Objective To compare effects of different prefilter types in series with virus filter Viresolve® Pro（Vpro） on throughput and flux decline during monoclonal antibody virus filtration in the context of industrial scale-up assessment, and to evaluate differences in process performance among prefiltration schemes under fresh and freeze-thawed feed conditions, so as to provide a reference for the exploratory selection of prefilters for manufacturing-scale virus filtration.Methods Three prefilters, Millistak+® X0HC Media （X0HC), Millistak+® HC Pro X0SP Media （X0SP), and Viresolve® Prefilter （VPF), were evaluated in series with the virus filter Vpro under constant-pressure conditions. Filter throughput, flux decline, and consumable costs for a predefined scenario of processing 1 000 L over 3 h were compared among different filter combinations. In addition, particle size characteristics and selected quality attributes of fresh and freeze-thawed feed materials were analyzed by dynamic light scattering, size exclusion chromatography, and capillary electrophoresis.Results Under the conditions of this study, the three prefilter combinations showed generally comparable performance for fresh feed, with throughputs of approximately 700 L/m² and flux declines of no more than 3%. For freeze-thawed feed, combinations with smaller pore size prefilters (X0HC+Vpro and X0SP+Vpro) showed better throughput and flux stability, whereas the VPF+Vpro combination exhibited a flux decline of up to 77% and an experimental throughput of only approximately 567 L/m². Based on a scenario of processing 1 000 L of feed within 3 h, X0HC+Vpro showed the lowest estimated consumable cost per batch.Conclusion Under the specific product and experimental conditions investigated in this study, appropriate prefilter selection may help improve flux performance and increase throughput per unit membrane area, providing a reference for cost reduction and process robustness enhancement at industrial scale.
Objective To explore the activation conditions of human prothrombin with healthy human plasma as raw material, and to improve the specific activity and yield of the product.Methods DEAE sephadex A-50 gel adsorption method was used to separate healthy human plasma mixture to prepare human prothrombin. Five factors including Ca2+ concentration, initial human prothrombin concentration, activation temperature, initial pH and activation duration were investigated to optimize the activation conditions of human prothrombin. Using the established activation conditions, 3 batches of final products were produced through purification of human prothrombin, activation of human prothrombin, cation exchange chromatography and other steps, followed by comprehensive quality testing.Results The average yield of human prothrombin in the DEAE sephadex A-50 gel adsorption step was 81.98%. Under the conditions of CaCl2 mass concentration 0.6%, initial human prothrombin concentration 10 international unit (IU)/mL, temperature 2-8 ℃, initial pH7.45 and incubation for 12 d, 209.50 IU of human thrombin was obtained per unit of human prothrombin. The average recovery rate of human thrombin in the cation exchange chromatography step of the final products was 77.57%, and the average specific activity of human thrombin products was 1 078.06 IU/mg. Theoretically, about 6.40×107 IU of human thrombin product could be prepared per ton of plasma, and all three batches of final products met the Chinese pharmacopoeia standards.Conclusion The established activation conditions can efficiently activate human prothrombin, and the human thrombin product prepared by cation exchange chromatography has high specific activity and high yield, which can greatly reduce the production cost, and is suitable for large-scale production.
Objective To establish and verify a determination method for the modification rate of polyethylene glycol (PEG) in the production process of PEG-uricase. Methods PEG modification rate was calculated by cysteine (Cys) concentration.The relationship between absorbance and Cys concentration was explored. The standard curve and measurement range were established. The amount of chromogenic agent and reaction time were optimized. The specificity， precision， accuracy， and durability of the method were verified. The method was applied for sample detection. Results Cys concentration and absorbance showed a good linear relationship in the range of 5-200 μmol/L,with coefficent of determination ≥0.99. When the dosage of the chromogenic agent was 10 μL (resulting in a final concentration of approximately 0.2 mg/mL), the Cys concentration was close to the theoretical concentration, and the recovery rate met the acceptable criteria. Temperature had little effect on the results, so room temperature was recommended. The method showed good precision and accuracy. The recovery rate for accuracy was between 90% and 110%, and all relative standard deviations (RSDs) values were less than 10.0%. Blank solvent and residual PEG had no effect on the detection system, indicating good specificity. When plate was read within 10-22 min after adding the chromogenic agent, the inter-batch RSD of Cys concentration was 5.1%, which met the acceptable criteria, indicating good durability of the method. The PEG modification rates of uricase samples from 3 different batches were 98.8%, 98.4% and 98.8%, respectively, and the RSD among batches was 0.2%. The difference among batches was small and the results met the quality requirements.Conclusions A spectrophotometric method for the determination of PEG modification rate of uricase in PEG-uricase production process is established. This method possesses good linearity, specificity, precision, accuracy, and durability.
Objective To construct a recombinant measles virus （MV） expressing rubella virus (RV) virus-like particle (VLP) using the MV Shanghai-191 vaccine strain (MVS191) as a vector.Methods The nucleic-acid sequence of RV open reading frame 2, containing capsid (C) and envelope (E) 1/2, was cloned into pT7-MVS191 vector to construct pT7-MVS191-RVS. To rescue the recombinant virus, pT7-MVS191-RVS was co-transfected into BSRT7 cells with 3 auxiliary plasmids expressing MV nucleoprotein, phosphoprotein, and large polymerase, respectively,and pCDIBP-T7RNAP expressing T7 RNA polymerase. Then, Vero cells were infected with the recombinant virus for amplification. Reverse transcription PCR and gene sequencing were used to identify the insertion of exogenous genes in the recombinant virus. Western blot was used to detect the expression of RV C protein. RV VLPs were purified by agarose gel filtration chromatography, and Western blot was used to analyze the assembly of VLPs in the elution peak. Kunming mice were immunized with the recombinant virus, and Western blot, ELISA, and micro-cytopathic effect inhibition assay were used to detect anti-RV antibodies, anti-RV antibody titers, and anti-MV neutralizing antibody titers in mouse serum, respectively.Results Gene sequencing demonstrated that the C-E2-E1 segment of 3 189 bp was inserted into the recombinant virus. Expression of RV C protein was detected by Western blot in the recombinant virus. RV C protein was detected in the void water elution peak. The anti-RV antibody titer induced by recombinant virus MVS191-RVS in the serum of immunized mice was equivalent to that of RA27/3 vaccine strain, which was 1∶32 000, and measles neutralization antibody titer was ＞1∶320.Conclusion A recombinant virus MVS191-RVS expressing RV VLP is successfully obtained using reverse genetics technology.
Objective To simulate the aseptic processing of self-prepared aluminum phosphate adjuvant in the formulation manufacturing process, perform secondary autoclaving on the adjuvant at different storage time points, and observe changes in the quality characteristics of the adjuvant before and after secondary autoclaving, so as to provide basic data for the storage, application, and shelf life setting of aluminum phosphate adjuvant.Methods Three batches of industrial-scale self-prepared aluminum phosphate adjuvants were stored at (25 ±2) ℃. Samples were taken regularly at 0, 1, 3, 6, 9, 12, 18, and 24 months for secondary autoclaving (121 ℃, 30 minutes). The quality characteristics of the adjuvant were tested before and after autoclaving, including appearance, pH, adsorption rate, particle size and distribution, point of zero charge (PZC), Zeta potential, phosphorus content in the supernatant, and P/Al molar ratio.Results When stored at (25±2) ℃ for 24 months, all quality characteristics of 3 batches of aluminum phosphate adjuvants remained stable. After secondary autoclaving at different storage time points, the appearance of 3 batches of the adjuvants met specifications, and the pHs were maintained at 6.33-6.44. The adsorption rates were maintained at 55.8%-65.9%. Particle sizes at the cumulative distribution of 10%, 50%, and 90% were kept within 2.166-2.574 μm, 3.866-4.539 μm, and 6.250-8.049 μm, respectively. PZCs were maintained at 4.33-4.75, and the absolute values of Zeta potential were maintained at 32.4-36.0 mV; the phosphorus contents in the supernatant were maintained at 0.15-0.16 mg/mL, and the P/Al molar ratios were maintained at 0.93-1.07.Conclusion The self-prepared aluminum phosphate adjuvant maintains stable properties during 24 months of storage at (25±2) ℃, and secondary autoclaving performed at different time points throughout the storage period does not affect its properties.
Objective To develop and validate a fluorescent PCR assay capable of synchronously detecting African swine fever virus (ASFV), porcine parvovirus (PPV) and pseudorabies virus (PRV) in immune swine plasma.Methods Specific primers and probes and standard plasmids were designed according to national standards of the above 3 viruses. The recombinant plasmids were identified and standard curves were plotted using these plasmids. The linearity, specificity, sensitivity and reproducibility of the developed method were validated and tested on 491 clinical samples of immune swine plasma.Results The coefficients of determination of the standard curves for ASFV, PPV, as well as PRV-glycoprotein H and PRV-glycoprotein E genes were 0.999 1, 0.999 8, 0.999 5,and 0.999 9, respectively, and the negative-positive compliance rate was 100%. The limits of detection for pig plasma samples were all 25 copies/μL, the detection limit of 5-sample pooled testing were 125 copies/μL for all targets, and the coefficients of variation for intra- and inter-batch were all <5%. No ASFV or PRV was detected in 491 samples, and PPV was detected in very few samples.Conclusion The fluorescent PCR assay developed in this study has good linearity, high specificity, high sensitivity and good reproducibility.
Objective To develop and validate a high performance liquid chromatography (HPLC) method for the detection of QS-21 content in adjuvants.Methods Adjuvant was demulsified by chloroform and isopropanol mixture(3∶7, volume ratio) with a demulsification ratio of 1∶1. Chromatographic separation was performed on a C18 column with gradient elution using aqueous solution containing 0.05%(volume ratio) trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile-water. The flow rate was 1.0 mL/min, injection volume was 10 μL, column temperature was 25 ℃, and detection wavelength was set at 210 nm. The system adaptability, linearity and range, accuracy, limits of detection and quantification, and specificity of established method were verified.Results This method demonstrated good system suitability. The relative standard deviations (RSDs) of both retention time and peak area of QS-21 chromatographic peak from 3 repeated tests were all less than 2.0%. The standard curve exhibited excellent linearity, with coefficients of determination ranging from 0.999 6 to 0.999 8. At 3 spiked concentrations levels (12.5, 25.0 and 37.5 μg/mL), the spiked recovery rates of QS-21 were between 96.0% and 114.4% with a RSD of 5.5%. The limit of detection was 1 μg/mL and the limit of quantification was 3 μg/mL.No QS-21 peak was observed in control solution. Conclusion The established QS-21 content detection method features simple operation, good linearity, high accuracy, and strong specificity, and can effectively detect the QS-21 content in adjuvants to support quality control of adjuvants.
Objective To establish an enterovirus 71 (EV71) working reference for quality control in the purity test of inactivated EV71 vaccine (Vero cell) bulk by high-performance liquid chromatography (HPLC), and to study the long-term stability of this working reference.Methods The EV71 working reference was prepared from the inactivated and purified virus bulk of EV71 inoculated in Vero cells, and was subjected to quality control tests, including sterility and protein content determination. The purity of EV71 working reference was calibrated using HPLC. To evaluate its long-term stability, the reference was stored at 2-8 °C for 72 months, with purity assays conducted at specified time points.Results The sterility and protein content of EV71 working reference met the specified requirements. Its calibrated purity was determined to be 98.02%-100.00%, meeting the quality control criteria for working reference. After storage at 2-8 °C for 72 months, the viral protein peak profile remained consistent, with retention time of 6.861-7.473 min and relative standard deviation (RSD) of 2.6%. The purity RSD was 0.1%, with no significant changes observed.Conclusion The established working reference for EV71 purity exhibits good long-term stability and can be used for the purity testing of inactivated EV71 vaccine (Vero cell) bulk.
The interaction between bone marrow mesenchymal stem cells (BMSCs) and hydrogel scaffolds is a research hotspot in the field of bone tissue engineering. In this paper, the molecular mechanism of hydrogel scaffold regulating osteogenic differentiation of BMSC is systematically reviewed, focusing on its role in coordinating signaling pathways, improving immune microenvironment and guiding cell behavior. Studies have shown that hydrogels regulate key signaling networks such as Wnt, adenosine monophosphate-activated protein kinase-UNC-51-like kinases 1-autophagy axis, Hedgehog and Nel-like molecule-1 through their physical and chemical properties such as stiffness and composition, thereby activating Runt-related transcription factor 2, Osterix, and other osteogenic core transcription factors. Functionalized hydrogels can also be loaded with immunomodulatory factors or bioactive ions such as IL-4 to induce macrophage polarization to M2 type, inhibit inflammation and release factors such as vascular endothelial growth factor and bone morphogenetic protein 2, reshape the pro-regenerative immune microenvironment, and enhance cell adhesion and directional migration. These mechanisms jointly overcome the problems of inflammation-osteogenesis negative feedback and low stem cell homing efficiency, and provide theoretical support for the clinical transformation of intelligent bone repair scaffolds. In this paper, the performance differences of natural, synthetic, and composite hydrogels as regulating carriers are further compared to provide a basis for material selection.
T cell-mediated antitumor immune responses play a central role in the treatment of solid tumors. In recent years, multiple T cell-based therapeutic strategies, involving the expansion and adoptive transfer of tumor-reactive T cells, the engineering of antigen recognition in transferred T cells, and the in vivo redirection of endogenous T cells, have achieved important clinical advances and regulatory milestones in solid tumors. This review summarizes the major technological approaches and representative evidence for T cell-based therapies for solid tumors, focusing on tumor-infiltrating lymphocyte therapy, T-cell receptor (TCR)-engineered T-cell therapy, TCR-based CD3 T-cell engagers, chimeric antigen receptor T-cell therapy, and antibody-based CD3 T-cell engagers. Based on products approved, re-approved, or under pivotal development, this review compares the clinical translation patterns of different modalities and highlights structural constraints in target population coverage, safety management, manufacturability, access, and evidence maturity. Overall, precise patient selection, manageable immune-related toxicities, and mature manufacturing and supply systems will be key determinants of sustainable clinical translation.
