Objective: To study the heavy metals and harmful elements and the arsenic (As) speciation in Rukuaixiao preparations and evaluate the As risk. Methods: Residues of Cr, Ni, Cu, As, Cd, Hg, and Pb in 78 batches of Rukuaixiao preparations were determined by microwave digestion-inductively coupled plasma-mass spectrometry (ICP-MS). High performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) was employed to analyze six As forms. The hazard index (HI) was adopted to evaluate the risk of As in 10 batches of samples. Results: The two methods demonstrated good linear relationship, precision, repeatability, and recovery. The content of seven heavy metals varied greatly among the samples from different manufacturers. The As speciation and valence states in 10 batches of samples from E company were analyzed. The results showed that total As content was high. HPLC-ICP-MS results showed that trivalent and pentavalent As exhibited high toxicity. The HI<1 indicated that the As risk was controllable. Conclusion: The established method for the determination of seven heavy metals and harmful elements and six forms of As demonstrates high efficiency, rapidity, accuracy, and stability. This study provides data support for the formulation of the limit standards for the heavy metals and harmful elements in Rukuaixiao preparations.

Objective: This paper aims to establish a single bioanalytical method applicable for detecting anti-drug antibodies to insulin glargine and its biosimilar. Methods: The enzyme-linked immunosorbent assay was established by using biosimilars as both the capture antigen and the competitive antigen in the confirmation assay, as well as peroxidase-conjugated recombinant protein A/G as the detection reagent. The method validation was carried out from the aspects of antigen equivalence, screening cutoff, titer cutoff, confirmatory cutoff, sensitivity, precision, selectivity, hook effect, stability, and system adaptability. Results: Antigen equivalence (titration similarity and inhibition similarity) cross validation showed that the binding ability of biosimilars and reference drugs exhibited similar binding ability to positive antibodies. The screening cutoff factor was 1.186; the titer cutoff factor was 1.454; the confirmatory cutoff was 10.7%. The sensitivities of the screening assay and confirmatory assay were 8.9 ng · mL^-1 and 7.7 ng · mL^-1, respectively. The intra-plate and inter-plate relative standard deviations (RSDs) were less than 20% (The RSD of intra-plate precision of inhibition rate in the negative samples in the confirmation assay was 20.0%-22.8%, excluded). There was no hook effect in the range of 0.8-6.4 μg · mL^-1. The samples were stable at room temperature for 3 h, refrigerated (2-8 ℃) for 1 d, and at -70 ℃ after four freeze-thaw cycles. Selectivity, drug tolerance, and system suitability were satisfactory. Conclusion: In this study, a single bioanalytical method applicable for detecting anti-drug antibodies to insulin glargine and its biosimilar is successfully established by the antigen equivalence verification of biosimilar and reference drugs.

Objective: To establish an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ MS/MS) method for the simultaneous determination of 10 aminoglycoside antibiotics in water. Methods: Following alkaline desorption of particulates in the water sample, heptafluorobutyric acid was added to form an ion pair with the target analytes, and then the ion pair was determined by the UPLC-QQQ MS/MS method. The analytes were separated on a Poroshell 120 SB-C₈ chromatographic column (100 mm×3.0 mm, 2.7 μm) by gradient elution, with the mobile phase of 1 mmol · L^-1 ammonium acetate and 0.1% (volume fraction) formic acid aqueous solution and acetonitrile. Then, the target analytes were ionized by electrospray ionization in positive ion mode, detected in multiple reaction monitoring mode, and quantified by the internal standard method. Key influencing factors, including sample stability, storage container, particulate matter in samples, solution pH, filter membranes, residual chlorine content in samples, and liquid chromatographic conditions, were systematically evaluated, and spiked water samples of five types of matrices were determined. Results: Samples can be stored sealed, refrigerated, and protected from light at below 4 ℃ for at least seven days, and plastic containers shall be used for storage. Aminoglycoside antibiotics tended to be adsorbed onto particulate matter, so alkaline desorption was required, and the filtrate must be adjusted to neutral before determination. A 0.22 μm polypropylene filter membrane shall be used for sample filtration. Residual chlorine can cause loss of the target analytes, and ascorbic acid must be added immediately after water sample collection. After optimizing instrumental conditions, limits of detection ranged from 1.0 to 1.7 μg · L^-1. Recovery tests were conducted using the standard addition method. Except for spectinomycin, which showed slightly lower recoveries in complex matrices (with average recoveries of 42.8% and 41.0% in domestic sewage and industrial wastewater, respectively), the recoveries of the other compounds in all matrices, as well as those of spectinomycin in pure water, groundwater, and surface water matrices, ranged from 60.0% to 127.0%. The relative standard deviations (RSDs) (n=6) were between 1.3% and 16.0%. Conclusion: All performance parameters of this method satisfy current quality-control requirements for environmental antibiotic monitoring and thus are of practical referential significance.

Objective: To develop a biological activity reference material for recombinant COVID-19 vaccine (CHO cell) for the evaluation of in-vitro relative potency and in-vivo potency in mice for recombinant COVID-19 vaccine (CHO cell). Methods: One qualified bulk of recombinant COVID-19 vaccine (CHO cell) was selected. After calibration of protein content, an aluminum adjuvant was added in the bulk to prepare one batch of vaccine as the candidate biological activity reference material. After passing the finished product test, the candidate biological activity reference material was subjected to homogeneity tests. Five independent experiments using one batch of phase Ⅲ clinical vaccine as the biological activity reference material were conducted to calibrate the in-vitro relative potency and in-vivo potency in mice for candidate biological activity reference material. The stability of the candidate biological activity reference material was analyzed by storage at (37±2) ℃ and (5±3) ℃. The phase Ⅲ clinical batch vaccine and the candidate biological activity reference material were used to test the applicability of the candidate biological activity reference material by evaluation of the in-vitro relative potency and in-vivo potency of six batches of vaccines. Results: All test items of the biological activity reference material for the recombinant COVID-19 vaccine (CHO cell) complied with the requirements. The filling volume was not less than the labeled amount, with the filling precision within ±1%. The RSD values of D10, D50, and D90 reflecting particle size distribution were 0.45%, 0.39%, and 0.54%, respectively, and that of antigen content was 2.7%. The phase Ⅲ clinical vaccine, when being calibrated as a candidate reference material, demonstrated the in vitro relative potency and in-vivo potency in mice no less than 0.5, with the geometric mean values of 1.0 and 1.0 and the geometric coefficients of variation of 8.9% and 31%, respectively. After storage at (37±2) ℃ for 28 days and (5±3) ℃ for 36 months, the in-vitro relative potency and the in-vivo potency in mice showed no significant upward or downward trends, indicating good stability. The results of the in-vitro relative potency and in-vivo potency in mice tests for the six batches of vaccines using phase Ⅲ clinical vaccine and the candidate biological activity reference material ranged from 0.9 to 1.0 and 0.7 to 1.4, respectively, indicating former and candidate biological activity reference materials had good consistency. Conclusion: The biological activity reference material for recombinant COVID-19 vaccine (CHO cell) can be used for evaluation of the in-vitro relative potency and the in-vivo potency in mice for this vaccine.

Objective: To conduct a qualitative study on the chemical components of Ganlu Xiaodu Dan and its blood- and brain-entering components using ultra-high-performance liquid chromatography-quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry (UHPLC-Q Exactive Orbitrap HRMS). Methods: UHPLC-Q Exactive Orbitrap HRMS was employed to identify the chemical components of Ganlu Xiaodu Dan. Twelve male Sprague-Dawley (SD) rats were randomly divided into a blank group and a Ganlu Xiaodu Dan group. The Ganlu Xiaodu Dan group received an intragastric administration of the aqueous decoction at a raw herb dose of 1.8 g · kg^-¹, while the blank group received an equivalent volume of purified water. Plasma samples were collected 1.5 h post-administration. Using an ACQUITY UPLC HSS T₃ column (100 mm×2.1 mm, 1.8 µm) at a column temperature of 40 ℃. The mobile phase consisted of 0.1% formic acid in water and acetonitrile with gradient elution at flow rate of 0.30 mL · min^-1. The injection volume was 6 µL. Electrospray ionization (ESI) was applied with a scanning range of m/z 100-1 500. Combined with information from a reference database, qualitative analysis of blood-entering components was performed in both positive and negative ion modes. Results: A total of 195 compounds were identified in the 80% methanol extract of Ganlu Xiaodu Dan, and 66 prototype components were detected in serum. The fragmentation patterns of baicalin, apigenin-7-glucoside, isorhamnetin-3-O-galactoside, biochanin A, 28-deacetylbelamcandal, plantamajoside A, phillyrin A, aurantio-obtusin, neochlorogenic acid, chrysosplenol, mangiferin, and chuanbeiketonine were analyzed. Conclusion: This study enabled the rapid characterization and identification of multi-category in vitro chemical components and blood-entering components of Ganlu Xiaodu Dan, providing a reference for research on its pharmacodynamic material basis and quality control.

In recent years, the abuse of etomidate has become increasingly serious. With etomidate being listed in the control catalog, the synthesis of non-scheduled analogues by modifying the chemical structure has become an emerging abuse trend to evade regulation, posing a serious threat to public health and safety. In the face of the increasing types of abuse, it is particularly important to strengthen the regulation and detection of etomidate and its analogues. Based on a systematic summary of the pharmacological and toxicological properties of etomidate, this paper focuses on the structure-activity relationships and detection methods of etomidate and its analogues, with the expectation of providing references for the establishment of control strategies and analytical methods for etomidate.

Objective: To establish a ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of 13 chemical components (terrestrosin K, phellodendrine hydrochloride, imperatorin, isoimperatorin, prim-O-glucosylcimifugin, buddleoside, verbascoside, liquiritin, wogonin, geniposide, luteolin-7-O-glucoside, paeoniflorin, and baicalein) in Mingmu Jili pills. Methods: The separation was performed through a Waters Acquity BEH C₁₈ column (100 mm×2.1 mm, 1.7 µm) with a column temperature at 40 ℃. The mobile phase consisted of acetonitrile-5 mmol · L^-1 ammonium formate (containing 0.1% formic acid) with gradient elution at a flow rate of 0.3 mL · min^-1. The injection volume was 1 µL. The detection was carried out by electrospray ionization (ESI) source combined with multiple reaction monitoring (MRM) in positive and negative ion modes. Results: The 13 components showed good linear relationships in their respective concentration ranges (r>0.999 0). The method demonstrated high precision, repeatability, and stability. The spiked recovery rates ranged from 93.7% to 110.0%, with the relative standard deviation (RSD) of 0.28%-9.7%. The content of 13 components in 13 batches of Mingmu Jili pills was 11.94-75.55, 66.88-156.32, 57.18-87.83, 18.57-27.68, 20.20-196.20, 230.73-485.25, 539.71-3 061.53, 24.30-87.02, 314.32-973.51, 297.45-1 736.38, 61.45-473.48, 397.44-1 275.18, and 331.81-4 091.77 µg · g^-1. Conclusion: The established method is efficient, simple, accurate, and reproducible, which can provide a reference for the quality control of Mingmu Jili pills.

Objective: To establish an ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ MS) method for the rapid and simple qualitative identification of Qizhi Tongluo capsules. Methods: Gradient elution was performed through a Waters ACQUITY^TM UPLC Hss T3 C₁₈ column (100 mm×2.1 mm, 1.8 μm) with the mobile phase composed of acetonitrile and 0.1% formic acid aqueous solution at the flow rate of 0.3 mL · min^-1 and the column temperature of 35 ℃. The chromatographic effluents were detected by mass spectrometry in positive and negative ion modes. Results: The specific chromatograms showed 32 common characteristic peaks from 13 Chinese herbal medicines. The structures of 23 characteristic components were confirmed by comparison with reference substances. RSDs of the relative retention time of 32 characteristic peaks in 10 batches of samples were all less than 1.0%, indicating that the 32 characteristic peaks were stable. Conclusion: The established liquid chromatography-mass spectrometry (LC-MS) specific chromatogram method is simple, sensitive, and accurate, comprehensively reflecting the chemical composition characteristics of Qizhi Tongluo capsules and serving as a new method for rapid identification of Qizhi Tongluo capsules.

Objective: To establish a rapid and sensitive method for determining As (Ⅲ) in Realgar, a commonly used Chinese medicinal material with safety mainly influenced by As (Ⅲ), thereby ensuring its safe use. Methods: An iron-cobalt layered double hydroxide/transition metal carbide (Fe-Co-LDH/MXene) nanocomposite was synthesized by a hydrothermal method. A glassy carbon electrode (GCE) was modified with this nanocomposite to fabricate a sensor (Fe-Co-LDH-MXene/GCE). The materials were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The sensor performance under different solution conditions was systematically evaluated by electrochemical methods. Furthermore, the stability and repeatability were assessed. Results: A nanocomposite with a distinct layered structure was successfully prepared. In the neutral aqueous solution, the sensor demonstrated excellent electrochemical responses to As (Ⅲ). A good linear relationship was observed between the response signal and As (Ⅲ) concentration within the range of 100-500 μg · mL^-1. The sensor showed good stability and repeatability. Moreover, it performed well in the determination of As (Ⅲ) in Realgar samples. Conclusion: The electrochemical sensor enables rapid and accurate determination of As (Ⅲ) in Realgar under neutral conditions, with simple operation and high sensitivity. It offers a new method for quality control of Realgar-containing Chinese medicine preparations.

Objective: To establish a rapid coculture and detection method for six pathogenic bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella paratyphi B, Pseudomonas aeruginosa, and Burkholderia cepacia) in cosmetics and raw materials based on the gene membrane chip technology and improve the biological safety assurance capability and risk screening efficiency of the product. Methods: The co-culture medium was used for co-culture of the six pathogenic bacteria, and specific primers and probes for the six pathogenic bacteria were designed. Multiplex PCR amplification was performed on the sample nucleic acid, and the gene membrane chip technology was employed for further product hybridization and analysis. Results: Specificity tests showed that the six pathogenic bacteria developed color only at their corresponding target spots, while the four negative template bacteria (Listeria monocytogenes, Bacillus cereus, Vibrio vulnificus, and Shigella spp.) showed no color development, indicating no cross-reaction and good probe specificity. At primer concentrations of 0.20/0.30 μmol · L^-1 (internal control) and 0.067-0.75 μmol · L^-1 (for the six targets), the color development intensities of all targets were uniform. The limit of detection (LOD) test showed that the established method achieved an LOD of 1.0×10⁵ copies · mL^-1 for all six pathogenic bacteria. The test results of nine batches of samples were all negative, consistent with those obtained by conventional methods. The established method specifically detected the corresponding targets in the spiked samples without cross-interference, meeting the requirements for routine testing. Conclusion: A co-culture method for six pathogenic bacteria in cosmetics and a multiplex detection method based on PCR-gene membrane chip technology were established. This method exhibits strong specificity, good stability, and high sensitivity, ensuring the quality and safety of cosmetics and raw materials. It can be further applied to the detection of cosmetics in factories and markets, enriching the detection methods for pathogenic bacteria in the cosmetics industry and providing a rapid and powerful technical means for detection.

Objective: To develop a LC-MS method for the quantitation of three free fatty acids—lauric acid, myristic acid, and oleic acid—in antibody pharmaceuticals based on the analytical quality by design (AQbD) principles outlined in the ICH Q14 guideline on analytical procedure development, and to perform subsequent method validation. Methods: The analytical target profile (ATP) was initially established. Based on literature review and laboratory experience, a 1290 UHPLC (ultra-high performance liquid chromatography) system coupled with an SQ- MS (single quadrupole mass spectrometer) was selected to develop the method. All operational steps, spanning from sample preparation to LC-MS detection, that could potentially influence analytical method performance were comprehensively identified, and their potential impact on method accuracy and precision was systematically assessed. With respect to the control strategy, factors associated with high risk or high operational cost were subjected to stringent control. Experimental investigations were conducted on chromatographic column selection, mobile phase composition, gradient or isocratic elution modes, column temperature, as well as mass spectrometric parameters including capillary voltage, fragmentor voltage, and nebulizer pressure. A quantitative analytical method suitable for the degradation products of polysorbate (Tween) was developed and subsequently validated in accordance with ICH Q2 requirements. Results: Methodvalidation demonstrated that the established LC-MS method for free fatty acid quantification exhibited spike recoveries ranging from 82.3% to 106.6% for lauric acid, myristic acid, and oleic acid. The linear range spanned from 0.01 to 2.00 μg · mL^-1, with a lower limit of quantitation (LLOQ) below 0.01 μg · mL^-1. No interference from proteinaceous components or excipients within the antibody formulation was observed. The method was applied to analyze the concentrations of lauric acid, myristic acid and oleic acid in both within-specification and expired antibody preparations, as well as in forced degradation samples from two investigational antibody formulations. Notably, significantly elevated levels of lauric acid, myristic acid, and oleic acid were detected in the forced degradation samples, indicative of polysorbate degradation. The method performance met the predefined requirements specified in the ATP, rendering it suitable for the quantitative detection of free fatty acids in diverse antibody pharmaceuticals. Conclusion: This study presents a case study of LC-MS method development employing AQbD principles. Through rigorous control of experimental materials, measurement procedures, and operational protocols, and via experimental verification of permissible ranges for mass spectrometer gas flow and voltage parameters, the final method specifies mass spectrometric parameters as acceptable operational ranges rather than discrete fixed values. This approach holds substantial value for the method lifecycle, particularly in the context of change management. The developed method is applicable to the quantification of free fatty acids derived from polysorbate degradation in antibody products. Furthermore, this case provides a meaningful reference for the application of LC-MS methodology in pharmaceutical quality control.

Objective: To conduct microbial monitoring and analysis of pharmaceutical water systems from three enterprises, and preliminarily explore culture media suitable for subculture of microorganisms isolated from these systems. Methods: A total of 130 microbial strains were isolated from the pharmaceutical water systems of three enterprises. Bacterial and fungal isolates were identified by 16S rDNA and ITS rDNA sequencing, respectively. Phylogenetic trees were constructed for frequently detected genera to analyze potential contamination sources. Furthermore, subculture studies were performed on all the 130 strains with three different culture media and the modified sterilization method. Results: The microorganisms most frequently detected in the water systems of the three pharmaceutical enterprises were Methylobacterium, Ralstonia, and Bradyrhizobium. Subculture results of the 130 strains showed that R3A and R2A media performed better than Tryptic Soy Agar (TSA). The sterilization method was modified as autoclaving phosphate and agar components separately before mixing in the R3A medium, which improved the growth rate of some slow growing microorganisms on RXA media. Conclusion: Microorganisms in pharmaceutical water systems show certain commonality. RXA media may be more suitable for subculture of microorganisms from pharmaceutical water systems. For some microorganisms susceptible to oxidative stress, modifying the sterilization method for the culture medium can promote their growth.

Objective: To optimize and validate the specific neutralization test method for the Corynebacterium diphtheriae strain C7 (β197) (strain C7), thereby improving the clarity of results and shortening the detection time. Methods: Strain C7 was inoculated in both wide and narrow patterns. A filter paper strip was placed either on the surface of culture medium or embedded within the culture medium. Seven types of culture media—peptone A Elek’s medium, peptone B Elek’s medium, peptone C Elek’s medium, peptone D Elek’s medium, peptone E Elek’s medium, nutrient agar medium, and commercially available Elek’s medium—were screened. Diphtheria antitoxin was prepared at four concentrations: 2 000, 1 500, 1 000, and 500 Lf · mL^-1. Petri dishes were poured with 15, 25, and 35 mL of culture medium, and strain C7 was inoculated at distances of 3, 5, 7, 9, and 11 mm from the filter paper strip. Equine serum or bovine serum was added to the basal culture medium. The key conditions for the specific neutralization test of strain C7 were systematically optimized and screened. Additionally, the specificity and repeatability of the optimized method were validated. Results: The optimal conditions for the specific neutralization test were determined as follows: strain C7 inoculated in a narrow pattern, filter paper strip placed on the surface of culture medium, peptone C Elek’s medium used as the neutralization medium, filter paper strip saturated with diphtheria antitoxin at 1 500 Lf · mL^-1, petri dish poured with 15 mL medium, strain C7 inoculated at a distance of 3 mm from the filter paper strip, and bovine serum added to the basal medium. The validation results demonstrated that the optimized method had good specificity and repeatability. Conclusion: The specific neutralization test method optimized in this study is stable and reliable. It significantly improves the clarity of results and detection efficiency for strain C7, providing more efficient and reliable technical support for the laboratory identification of this bacterium and the quality control of related vaccines.

Objective: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of 31 chemical drugs in disinfection products. Methods: A Waters ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) with the column temperature of 30 ℃ was used for gradient elution with the mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL · min^-1. The injection volume was 2 μL. Mass spectrometry acquisition was conducted in positive and negative ion modes with electrospray ionization (ESI) and analyses were detected in the multiple reaction monitoring (MRM) mode. Results: The 31 chemical drugs were effectively separated within 13 min, and the limit of detection was 0.05 mg · kg^-1. A good linear relationship (r>0.99) was observed between peak area and concentration within the range of 0.125-2.5 mg · kg^-1. The spiked recovery rate ranged from 73.2% to 114.0%, with the relative standard deviation (RSD)≤12.2% (n=6). Conclusion: This method is rapid, accurate, sensitive, and specific, allowing the simultaneous detection of 31 chemical drugs in disinfection products and providing technical reference for the regulation of disinfection products.

Focusing on the quality control items specified in the quality standards for antibody products, this paper systematically reviews the latest advances in analytical technologies, encompassing the detection of key quality attributes such as size heterogeneity, charge heterogeneity, glycan heterogeneity, biological activity (including binding/functional activity assays and reporter gene-based methods), excipient content, protein content, peptide mapping analysis, and the quantification of host-related residual impurities. The detection principles, key considerations for method development, and applicable scenarios of the aforementioned technologies are detailed. In addition, the technical advantages, core development points, and application progress of multi-attribute monitoring (MAM) technology for the characterization of post-translational modifications are expounded. The quality control methodology system and technical advances of antibody products summarized in this study can provide a valuable reference for the establishment of quality standards, optimization of quality control methods, and stability studies of antibody drugs, and further facilitate the improvement of quality control and regulatory standards in the research, development, and manufacturing of antibody products.

Objective: To determine the metabolic profiles of etomidate and its structural analogues metomidate, propoxate, and isopropoxate in human liver microsomes (HLMs). Methods: In a human liver microsome incubation model, four target compounds were separately added and incubated at 37 ℃ for 1 hour. The reaction was terminated by adding ice-cold acetonitrile. After centrifugation, the supernatant was collected, evaporated to dryness under nitrogen, and reconstituted. The processed samples were analyzed using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry in ESI⁺ mode. The mobile phases consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, with a Waters HSS T3 column used for separation. The IDA scan mode was employed to detect the parent compounds and their metabolites, aiming to investigate the metabolic pathways. Results: The biotransformation of the four compounds primarily involved pathways such as dealkylation, dehydrogenation, oxidation, and glucuronidation. In three real hair samples with positive results, the unchanged forms of etomidate, metomidate, and isopropoxate were detected, along with the same dealkylation metabolites and carboxylation products. Additionally, the characteristic metabolites of two compounds were detected (metabolites M3 yielded from etomidate and isopropoxate after the loss of the phenylethyl group). Conclusion: It is recommended that the unchanged form of etomidate and its metabolite M3, the unchanged form of metomidate, the unchanged form of propoxate and its metabolite M3, as well as the unchanged form of isopropoxate and its metabolite M3 can be used as biomarkers for the intake of the four target compounds.

Objective: To establish a cell line and detection method based on the reporter gene assay (RGA) for evaluating the biological activity of denosumab. Methods: The RANK-GloResponse NF-κB-RE-luc2P HEK293 cell line was constructed by transfecting HEK293 cells with plasmids carrying the RANK gene and the NF-κB response element (NF-κB-RE) coupled with a luciferase reporter gene (Luc2P) via transgenic technology. After optimizing experimental conditions including cell density, chromogenic assay conditions, quantity of RANK ligand (RANKL), and denosumab dilution gradient, the detection method was established. The accuracy, precision, linear range, specificity, and robustness of the method were validated in accordance with 9101 and 9401 of General Rules of the Pharmacopoeia of the People’s Republic of China, Four Volumes. Results: The RGA for detecting the biological activity of denosumab was successfully validated. Within the potency level range of 64%-156%, the relative bias (RB) of the method was within±12.0%. The regression equation had a slope of 1.047 6 and a correlation coefficient of 0.988 1. The maximum coefficient of variation (RSD) for precision and accuracy was 15.0%, and the maximum geometric coefficient of variation (GCV) for robustness across different cell passages, cell densities, and culture times was 7.9%. Cell passage stability tests confirmed that the probability (P) value exceeded 0.05 for up to 30 generations. Conclusion: This study successfully constructed a reporter gene-based cell line suitable for detecting the biological activity of denosumab. Methodological validation demonstrated its reliability, providing a robust technical support for the quality control of related biological products.

Objective: To establish an optimized bioassay for bFGF, namely the heparin sodium dilution method, aimed at improving the physiological relevance and stability of testing for heparin sodium-containing preparations. Methods: Based on the method described in General Chapter 3527 of the Chinese Pharmacopoeia (2025 Edition, Part IV), the assay was modified by introducing a maintenance medium supplemented with heparin sodium during the sample dilution step and shortening the cell starvation duration to 5 h. Both the optimized method and the standard pharmacopoeia method were employed to assay bFGF preparations with and without heparin sodium to compare their performance. Results: For heparin-containing preparations, the heparin sodium dilution method showed a significantly higher curve-fitting quality (0.998±0.001) and a wider dose-response interval (0.499±0.070) compared to the pharmacopoeia method (0.995±0.002, p<0.01, and 0.295±0.039, p<0.000 1, respectively). The EC₅₀ value (0.419±0.088) was significantly lower than that of the pharmacopoeia method (0.627±0.154) (p<0.000 1). No significant difference was observed in labeled potency between the two methods, and the optimized method demonstrated a satisfactory linear range. For heparin-free preparations, both methods met the correlation coefficient requirement (r>0.9) and showed good parallelism; however, the labeled potency measured by the heparin sodium dilution method (146.78%±10.0%) was significantly higher than that of the pharmacopoeial method (90.65%±2.8%) (p<0.001). Conclusion: The heparin sodium dilution method improves the fitting quality, sensitivity, and method stability of the four-parameter regression assay for heparin sodium-containing preparations. It serves as a valuable supplement to the pharmacopoeia method. These findings provide a more scientific and reliable approach for the quality control of bFGF preparations.

Objective: To establish reversed-phase high-performance liquid chromatography (RP-HPLC) and dual-wavelength size exclusion high-performance liquid chromatography (SE-HPLC) methods for effectively analyzing the conjugation status of simulated deconjugated antibody-drug conjugate (ADC) formulations, and to combine these methods with ultraviolet (UV) spectrophotometry for protein concentration determination, aiming to meet the requirements of formulation analysis. Methods: Group A formulations simulating partially deconjugated ADC were prepared by physically mixing intact ADC, small-molecule drug, and antibody. Corresponding control Group B formulations, without simulated deconjugation but with matched total antibody and total small-molecule drug concentrations, were also prepared. A UV spectrophotometric method was established to measure absorbance of Group A and B formulations at 252 nm and 280 nm. The drug-to-antibody ratio (DAR) was calculated according to the Beer-Lambert law to reflect the total antibody concentration and conjugation status. An RP-HPLC method was developed by gradient elution through a Zorbax Ellipse XDB-C₁₈ column (50 mm×2.1 mm, 3.5 μm) at 35 ℃ with 0.1% trifluoroacetic acid in water as mobile phase A and 0.08% trifluoroacetic acid in acetonitrile as mobile phase B at the flow rate of 0.5 mL · min^-1, autosampler temperature of 5 ℃, and detection wavelength of 252 nm. This method was used to detect free small molecules generated from deconjugation, indirectly analyzing the conjugation status of ADC. A dual-wavelength SE-HPLC method was established by elution through a TSK G3000SWXL gel column (7.8 mm×30 cm, 5 μm) with the mobile phase consisting of 15% isopropanol and 85% phosphate buffer (0.2 mol · L^-1 potassium phosphate, 0.25 mol · L^-1 potassium chloride, pH 7.0) at room temperature, the flow rate of 0.5 mL · min^-1, autosampler temperature of 5 ℃, and detector wavelengths of 252 nm and 280 nm. This method was used to directly calculate the DAR of ADC formulations by measuring the corresponding peak areas. Results: The total antibody concentrations in Group A and B formulations determined by UV spectrophotometry (n=3) were 5.14 mg · mL^-1 and 5.04 mg · mL^-1, respectively, with corresponding DAR values (n=3) of 3.45±0.03 and 3.47±0.02. The RP-HPLC method effectively detected the simulated free small molecules in Group A formulations, which were absent in Group B. The DAR values determined by dual-wavelength SE-HPLC for Group A and B formulations were 1.83 and 3.65, respectively. Conclusion: UV spectrophotometry can accurately determine the antibody concentration in this simulated deconjugated ADC formulation but cannot effectively determine its conjugation status. RP-HPLC can detect free small molecules in this simulated deconjugated formulation, indirectly proving the occurrence of simulated deconjugation. Dual-wavelength SE-HPLC can effectively and accurately determine the conjugation status of this simulated deconjugated formulation. Its combination with UV spectrophotometry for protein concentration determination shows promise in meeting the requirements of formulation analysis.