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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (12): 59-66    DOI: 10.13523/j.cb.20171209
Orginal Article     
Comparison of the Biological Activity of mPEG20k Modified and Transferrin Coupled Ciliary Neurotrophic Factor
Sheng-nan CHI1,2,Zeng-lan LI1,2,Chun ZHANG2(),Shuang YIN2,Cui FENG2,Qi WANG1,2,Yong-dong LIU1,2(),Zhi-guo SU1,2
1 University of Chinese Academy of Sciences, Beijing 100049, China
2 State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing　100190,China
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Abstract  

In order to prolong the blood half-life of the recombinant ciliary neurotrophic factor in vivo, CNTF was previously formulated by mPEG20k modification and transferrin coupling based on the natural free thiol of Cys17 residue. These two conjugates of PEG20k-CNTF and Tf-PEG5k-CNTF were charicterized and compared using conventional analysis. The HP-SEC and DLS results showed that the conjugates exhibited a similar apparent molecular size. TF-1(CN5α-1) cell survival test showed significant decreased activity of 50.6% (mPEG20k-CNTF) and 65.8% (Tf-PEG5k-CNTF) compared with the unmodified rhCNTF, respectively. Antibody binding affinity revealed that PEG20k-CNTF decreased to 3.8% of the rhCNTF, and Tf-PEG5k-CNTF retained 89.9% of the rhCNTF. Pharmacokinetics study showed that the blood half-lives of PEG20k-CNTF and Tf-PEG5k-CNTF conjugates in vivo were 5.34 ± 0.26 and 8.65 ± 0.60 hours, respectively, which were about 21.4 times and 34.6 times higher than that of rhCNTF. Pharmacodynamics study showed twice weekly administration of 1.0 mg/kg (rhCNTF equivalent) both conjugates led to significant weight-losing, and the efficacy of PEG20k-CNTF group was more potent than that of Tf-PEG5k-CNTF group.

Key words： PEGylation      Transferrin coupling      Ciliary neurotrophic factor      Losing weight     
Received: 14 July 2017      Published: 16 December 2017
ZTFLH:  Q819  
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Sheng-nan CHI
Zeng-lan LI
Chun ZHANG
Shuang YIN
Cui FENG
Qi WANG
Yong-dong LIU
Zhi-guo SU
Cite this article:

Sheng-nan CHI,Zeng-lan LI,Chun ZHANG,Shuang YIN,Cui FENG,Qi WANG,Yong-dong LIU,Zhi-guo SU. Comparison of the Biological Activity of mPEG20k Modified and Transferrin Coupled Ciliary Neurotrophic Factor. China Biotechnology, 2017, 37(12): 59-66.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20171209     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I12/59

Fig.1 RP-HPLC analysis of rhCNTF and modified rhCNTF (a) 12% SDS-PAGE analysis of rhCNTF and modified rhCNTF (b)
Fig.2 HP-SEC(a) and DLS(b) analysis of the rhCNTF and the conjugates
*: P>0.05; ** : P<0.05
Fig.3 Measurement of the TF-1(CN5α-1) cell survival activity and the anti-CNTF antibody binding affinity
** :P<0.05
Fig.4 Pharmacokinetics of the rhCNTF and conjugates in SD male rats
Fig. 5 Pharmacodynamics of rhCNTF and mPEG20k-CNTF, Tf-PEG5k-CNTF in C57 male mouse
Fig.6 In vivo imaging of sulfo-Cy5 labeled CNTF,mPEG20k-CNTF and Tf-PEG5k-CNTF on BALB/c nude mice
Fig.7 Schematic presentation of the spatially morphological difference between mPEG20k-CNTF and Tf-PEG5k-CNTF conjugates
Fig.8 Schematic presentation of the transporting routes difference between mPEG20k-CNTF and Tf-PEG5k-CNTF conjugates
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