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Objective:To study the effect of physical therapy and heel sound feedback on lower limbs motor function, mobility and activities of daily living (ADL) for stroke patients based on International Classification of Functioning, Disability and Health (ICF) core set. Methods:From April, 2018 to May, 2020, 113 stroke patients with motor dysfunction were divided into ischemia group (n = 67) and hemorrhagia group (n = 46) according to the cause of stroke. They received physical therapy for lower limbs and heel sound feedback for eight weeks, and assessed with ICF core set for stroke-gait, Fugl-Meyer Assessment-Lower Extremities (FMA-LE), Timed 'Up and Go' Test (TUGT), and modified Barthel index (MBI) before and after intervention. Results:The main effect of time was significant for qualifiers of ICF core set for stroke-gait, the scores of FMA-LE and MBI, and TUGT time (F > 100.59, P < 0.001), and it improved time by time as Post Hoc test. The main effect of groups was not significant (F < 2.29, P > 0.05), nor as Post Hoc test. The interactive effect between time and groups was significant for TUGT time (F = 6.45, P < 0.01), perhaps improved more in the hemorrhagia group, however, the interactive effect was not significant for the others. Conclusion:Physical therapy and heel sound feedback can improve motor function of lower limb, mobility and ADL for stroke patients.
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OBJECTIVE: To conduct an inter-laboratory comparison of UPLC method for polymer determination in human serum albumin and verify the method applicability. METHODS: National reference of human serum albumin and 20 samples of human serum albumin from domestic and foreign manufactures were distributed to six laboratories in order to carry out inter-laboratory comparison and demonstration of applicability. UPLC was used to determine the content of polymer and HPLC method was used for parallel comparison. RESULTS: There was no significant difference in the determination results between the UPLC method and current HPLC method in four laboratories (P>0.05) equipped with both HPLC and UPLC. The mean values of 21 samples measured with UPLC method by six laboratories were matched with those measured with HPLC method by four laboratories (t test P>0.05). The mean values of standard deviation (SD) and relative standard deviation (RSD) for 21 samples by UPLC method were only 0.06 and 1.02% respectively. The mean values of standard deviation (SD) and relative standard deviation (RSD) were 0.14 and 2.33% for parallel 21 samples determination by HPLC method, suggesting that the difference of UPLC test results between laboratories was smaller. CONCLUSION: The results of UPLC method are in good agreement with those of HPLC method. UPLC method is more effective and efficient, with smaller inter-laboratory difference, thus is significantly better than the HPLC method.
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OBJECTIVE: To evaluate the quality of human albumin products, and determine the content of aluminumion in the upcoming expired human albumin samples of both domestic and imported products for batch release. METHODS: Statistics was carried out for domestic and imported human albumin products in 2017 about the approved registration, import approval number, storage conditions, aluminumion content of batch release and other information. Aluminumion contents of the samples were detected by National Institutes for Food and Drug Control which banded with 7 authorized agencies for batch release of blood products according to the atomic absorption spectrometry or verified inductively coupled plasma mass spectrometry (ICP-MS)method for the determination of aluminumion content in China Pharmacopoeia 2015. RESULTS: There were a total of 28 domestic manufacturers of blood products and 158 human albumin drug approval numbers (totally 40 for daily production, 2017), among which 21 were approved for the storage condition of "room temperature" and 7 for "refrigerate". A total of 185 batches of human albumin samples from 25 domestic manufacturers were sampled. The mean aluminumion content in the batch release reports was 61 μg·L-1(8-134 μg·L-1), while that in the samples of the upcoming expired products was 137 μg·L-1(20-487 μg·L-1). The mean aluminumion content increased by about 1 time after the storage period. There were about 17.8% (33/185)samples having aluminumion content over 200 μg·L-1. There were a total of 13 manufacturers of imported human albumin with a total of 17 import specifications in 2017. The approved storage condition was all "room temperature". A total of 78 batches of human albumin products for batch release from 11 import enterprises were sampled. The mean aluminumion content in the batch release reports was 27 μg·L-1(7-60 μg·L-1), while that in the samples of the upcoming expired products was 50 μg·L-1(1-175 μg·L-1). The overall mean increased about 1 time after the storage period, which was consistent with the domestic human albumin products. But the two parameters of the imported products were both lower than the domestic ones. None (0/78)of the samples having aluminumion content over 200 μg·L-1. CONCLUSION: The aluminumion contents of some batches of upcoming expired domestic human albumin products are over 200 μg·L-1. The overall aluminumion contents in the initial batch release and the upcoming expired products in the import albumin products are lower than the domestic ones.
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OBJECTIVE: To determine the citrate ion and aluminum contents in human albumin products and investigate the related factors of aluminum content. METHODS: Sixty-five batches of human albumin products which were very close to the end of shelf life or would be expired within not more than 2 m, including 54 batches of imported ones and 11 batches of domestic ones, were analyzed. The content of citrate ion was determined using the enzyme reaction method, and aluminum content was determined using the atomic absorption method. Factors related to human albumin production, such as ultrafiltration time, glass bottles, sample storage conditions, and the initial value of aluminum content were investigated and analyzed, especially the relationship with the aluminum content at the end of the shelf life. RESULTS: The overall mean content of citrate ion in 65 batches of human albumin was 24 μmol•L-1 (0 - 144 μmol• L-1), the mean content of citrate ion in 54 batches of domestic human albumin was 24 μmol•L-1, and that in 11 batches of imported human albumin was 23 μmol•L-1. The linear correlation coefficients between final aluminum content and citrate ion content of 65 batches of human albumin, 54 batches of domestic human albumin and 11 imported human albumin were 0.315 5, 0.331 8 and 0.746 6, respectively. The linear correlation coefficients between the final aluminum content and storage time of 65 batches of human albumin, 54 batches of domestic human albumin and 11 imported human albumin were 0.102 6, 0.059 3 and - 0.037 4, respectively. The linear correlation coefficient between the final and initial aluminum contents of 54 batches of domestic human albumin was 0.325 5. The linear correlation coefficients between final aluminum content and ultrafiltration process time, citrate ions and ultrafiltration process time of 54 batches of domestic human albumin were 0.011 8 and - 0.108 2, respectively. CONCLUSION: Citrate ion content in human albumin retention samples are lower than 150 μmol•L-1. Citrate ion content and final aluminum content are weakly correlated, however, the correlation coefficient between the two indexes of imported human albumin is much higher than that of domestic samples. The initial and final aluminum contents shows low correlation, and ultrafiltration time shows very weak correlation with storage time.
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OBJECTIVE: To determine glycoprotein content in recombinant human albumin from different expression systems with different methods. METHODS: Recombinant human albumin samples from Saccharomyces cerevisiae expression system, Pichia pastoris expression system, Oryza sativa expression system as well as plasma derived human albumin were investigated by phenol sulfuric acid method, HPLC peak area method and ConA combining elution HPLC method. RESULTS: For 10 batches of samples expressed in pichia pastoris expression system, the total contents of mannose were 2.7 mg•g(Pro)-1 (A manufacturer, n = 4) and 1.7 mg•g (Pro)-1 (B manufacturer, n = 6), respectively. The HPLC peak area percentages of ConA binding protein in recombinant human albumin from Pichia pastoris expression system were the highest, which showed 2.65% (A manufacturer) and 0.71% (B manufacturer) respectively, the peak area percentage of ConA binding protein in Oryza sativa expression system was 0.05% (E manufacturer, n = 3), and the ConA binding protein peak area of plasma derived human albumin was 0.01% (W manufacturer). The results of ConA binding and elution analysis with HPLC method for Quantitative determination of ConA binding protein showed that the ConA binding protein contents in the samples from pichia pastoris expression system were much higher: 27.58 mg•g(Pro)-1 (A manufacturer), 21.48 mg•g(Pro)-1 (B manufacturer), 32.02 mg•g(Pro)-1 (C manufacturer); the ConA binding protein content in the sample from Saccharomyces cerevisiae expression system was lower, 2.29 mg•g(Pro)-1 (D manufacturer); the ConA binding protein content in the samples from oryza sativa expression system was the lowest, 1.27 mg•g(Pro)-1 (E manufacturer); the plasma derived human albumin ConA binding protein content was 31.16 mg•g(Pro)-1 (S manufacturer). CONCLUSION: In terms of the results of the samples and methods involved in this study, there were glycosylated or glycol forms of protein in all recombinant human albumin samples from different expression systems; the glycosylated protein content in samples of Pichia pastoris expression system is higher than Saccharomyces cerevisiae expression system, while the glycoformed protein in samples of Oryza sativa expression system is the lowest. Plasma derived human albumin also contains glycoprotein or glycosylated protein.
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OBJECTIVE: To evaluate the applicability of UPLC/MS method for the identification test of human serum albumin (HSA) products including plasma derived and recombinant HSA samples. METHODS: ACQUITY UPLC with Vion IMS QT of LC/MS system was used combined with on-line HSA sample desalting with ACQUITY UPLC BEH C18 column. The acquired multiplycharged mass spectrum was processed with MaxEnt1 automatic protein deconvolution software in UNIFI, which can transfer the raw mass spectrometry data to zero charge molecular mass or mass distribution of the intact protein. RESULTS: Intact protein mass analysis not only provided the accurate mass of HSA, but also provided an overall view of the heterogeneity of HSA and the relative amounts of various forms. From this study, a very specific mass signal [(66 437 ± 1), which is the theoretical average MW of human serum albumin ]was obtained from all the six HSA samples. And the characteristic spectra of different samples were also got. CONCLUSION: UPLC/MS method has very good specificity and high sensitivity and can distinguish HSA products made by different manufacturers and processes. The total analytical time is 10 min, which is ideal for the QC identification test of HSA products.
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OBJECTIVE: To develop and verify a magnetic beads-based extraction combined with quantitative PCR(q-PCR)method for determination of residual host cell DNA in recombinant human albumin products expressed in Pichia pastoris. METHODS: The residual Pichia pastoris host cell DNA in samples were extracted by magnetic beads-based extraction method and then determined by Taqman probe-based q-PCR. The residual DNA content was calculated according to the standard curve. The developed method was verified for accuracy and precision with different derivation albumin matrixes and concentrations, and the residual DNA of 3 batches of recombinant human albumin products expressed in Pichia pastoris were detected. RESULTS: The minimum detection limit of Pichia pastoris residual DNA by the developed method was 3 fgμL-1, the linear range was 3 fgμL-1-300 pgμL-1, and the correlation coefficient(r2) was 0.998 3. The recovery rates of spiked samples in rHA matrix were 93.58%(RSD 19.6%, n=4)at 100 fgμL-1 and 215.56%(RSD 42.9%, n=4) at 10 fgμL-1, respectively. The recovery rates of spiked samples in HSA matrix were 67.09%(RSD 6.9%,n=3)at 100 fgμL-1 and 113.40%(RSD 11.1%, n=3) at 10 fgμL-1, respectively. The residual Pichia pastoris DNA contents in 3 batches of recombinant human albumin products expressed in Pichia pastoris determined by the developed method were 5.98, 4.16, 4.49 fgμL-1(n=7) respectively and not more than 1 ng per 10 g protein. CONCLUSION: Magnetic beads extraction method combined with fluorescence quantitative PCR method solves the technical problem of quantitative determination of trace DNA in recombinant human albumin products with ultra-high concentration protein. The method is accurate and reproducible, and can be used for quantitative determination of DNA residue in recombinant human albumin expressed by Pichia pastoris.
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OBJECTIVE: To establish a determination method of molecular-size distribution of human serum albumin (HSA) by ultra performance liquid chromatography (UPLC). METHODS: An UPLC method was developed to specifically determine the polymers and other components in HSA on UPLC PROTEIN BEH SEC analysis column with Waters Alliance UPLC system and Waters UPLC TUV detector. The separation was performed using a mobile phase consisting of PBS at a flow rate of 0.6 mL•min-1 and the UV detection wavelength was set at 280 nm. HSA samples were diluted to different concentrations (0.5-20 mg•mL-1) to confirm the optimal concentration range of the injection. The change of component percentage and the linear relationship between HSA concentration and chromatographic peak height were confirmed and the molecular-size distribution was calculated by area normalization method. Within the optimum injection concentration range, the national control sample for HSA was diluted to 12 mg•mL-1 and tested by UPLC method and the methodology was confirmed. Twenty batches of HSA samples were determined by both UPLC and existing HPLC methods, and the samples were determined in parallel. The consistency of the methods was compared and the method was reconfirmed. RESULTS: The UPLC retention time of HSA polymer was 1.469 min, of dimer was 1.972 min, and of monomer was 2.267 min, respectively. The resolution of dimer and monomer was 2.20 and the USP tailing of monomer peak was 1.18 respectively. In the range of the injection concentrations, 0.5-20 mg•mL-1, there was linear relationship between the concentrations of the components of 11 HAS samples, including polymer, dimer and monomer peak, and the peak area%, peak height, peak area, and the squares of linear correlation coefficient were all greater than 0.997 0. The components peak area percentage of HSA samples remained relatively stable within the concentration range of 10-16 mg•mL-1 (total injection amount of 100-160 μg). The RSDs of the percentage of polymers were 0.00% (n=3, 10 mg•mL-1), 0.10% (n=3, 12 mg•mL-1), and 0.10% (n=3, 16 mg•mL-1), respectively. The UPLC method was used to determine the national control sample for human albumin of 12 mg•mL-1, and the mean value of peak area% was 5.62% (n=10). The results were consistent with those of the parallel determination by HPLC (5.58%), both of which were in accordance with the quality control range of the national standard for human albumin. The RSD of the percentage of the peak area of the polymers in national standard for human albumin by UPLC was 0.40% (n=10). The HPLC and UPLC methods were used to determine the polymer peak area percentage of 20 batches of HSA samples from 7 domestic and foreign enterprises at the concentration of 12 mg•mL-1. The correlation coefficient of the two methods was 0.996 0 (P> 0.05) and there was no significant difference between the two methods (P>0.05). CONCLUSION: Compared with the traditional HPLC method, the detection time of HSA SEC by the proposed UPLC method is shortened by at least 10 times, and the accuracy and repeatability of the determination are satisfactory. UPLC method can save much more analysis time, is simple and much faster, and can be used for high-throughput determination of molecular-size distribution of human serum albumin.
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OBJECTIVE: To prepare the first batch of Chinese national standard for human albumin used for the test of batch release or other quality control of human albumin products. METHODS: The first batch of national standard for human albumin was prepared with certificated human albumin products, mixed and filled under aseptic conditions. The standards were distributed to 11 laboratories for cooperative calibration according to the unified methods published on China Pharmacopeia. Seven test items including total protein content, sodium caprylate, polymers content, pH, absorbance, sodium content, and aluminium content were detected. RESULTS: The limits of the six test items except aluminium content were established as follows after the data from 11 collaboration laboratories were received and statistically analyzed: total protein(193.30 ± 5.08)g·L-1;sodium caprylate (0.162 4 ± 0.009 2) mmol· g(Pro)-1; polymers content (2.72 ± 0.29)% (HPLC-SEC);pH(6.71 ± 0.08) [temperature (22 ± 3)℃];absorbance 0.030 ± 0.005;sodium content (134.9 ± 23.6)mmol ·L-1. The range of initially established aluminium content was (88.4 ± 30.5)μg·L-1. However, it was observed to increase obviously after 21 months according to the trend analysis, so it was deleted from the test items for the national standard for human albumin ultimately. CONCLUSION: The prepared national standard for human albumin met the relevant requirements and may be served as the first generation of national standard for human albumin products.
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OBJECTIVE: To prepare the first batch of national standard for enterovirus 71 immunoglobulin for the efficacy test of EV71 human immunology products. METHODS: The domestic intravenous immunoglobulin products with batch release certification and high efficacy EV71 immunoglobulin products were mixed, filled, and lyophilized under aseptic conditions to get the first batch of national standard for enterovirus 71 immunoglobulin. The standards were distributed to five laboratories for cooperative calibration according to the unified SOP for microneutralization test. Neutralizing titer which corresponded to the reciprocal of the highest serum dilution that neutralized enterovirus 71 was defined as the efficacy (reported as unit) of the national standard. Sterility test, moisture determination, precision for filling test, and stability of potency were verified. RESULTS: A total of 63 calibration tests were earned out by the five collaboration laboratories, and the results were statistically analyzed after logistic convertion. The inter-laboratories variations varied from 1.5%-4.1% and the intra-laboratories variation was 3.1%. The geometric mean of the prepared national EV71 immunoglobulin standard was 327 U and defined as 330 U for convenience of use. The potency of the prepared standard was stable after 22 m and the contents of monomer plus dimer determined by HPLC-SEC were more than 98.0% during storage at a wide range of temperatures. The prepared national EV71 immunoglobulin standard was qualified in the sterility test, and the moisture content and precision for filling were 0.6% and 0.56%, respectively. CONCLUSION: The prepared national EV71 immunoglobulin standard met all the relevant requirements and may be served as the first generation of national standard for the potency test of EV71 immunoglobulin products.
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OBJECTIVE: To establish a quantitative determination method of sucrose in human fibrinogen by HPLC. METHODS: An HPLC method was developed to specifically determine sucrose on Zorbax carbohydrate analysis column with Waters Alliance system and 2414refractive index detector. The separation was performed at 30°C using a mobile phase consisting of acetonitrile and water (70:30) at a flow rate of 1.4 mL · min-1. Thirteen batches of recombinant human coagulation factor VM samples were selected for methodology comparison of the established HPLC method with the IEC-HPLC method adopted by Ch. P 2010, because these samples only contained sucrose and did not receive dry-heat treatment. RESULTS: The RSDs (n=6) of the retention time and peak area were 0.17% and 0.09%, respectively. The recoveries of sucrose at low (5 mg · mL-1), middle (10 mg · mL-1), high (15 mg · mL-1) concentration were 96.2%, 98.8% and 100.3%, respectively. The average recovery was 98.4% and the linear correlation coefficient r was 1.0000 in the ranges of 2-20 mg · mL-1. Statistical analysis showed that the amounts of sucrose in 13 batches of recombinant human coagulation factor VIII samples determined by the HPLC method and IEC-HPLC method had no significant difference (P≤0.05) and the correlation coefficient r was 1.0000. CONCLUSION: The proposed HPLC method is simple, accurate and re-peatable for determination of sucrose in dry-heat treated human fibrinogen product. The HPLC method showed good accordance with IEC-HPLC method for assay results of sucrose, thus can be widely applied.
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OBJECTIVE: To determine the insoluble particles in human immunoglobulin (pH 4) for intravenous injection and human hepatitis B immunoglobulin(pH 4) for intravenous injection in both liquid and freeze-dried forms from 14 domestic manufacturers. METHODS: Thirty-two batches of human immunoglobulin products including 19 batches of human immunoglobulin (pH 4) for intravenous injection, 5 batches of human immunoglobulin (pH 4) for intravenous injection (freeze-dried), 5 batches of human hepatitis B immunoglobulin (pH 4) for intravenous injection, and 3 batches of human hepatitis B immunoglobulin(pH 4) for intravenous injection (freeze-dried) were tested by light obscuration particle count test method recommend by appendix of 2010 CHP by GWF-8JA laser particle size analyzers. Insoluble particles greater than 10 and 25 μm were counted, respectively. Microscopy counting was carried out if the test results from light blockage method did not meet the qualification criteria in 2010 ChP. Trend analysis and comparison of the results from NIFDC and enterprises were done. RESULTS: The test results of insoluble particles in 32 batches of human immunoglobulin products indicate that 90.6%(29/32) of the products complied with the requirement of 2010 ChP and the results were in agreement between NIFDC and enterprises. Unfortunately, 3 batches of human immunoglobulin(pH 4) for intravenous injection(freeze-dried) failed in light obscuration particle count test, however, they met the requirement of 2010 ChP when microscopy counting method was used. CONCLUSION: The quality control of human immunoglobulin products in China market is generally fine, and the test results from different laboratories are basically consistent. Test result of insoluble particles in freeze-dried IVIG by microscopy counting method and light obscuration particle count test method show significant difference.