Establishment And Standardization Of Detection Methods Of Residues Of Bacterial Endotoxin And Cryopreservation Solution Common Component DMSO In Cell Preparations

With the in-depth research of cell therapy technology,stem cells and their functional cell preparations have become the hope of clinical treatment of major and difficult diseases.However,due to the particularity of cell preparations,stem cells and their functional cell preparations have not been approved in China,and the lack of reliable quality control standards for cell preparations is one of the main reasons.This project is dedicated to the research on the quality control standards of cell preparations,mainly aiming at the harm of dimethyl sulfoxide（DMSO）,a common component of cryopreservation solution in cell preparations,and endotoxin residues caused by bacterial contamination during culture,and establishing the detection method suitable for cell preparations and carrying out the method standardization research.In this study,the cell supernatant at the time of recovery,medium change and full time during cell culture was mainly used as the detection object.Establish a Raman spectroscopy method for the detection of DMSO residue in cell preparations,and carry out methodological verification,and then use gas chromatography to compare and verify the Raman spectroscopy method;Establish a recombinant factor C detection method for detecting endotoxin residues in cell preparations and verify it with limulus amebocyte lysate test method included in Pharmacopoeia.（1）Establishment and standardization of Raman spectroscopy detection method for DMSO residues in cell preparationsRaman spectroscopy was established to detect the residual DMSO of cryopreservation solution in cell preparations,and the cell supernatants of three stages was used as the research object,and the Raman spectra of DMSO reference substances with different concentrations were collected,Taking the concentration as X and the Raman intensity difference between 679 cm^-1and 701 cm^-1as the Raman relative intensity Y,establish a linear equation,the linear relationship is Y=662.12X+259.55,R²=0.9996,which means a good linear relationship in the concentration range of 1-100 mg/m L.The precision test showed that the RSD of the relative peak intensity of DMSO was 2.47%,indicating good precision;the repeatability test showed that the average DMSO concentration was 1.6482mg/m L,and the RSD was 1.77%,indicating good repeatability;In the spiked recovery test,the sample recovery was 97.6%-104.3%,the average recovery was101.9%,and the RSD was 1.98%,indicating that the spiked recovery was good and the accuracy of the method was high.The medium has fluorescence background interference,but it does not affect the determination of the sample at679 cm^-1,and the method validation results were all good.The average content of DMSO during resuscitation measured by Raman method was 9.8408 mg/m L,and the average residual amount was 0.984%.When the medium was changed and the cells were overgrown,its content was lower than the detection limit of Raman spectroscopy by 1 mg/m L,and the corresponding residual amount was 0.1%,which is lower than the limit of 0.5%specified in the Pharmacopoeia.Gas chromatography was used to verify the detection results of Raman spectroscopy,and the supernatants of the three culture stages were also used as samples for detection.In the methodological experiment of gas chromatography,the system suitability experiment met the requirements,and the solvent ethanol did not interfere with the determination,the linearity,precision,repeatability,and standard addition recovery were all good,the limit of quantification was 8.0297μg/m L,and the limit of detection was 2.0074μg/m L.The entire methodology is well validated.The detection results of Raman spectroscopy were well verified by gas chromatography,and the differences in the results of the two methods were very small,between 0.0545-0.7303mg.Therefore,the Raman spectroscopy method established in this study can be used for rapid detection of DMSO residues in cell supernatants,and the results are accurate and reliable,and relevant draft standards have been submitted.（2）Establishment and standardization of recombinant factor C detection method for endotoxin residues in cell preparationsRecombinant factor C method was established to detect endotoxin residues in cell preparations.The cell supernatant was used as the research object.The endotoxin standard reacted with the fluorescent substrate.Take the logarithm of the concentration of the standard solution as X,and the logarithm of the netΔRFU as the ordinate Y,and perform linear fitting.The value of R²should not be less than 0.980.Use the established linear equation for quantitative detection and explore the dilution factor of the sample,and examine the recovery rate of standard addition.Detect induced pluripotent stem cell（i PSC）supernatant diluted5-fold,10-fold,20-fold and parallel spiked samples,and finally determine 20-fold as the final dilution factor.i PSC medium does not interfere with sample detection.The results showed that the undiluted concentration in the 10-fold diluted supernatant was 0.1202 EU/m L;the undiluted concentration in the 20-fold diluted supernatant was 0.1123 EU/m L;the detection results of the undiluted samples converted after 10-fold dilution and 20-fold dilution were almost consistent;The endotoxin concentration of the undiluted medium after 10-fold dilution of i PSC medium was 0.0778 EU/m L,and the endotoxin concentration of undiluted medium after 20-fold dilution of i PSC medium was 0.0758 EU/m L,diluted10-fold and 20-fold of the endotoxin concentrations in the undiluted medium after conversion were also consistent with each other;The endotoxin concentration of10-fold dilution in PBS is 0.006 EU/m L,and the concentration of undiluted endotoxin is 0.06 EU/m L.Escherichia coli was used as a peripheral positive control for bacterial endotoxin detection,and the result was higher than the upper limit of the linear range,but it was used as a strong positive control to verify the reliability of the detection method.Finally,when the optimal dilution factor is 20times,the concentrations of endotoxin in the supernatant of i PSC resuscitation,medium exchange and overgrowth are 0.51EU/m L,0.60EU/m L,and 0.56EU/m L,respectively.The results of the experimental are consistent with the results of the Limulus reagent gel method for endotoxin detection stipulated in the Pharmacopoeia,and they are all negative,that is,the concentration is less than1.2EU/m L.The validation experiment of the gel method promoted the standardization of the recombinant factor C method for the detection of bacterial endotoxin.Cell cryopreservation is an important part of the preparation and application of cell preparations,and the endotoxin residues produced by the common component DMSO residue of the cryopreservation solution and the endotoxin residue caused serious harm to human health,and it is of great significance to establish the Raman spectroscopy of DMSO residues in cell preparations and the recombinant C factor detection method of endotoxin residues.Raman spectroscopy has the advantages of fast,non-destructive,in situ detection,no pretreatment required,portability.Therefore,the Raman spectroscopy established in this study is very suitable for the rapid detection of cell preparations.The recombinant factor C method has the advantages of high sensitivity and no animal-derived components,which is superior to the traditional Limulus reagent detection method,and is suitable for the detection of endotoxin residues in cell preparations.In conclusion,the two detection methods established in this study provide a new means for the establishment of the cell preparation quality control system.