Study And Application Of The Analysis Methods For Trace Protein Macromolecules In Drug

Allergic reactions often occur in clinic,residual protein macromolecules from the drugs such as Chinese medicine injection,pharmaceutical necessities for injection and?-lactam antibiotics can cause allergic reactions.Numerous studies have shown protein macromolecules in the drug to be a trigger for allergic reactions,due to this safety concern,removal of these macromolecule impurities is significant to reduce the incidence of allergy.Drug manufacturers also tried to remove the protein macromolecule impurities in the drug production by improving production technology continuously.Hence,a highly sensitive and selective analytical method is greatly required to conduct process validation and adequately monitor the specific manufacturing process.However,due to the complicated production process,a series of changes have occurred in the protein macromolecules,including polymerization and degradation,several immune technique are based on the antigen-antibody reaction and need antibodies with high specificity,affinity and sensitivity to the target analyte,thus they can only detect one or the same category analytes and they are not suitable for the detection of our research protein.Traditional detecting of proteins methods are classical methods and used for the determination of total proteins.however,because of their large sample matrix interference and limited detection ranges,they are not sufficient to provide adequate concentration levels needed in the analysis of pharmaceutical industry.Thus,it is very difficult for the researchers to establish a high recovery,high sensitivity and high selective method to control these impurities.Vegetable protein macromolecules from the Chinese herb most likely to arise in the Chinese medicine injection during the extraction process,a gel exclusion chromatography method was established to develop for the determination of the residual protein in Yinzhihuang injection,the method based on size,it could potentially be used to separate the macromolecules from the complex micromolecules of Chinese medicine injection.Meanwhile,to further increase the sensitivity,we selected the enhanced end absorption of protein as detection wavelength.A TSK gel SuperSW2000 column?4?m,4.6×300 mm?was used with the mobile phase consisted of phosphate buffer solution at a flow rate of 0.3 mL/min,and the detection wavelength was at 220nm.The linear range of BSA was 0.312⁴0.0?g/mL?R²=0.999?,the average recovery was morn than 81%,the repeatability was good and RSD was 0.7%,the lowest limit of detection was 0.104?g/mL and is about 10 times higher than that of Bradford.The proposed method was proven to be simple,accurate and good repeatability.This study provided a new approach for the analysis of residual protein in other similar drugs.Lactose for injection is a good pharmaceutical excipient for drug for injection,but because of the whey in the lactose may cause allergic reaction,therefore,it is great importance to strictly control the whey protein.In this work,two methods were developed to determine the trace amounts of?-lactalbumin and?-lactoglobulin remained in lactose.HPSEC method used a TSK gel SuperSW2000 column?4?m,4.6×300 mm?,the mobile phase was phosphate buffer solution and the flow rate was 0.3 mL/min,the detection wavelength was at 220 nm.The linear range of total protein was 3.40⁶8.0?g/mL?R²=0.999?,the average recovery was morn than 87%,the repeatability was good and RSD was 0.5%,the lowest limit of detection was 1.13?g/mL.The results show that the sensitivity of HPSEC method was slightly higher than UPLC-Q-TOF method,and the HPSEC method is simple,rapid,low cost,applicability,easy to spread,but due to the close molecular weight of?-lactalbumin and?-lactoglobulin,they could not be separated completely in HPSEC column,thus the method could not used for quantitative respectively.The UPLC-Q-TOF method for the simultaneous determination of?-lactalbumin and?-lactoglobulin was developed.The separation of analytes was made on a C18 protein column using a mobile phase of 0.1%?V/V?TFA-water and 0.1%?V/V?TFA-acetonitrile by gradient elution.The TOF scan was employed for the quantification.By Peakview 1.2 software,the target compound was confirmed by the comparison of theoretical accurate moleculer mass,isotopic pattern abundance and product ion characteristics.In this condition,the linear range for?-lactalbumin and?-lactoglobulin were2.00⁸0.0?g/mL?R₁=0.997?and 6.00²40.0?g/mL?R₂=0.996?,the recovery were 82.9%⁸7.2%and 96.1%⁹8.4%,the limit of detection were 1.00 and3.00?g/mL for?-lactalbumin and?-lactoglobulin respectively.This method is accurate and selectivity,but the sensitivity is not high.Therefore,in the actual detection,if we need the operation simple,fast,low cost,high sensitivity and the total protein of?-lactalbumin and?-lactoglobulin,we can choose HPSEC method;If we need the method has a good specificity and selectivity,we can choose UPLC-Q-TOF;If we need higher specificity and sensitivity,since the ionization efficiency of the MS ion source for proteins injected directly are not high like other micromolecules,we could make the protein macromolecules into micromolecules by enzymolysis,it need further study.Residual protein macromolecules from the?-lactam antibiotic fermentation process can cause allergic reactions,In the present paper,we used Phenoxymethylpenicillin potassium as a model drug and tried to develop a HFCF-UF device to enrich macromolecules from a sample solution,followed by its determination using HPSEC which selected the enhanced end absorption of protein as detection wavelength.A TSK gel SuperSW2000column?4?m,4.6×300 mm?was used with the mobile phase consisted of phosphate buffer solution at a flow rate of 0.3 mL/min,and the detection wavelength was at 220 nm.In this technique,the sample solution,to be concentrated,is transferred to the reservoir,after by a simple spinning in a centrifuge,the protein macromolecules are enriched in the hollow fiber.The phenomenon of NSB were investigated and solved.The EF can reach 99,the average recovery was more than 87%,after concentration by the device of HFCFUF,we could detect the quantity of protein macromolecule low to 30.3ng/g.The proposed method was proven to be simple,accurate and sensitive.This study could be used as a alternative way for the determination of other similar macromolecule impurities in drugs.