in the amounts of anti-mPEG-rCU IgM antibodies measured between the normal rats in group one and the rats in group two, which displayed RLEA (Figure 9A). The amount of IgM antibody reactive to mPEG-rCU in all cases reached its maximum level before the second injection (at day 7) and decreased in later injections. Only the rats in group two that displayed RLEA gave a positive response during the injections (where anti-mPEG-rCU IgM was regarded as positive if the optical density at 490 nm wavelength (OD490) was more than 2.1 times that of the same rats before injection). The amounts of IgM antibody in group one rats showed a slight increase but they nevertheless showed a negative response after the first injection. Similarly positive IgM antibodies were observed in one rat of group three, which also showed RLEA (data not shown).

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