INVESTIGATION ON BIOSYNTHESIS OF SOME BIOSIMILAR DRUGS USING VARIOUS CULTURE TECHNIQUES – RESEARCH ARTICLE

Plant tissue culture technique play very significant role in the investigation on biosynthesis of biopharmaceuticals, plant metabolites etc. The present investigation based on the production of biosimilar drugs by the help of plant tissue culture technique. In this work, D.carota cell line developed in nutrient media. MS media was used as nutrient medium. Several types of modification also done in MS media with a view to enhance initiation of callus, like modified MS media, MS media with coconut water and MS media with sugar solution. MS media with coconut water (74%) show maximum initiation of callus. The biomass production in D.carota was recorded 126.48 g/L, 129.80 g/L and 121.50 g/L with MS media, MS media with coconut water and MS media with sugar solution respectively. Callus obtained from static MS media used for establishment of suspension culture. The cells were grown in culture vessels on a rotary shaker incubator. The growth of cells was studied by packed cell volume and viability of cell. The packed cell volume was recorded 13.5%, 14.5% and 10.2% in callus of MS media, MS media with coconut water and MS media with sugar solution with viability 89%, 91% and 85% respectively. The growth of D.carota cells was also studied with fix concentration of inoculum i.e. 10% packed cell volume. After 16 th day of inoculation 26.17%, 29.20% and 23.20% packed cell volume was recorded in MS media, MS media with coconut water and MS media with sugar solution respectively. After the establishment of well-developed cell line, substrate solutions (Benzoic acid and chloroquine) were fed into media in different concentration i.e. 10,000 µg/ml, 15,000 µg/ml, 20,000 µg/ml and 25,000 µg/ml. The viability of the cells of culture was recorded with different concentration of substrates during the experiment. The percentage viability of D.carota cell in suspension culture was recorded 90%, 91%, 87% and 89% with 10,000 µg/ml, 15,000 µg/ml, 20,000 µg/ml and 25,000 µg/ml of benzoic acid fed respectively. In chloroquine fed, the percentage viability of D.carota cell in suspension culture was recorded 92%, 90%, 90% and 89% with 10,000 µg/ml, 15,000 µg/ml, 20,000 µg/ml and 25,000 µg/ml respectively. The samples were analysed periodically and metabolites were identified by matching with known standards using thin layer chromatography and high-performance liquid chromatography. Thin layer chromatographic studies revealed that D. carota cell culture gave one metabolite each with benzoic acid and chloroquine. The metabolites formed were identified with authentic samples by Co-TLC which illustrates that D. carota transformed benzoic acid into p-hydroxybenzoic acid and chloroquine into hydroxychloroquine. The identity of metabolites was further confirmed by HPLC. The ethyl acetate fraction and chloroform fraction of D.carota were subjected to HPLC. The identification and quantitative estimation of peaks was carried out with known standards of p-hydroxybenzoic acid and hydroxychloroquine. The peaks of metabolites of ethyl acetate was found identical with known standards of p-hydroxybenzoic acid and the peaks of metabolites of chloroform fraction was found very close with known standards of hydroxychloroquine, i.e. it was assumed that chloroquine converted into hydroxychloroquine. On 20 th day of incubation, accumulation of P-hydroxybenzoic acid in suspension culture was recorded 70%, 76.66%, 53.5% and 47.6% with 10,000 µg/ml, 15,000 µg/ml, 20,000 µg/ml and 25,000 µg/ml respectively. The yield of hydroxychloroquine in suspension culture was recorded 61%, 57.33%, 44.45% and 42.8% with 10,000 µg/ml, 15,000 µg/ml, 20,000 µg/ml and 25,000 µg/ml respectively after 20 th day of incubation. The studies performed with media of suspension culture and control sample were showed no accumulation of p-hydroxybenzoic acid and hydroxychloroquine.