Effects Of Pine Needle Extract On The Growth Of Chinese Hamster Ovary Cells And Analysis Of Its Protein Products

Modern research has found that pine needles are extremely rich in flavonoids and vitamins(vitamin C,vitamin A,vitamin E etc.),a variety of amino acids and mineral elements,which can prevent and treat influenza,night blindness,rheumatism and improve health.The extract of pine needles is rich in natural water-soluble bioflavonoids concentrate,a variety of amino acids,vitamins,polysaccharides,fatty acids,trace elements,etc.,which are essential elements for cell growth,and active substances such as enzymes and coenzyme.Pine needle extract are beneficial to the growth of Chinese Hamster Ovary(CHO)cells by eliminating free radicals,resisting genetic mutation and promoting cell proliferation.To understand the mechanism of pine needles at the cellular level by studying the changes in objective indexes of cell culture.And to develop the cell medium with special benefit and improve its comprehensive utilization value.Objective: To study the composition and content of the extracts from different Pinus massoniana needles through observing the growth of CHO cells by feeding in batches.The monoclonal antibodies(m Ab)produced by CHO cells were analyzed from multiple angles to indicate the effects of Pinus massoniana extract on CHO cells cultured in vitro.Methods:(1)Some compounds in the extracts of pinus massoniana needle were quantitatively and qualitatively identified by ultraviolet spectrophotometer and mass spectrometry.(2)The effects of different Pinus massoniana needle extracts on the growth of CHO cells by batch feeding culture technique were analyzed.(3)Affinity chromatography was used to purify the target protein products,and HPLC was used to determine the titer,purity and glycosylation of m Ab,and to analyze the effects of different pinus massoniana needle extracts.The effects of different pinus massoniana needle extracts on the binding activity of m Ab were tested by enzyme-related immunosorbent assay.Results:(1)The total sugar content and total flavonoids content of pine needle water extract(PNWE)were 46.69% and 3.45% respectively.The total sugar content and total flavonoids content of pine needle ethanol extract(PNEE)were 51.43% and 51.43%respectively.The total sugar content and total flavonoids content of pine needle polysaccharide extract(PNPE)were 96.03% and 2.47% respectively.38 compounds were identified from PNPE by LC-MS technique.(2)Three kinds of pinus massoniana needle extracts could delay the death of CHO cells at specific concentrations by fed-batch culture.The cell density and cell viability of CHO cells were increased to varying degrees.(3)The m Ab titer of the three pinus massoniana extracts was higher than that of the control group(P < 0.05),with statistical significance,and the antibody titer of PNPE was the highest,reaching 2.8g/L.The three pinus massoniana extracts had no effect on m Ab at the molecular size level.For charge variants,on the whole,the acid charge variants of m Ab were significantly decreased(P < 0.05)and the basic charge variants were significantly increased(P < 0.05),and the main substances increased within a certain concentration range.PNWE and PNEE had little effect on glycosylation,and only G0 glycotype level increased with the increase of extract concentration.PNPE has a great effect on glycosylation,reducing the levels of G0 F,G0 minus Glc NAc,G0 F minus Glc NAc at specific concentrations.The glucotype levels of G1 Fa,G1Fb,G0,G2 F,G1F Glc NAc,Man5,Man6 were increased.The three kinds of pinus massoniana extracts had little effect on the binding activity of m Ab.Conclusions: In this study,the contents of total sugar and total flavonoids in three extracts of pine needles were determined,and 38 compounds were identified in pine needle polysaccharide extracts.It has been shown that pinus massoniana pine needle extract can be used to increase the live cell density and viability of CHO cells and increase m Ab production.In addition,the polysaccharide extract of pinus massoniana can significantly reduce the level of acid charge variant and regulate the glycosylation spectrum.It is a new research field of biosimilars to develop new raw materials using natural plant extracts to improve the quality of biopharmaceutical products.Our pioneering exploration provides new directions for the optimization of cell culture processes and the development of culture medium systems in the future.