| Human serum albumin (HSA) is the most abundant protein in plasma, the level of which reaches40-50g/L. It plays an important role in maintaining osmotic pressure in the body, as well as transporting lipids, hormones and medicine molecules. In clinic, this protein has been widely used to treat burns, liver ascites, low albumin disease etc. Besides, HSA can also be applied as pharmaceutical excipient, stabilizer and additive in cell culture. Due to the extensive application, currently, HSA has been not able to meet the demand of market. Our lab developed a new rice-seed expression system to produce recombinant HSA (rHSA), and large-scale production of rice-derived rHSA in a cost-effective way has been realized. Generally, HSA is used clinically at a high dosage of10-20g per person, hence, it is urgent to assess the safety of the impurities from host cells when rice-derived rHSA was produced, and the effective quality control is necessary. Host cell proteins (HCPs) and host cell DNA is the two main impurities from host cells and considered the most important factors in recombinant protein drug quality control. EMEA make it clear that these two kinds of impurities should be characterized distinctly to ensure the safety of the products in clinical use. The main results of this dissertation are as follows:1. The establishment of residual host cell DNA quantative method:,A quantitative real-time polymerase chain reaction (qPCR) assay, based on the5S ribosomal RNA (rRNA) genes, was used to develop a quantitative method of rice residual DNAs. The two common qPCR assays, S YBR Greean I and TaqMan, were compared. In the case of no significant differences existing between the two reaction systems, SYBR Green I was chosen as the residual DNA quantitative method because of its cost-effectivity. Through the choice of appropriate primers and the optimization of primer concentration, the established residual DNA quantitative method exhibited a high degree of linear relationship with a R2of0.9991, when the template concentration was in the range of2×104pg to0.2pg per reaction system. The amplification efficiency was close to100%and the detective limit lowed to0.2pgper reaction system. The established qPCR assay had a good repeatability and specificity, showing a high recovery of80%to122.2%in the recovery experiments, which was also not affected by the choice of standard samples. The amount of residual DNA in a typical rHSA product was quantified to be3.5ng per dosage (10g) by this method,lower than the requirements of10ng per dosage by regulations.2. The establishment of residual HCPs quantitative method:An enzyme-linked immune sorbent (ELISA) assay was developed using antibodies prepared by rice seed antigens. The produced antibody gave high antibody coverage and did not cross react with the target protein, HSA. The sensitivity of two detecting antibodies coupled with HRP or Biotin respectively was compared, and the results demonstrated that the antibody with Biotin gave higher sensitivity, which was chosen to be the detecting antibody in the ELISA method. Following the optimization of concentrations of capture and detecting antibodies, the established ELISA assay had a good non-linear relationship with a R2of0.99. The quantitative range was from25ng/ml to1.25ng/ml, and the minimum quantitative limit was1.25ng/mL. The ELISA method also had a robust repeatability and accuracy. The coefficient of variation (CV) of quantitative results was lower than11.5%, and the recovery of spiked standard HCPs in the recovery experiments ranged from86%to110.6%. By quantifying the residual HCPs using the established method, the purity of an rHSA product could be calculated to be99.9998%.3. The identification of residual HCPs:To avoid the interference from rHSA in HCPs identification, rHSA wasremoved and the HCPs were concentrated by an immune chromatography method. Anti-human serum albumin polyclonal antibodywas produced in rabbits through human serum albumin as antigen. The immune chromatography with a capacity of100mg HSA was achieved by coupling anti-HSA antibodies with CNBr-activatived SepharoseTM4Fast Flow, which was used to seperate rHSA from HCPs. The recovery of HCPs from rHSA with a purity of99.8%was higher than90%in this seperation technique. The collected HCPs were concentrated and subsequently characterized by label free NanoLC-ESI-MS/MS, showing that at least seven-orders of magnitude identification range was achieved. Almost all impurities were identified effectively by this method.4. The toxicity research of residual HCPs by repeated administration:The collected HCPs from products with the purity of99.9%were intravenously injected into mice at a clinical dosage of1-fold (0.2mg),5-fold (1mg) and25-fold (5mg) for28-day toxisity test. During the whole testing, mice did not die from the deadly toxicity caused by samples. The major toxic symptoms included anemia and hypersplenism, which were preliminarily speculated to be related to low permeability hemolysis or immuno hemolytic disease.. |
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