SDS-PAGE analysis of seed albumins and globulins from two pigeonpea, Cajanus cajan, cultivars (DSLR-17 and BDN-2) and ten wild species, including C. cajanifolius, C. lineatus, C. sericeus, C. acutifolius, C. lanceolatus, C. reticulates, C. albicans, C. scarabaeoides, C. volubilis and C. platycarpus, resulted in 34 albumin and 27 globulin polypeptides. Proximity matrix analysis based on electrophoretic banding patterns of albumins and globulins jointly revealed C. cajanifolius to be closest to C. cajan having similarity coefficients of 0.595 and 0.676, respectively. Cluster analysis also exhibited the grouping of C. cajanifolius with C. cajan in one cluster. C. platycarpus has been out grouped. The present analysis more or less agreed with the sectional classification of the genus Cajanus and it has been hypothesized that cultivated pigeonpea has evolved through multi-genomic interaction involving C. cajanifolius and that it has experienced minor genomic reorganization during its divergence.

(2015) A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line, Pharmaceutical Biology, 53:6, 849-854, DOI: 10.3109/13880209.2014 Context: Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG).Objective: The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF. Materials and methods:The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 Â 10 5 cells/ ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations. Results: The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line. Conclusion: Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.

Granulocyte-colony stimulating factor (G-CSF) has commonly been used to help the patients to recover from neutropenia inflicted due to radiotherapy, organ transplants and chemotherapy. As the number of people undergoing these therapies and procedures are increasing world-wide, the need for more economical ways of G-CSF production and improvement in its efficacy has become increasingly crucial. In the present study, recombinant human G-CSF (rhG-CSF) was expressed in E. coli and its purification process was optimized by demonstrating better efficiency and higher recoveries (upto 54%) in a multi-step chromatographic purification process, which is greater than the existing reports. Additionally, the efficacy of rhG-CSF was increased by derivatizing with polyethylene glycol (PEG; upto 85% PEGylation), which increases the plasma clearance time, reduces the immunogenicity and requires less frequent administration to the patient. Overall, the present study suggests a cost-effective purification process of rhG-CSF and also proposes its efficient conjugation with PEG for enhanced efficacy as compared to the existing commercially available forms.