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Special Column for Quality Analysis of Biochemical
Editor: Invited Column Editor-in-chief:ZHENG Lu-xia
Biochemical drugs refer to a class of drugs for clinical prevention, treatment and diagnosis that are obtained from organisms such as animals, plants and microorganisms by means of extraction, isolation and purification, or by means of biochemical semi-synthesis/chemical synthesis, microbial synthesis or modern biotechnology, mainly including： amino acids and their derivatives, nucleotides and their derivatives polypeptides, proteins, enzymes and coenzymes, polysaccharides, lipids, etc Due to the basic biochemical components or analogs from living organisms, biochemical drugs have the characteristics of easy absorption by human body, good curative effect, small medicinal dose and low toxicity, and have been widely used in clinical practice for many years, playing an important role in the prevention and treatment of diseases Especially for diseases doing serious harm to human health and life, such as heart cerebrovascular diseases, tumors, diabetes and so on, biochemical drugs have their unique therapeutic effects They have also played an effective role in the fight against COVID-19 over the past two years Over the years, from animal organ extraction, chemical synthesis to today’s gene recombination technology, the biochemical pharmaceutical industry of our country has experienced tremendous development and changes, and has become an important part of the pharmaceutical industry Outputs of heparin and urinary extracts have been in the forefront of the world and exported to foreign countries, achieving considerable economic benefits Compared with the whole pharmaceutical industry, the biochemical pharmaceutical market share is relatively low in China However, with the increase of support for the development of biopharmaceutical industry in recent years, the development potential of biochemical drugs is huge, especially in the fields of peptides, proteins, enzymes, polysaccharides and so on, which are the important direction of current international new drug research and development Meanwhile, biochemical pharmaceutical enterprises are also facing many difficulties and challenges Facing the pressure of generic drug consistency evaluation policy and the new situation of innovative drug development, increasement of investment in research and development investment is needed in order to improve their product quality to meet the needs of the new market environment All these situations make scientific and reasonable technical standards and evaluation methods become more and more important.
The characteristics of biochemical drugs are different from the general chemical drugs, and the dosage form is mainly injection, which is a class of drugs with high risk and difficult quality control of biochemical drugs Their source is biologically diverse, the structure is difficult to confirm, the molecular weight is often not fixed, and the biochemical drugs extracted from organisms usually have composition uncertainty At the same time, the production process is complex, easy to introduce special impurities These characteristics increase the difficulty of biochemical drug quality research and control How to adhere to the global perspective, based on China’s national conditions, develop new detection technologies and methods, improve the technical support for the quality control of biochemical drugs, has become an urgent need to promote the production manufacturers to improve product quality and realize the scientific supervision of post-market drugs.
With the prosperous development of new modern analytical techniques in analytical chemistry, biology, immunology and other related fields, the combined application of multidisciplinary knowledge and techniques in the quality control of biochemical drugs has been realized Due to the particularity and complexity of biochemical drugs, their quality cannot be fully controlled by using a single technology It is necessary to use a combination of analysis methods including chemistry, spectroscopy, chromatography, mass spectrometry, polymer material science, enzyme kinetics, molecular biology, cell biology, immunochemistry and other disciplines In particular, according to the different characteristics of specific varieties and dosage forms, the development and establishment of some new technologies and methods with strong pertinence to ensure the biological activity and safety of their use is the development direction of improving the quality control level of biochemical drugs At the same time, considering the complex source of biochemical drugs, especially for the production of varieties involving the treatment, extraction and purification of the initial organs, tissues or secretions, the source and quality of the starting materials is also important, such as the origin of species and the risk of virus contamination Therefore, it is necessary to carry out strict control the quality of biochemical drugs from the source, to move forward the gate of quality risk control to the production process and source, and to strengthen the supervision concept of the whole life cycle, so as to comprehensively guarantee the quality of biochemical drugs.
Division of Biochemical Drugs and Biological Products, Shanghai Institute for Food and Drug Control (SIFDC), has applied for and undertook a number of research projects on the quality control of biochemical drugs during the 12th and 13th “Five-Year” plans, which included standardization study on the application of nucleic acid molecular identification technology in drug quality control, study on the effective substance basis of bone peptides drugs, demonstration study on the cause and control of visible particulates in injection of special varieties from animals or plants, revision of the tests of fats and fatty oils in the appendix of pharmacopoeia, determination of protein content (integration of pharmacopoeia part Ⅰ, Ⅱ and Ⅲ), etc , and the quality standard improvement project of raw materials and preparations of more than twenty biochemical varieties such as protamine sulfate and posterior pituitary We also participated in the completion of the national evaluation of nine biochemical drugs such as lysozyme and atosiban acetate. We are continuously committed to the development and application of a variety of new detection technologies and methods, providing more technical support for the quality control and scientific supervision of biochemical drugs, and promoting the high-quality and innovative development of the biochemical pharmaceutical industry.
Chinese Journal of Pharmaceutical Analysis has set up a special column of quality analysis of biochemical drugs in this period, specially organized for a number of professionals from, SIFDC to write 5 related papers This column focuses on new techniques and methods for the physical and chemical analysis of biochemical drugs, which include ion-exchange column chromatography, chromatography, mass spectrometry, micro-flow imaging, etc., aims to introduce the application of new instrumental analysis technology in the quality control of biochemical drugs by taking representative biochemical varieties as research examples, such as protamine sulfate, urokinase, lysozyme and atosiban acetate, and solve the technical difficulties in the quality control of biochemical drugs With the help of the professional platform and column contents of Chinese Journal of Pharmaceutical Analysis , we hope to provide novel quality control concepts and beneficial solutions for the quality research of biochemical drugs of our country, strengthen the role of new instrumental analysis methods to support supervision, promote the development of biomedicine with practical actions, and help the construction of healthy China.
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  • Special Column for Quality Analysis of Biochemical Drugs
    Determination of molecular fraction ratio in urokinase for injection by ion exchange chromatography combined with size exclusion chromatography*
    YAN Cui-xia, YIN Hong-rui, SHI Fang-liang, CHEN Gang, ZHENG Lu-xia, SHAO Hong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(1): 4-11. https://doi.org/10.16155/j.0254-1793.2023.01.01
    Abstract (25) PDF (6)   Knowledge map   Save
    Objective: To establish a determination method for molecular fraction ratio in the sample pretreatment of urokinase for injection using ion exchange chromatography(IEC) combined with size exclusion chromatography(SEC) so as to find a solution to fix the ratio of molecular fraction could not be controlled in the preparation's quality standard for a long time, the product quality of six domestic companies was investigated. Methods: Human serum albumin was removed by IEC for its distinguishing isoelectric point, and then the urokinase in the rest sample were measured by SEC. The chromatographic column was TSKgel G2000SWXL column(300 mm×7.8 mm, 5 μm) with 0.1 mol·L-1 sodium dihydrogen phosphate buffer(pH 3.0) as mobile phase, the flow rate was 0.5 mL·min-1, the column temperature was 35 ℃. The detection wavelength was 280 nm and the injection volume was 50 μL. Results: The urokinase at high and low molecular mass showed good linear relationship (r≥0.998 0) when the concentration of urokinase was in the range of 0.05 to 5.1 mg·mL-1. The detection limit of high molecular mass urokinase and low molecular mass urokinase were 1.5 μg·mL-1 and 5.1 μg·mL-1, respectively, and the limit of quantification were 5.1 μg·mL-1 and 16.9 μg·mL-1,respectively.The RSDs of precision, repeatability, stability test in 24 h were less than 4.0%. The recovery was 92.5%-97.0%, the relative content range of high molecular mass urokinase in all 18 batches of samples was 82.8%-96.5%, and low molecular mass urokinase was 3.5%-17.2%. Conclusion: The established ion exchange chromatography combined with size exclusion chromatography is applicable for the determination of molecular fraction in the preparation of urokinase for injection, and fills the gap in the current quality standards of urokinase for injection.
  • Special Column for Quality Analysis of Biochemical Drugs
    Analysis and study on the primary structure of the lysozyme using UPLC-Q TOF MS*
    YIN Hong-rui, ZHANG Ying, FANG Xin-xin, CHEN Gang, SHAO Hong, ZHENG Lu-xia
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(1): 12-19. https://doi.org/10.16155/j.0254-1793.2023.01.02
    Abstract (27) PDF (9)   Knowledge map   Save
    Objective: To analyze the primary structure of the lysozyme from three different domestic and foreign manufacturers using UPLC-Q TOF MS. Methods: The UPLC-Q TOF MS analysis was performed on a BEH300 C4(50 mm×2.1 mm, 1.7 μm) column and a BEH300 C18(150 mm×2.1 mm, 1.7 μm) column with 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid-acetonitrile solution as mobile phase B, with gradient elution at a flow rate of 0.2 mL·min-1. Q TOF MS electrospray positive ion mode was used, and for relative molecular mass detection, MS scan mode was applied. For amino acid sequence and disulfide bond connection, MSE scan mode was applied. Results: The measured relative molecular mass of the three lysozyme samples was consistent with theory; the results of peptide map showed that the amino acid sequences of all samples were consistent with the theory; the connection modes of disulfide bonds of all lysozymes were Cys6-Cys127, Cys30-Cys115,Cys64-Cys80 and Cys76-Cys94. Conclusion: UPLC-Q TOF MS method can be used to analyze the primary structure of the lysozyme.
  • Special Column for Quality Analysis of Biochemical Drugs
    Structure identification of the impurities in atosiban acetate injection using UPLC-MS*
    YIN Hong-rui, ZHANG Ying, YU Ji-wei, ZHENG Lu-xia, SHAO Hong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(1): 20-28. https://doi.org/10.16155/j.0254-1793.2023.01.03
    Abstract (25) PDF (6)   Knowledge map   Save
    Objective: To identify the structure of impurities in atosiban acetate injection using high performance liquid chromatography(HPLC), ultra-high performance liquid chromatography coupled with quadrupole tandem orbitrap mass spectrometry(UPLC-Q Orbitrap MS). Methods: The HPLC analysis was performed on an Inertsil ODS-2 C18(250 mm×4.6 mm, 5 μm) column with the mobile phase A consisted of acetonitrile-methanol-trifluoroacetic acid solution(pH 3.2) (15∶10∶75) and mobile phase B consisted of acetonitrile-methanol (60∶40), with gradient elution at a flow rate of 1.2 mL·min-1, and the detection wavelength was 220 nm. The UPLC Q-Orbitrap MS analysis was performed on a BEH300 C18(150 mm×2.1 mm, 1.7 μm) column with 0.1% formic acid solution as mobile phase A and 0.1% formic acid-acetonitrile solution as mobile phase B, with gradient elution at a flow rate of 0.2 mL·min-1. Samples were analyzed by electrospray ionization source with positive ion mode and Full MS/dd-MS2 scan mode. Results: 5 batches of atosiban acetate injection produced by five different manufactures were analyzed of related substances by area normalization method. The number of impurity peaks with content >0.1% was 9, 10, 13, 9 and 6, respectively, and the total impurity content was as follows: 0.35%, 0.63%, 0.63%, 0.32% and 0.18%. The impurity peaks in the samples of two enterprises with a large number of impurities were collected and identified. Among them, 9 impurity distillate were collected in the sample of one enterprise, and the structures of 12 impurities were identified, 13 impurity distillate were collected and in the sample of another enterprise, and the structures of 15 impurities were identified.The identified impurities mainly included deletion, insertion, misconnection, modification of peptide and so on. Conclusion: The impurity structure in atosiban acetate injection can be effectively identified by using UPLC-MS, the determination of impurity structure is helpful for the manufacturers to analyze the origin of their product's various impurity occurrence.
  • Special Column for Quality Analysis of Biochemical Drugs
    Determination of protamine sulfate content and identification of peptides by HPLC combined with LC-MS*
    HU Chuan-mei, LING Jin, YIN Hong-rui, ZHENG Lu-xia, CHEN Gang, SHAO Hong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(1): 37-44. https://doi.org/10.16155/j.0254-1793.2023.01.05
    Abstract (45) PDF (11)   Knowledge map   Save
    Objective: To establish a high performance liquid chromatography (HPLC) method for the simultaneous determination of principal components in protamine sulfate, and to employ LC-MS for the identification for the amino acids sequences of main peptides. Methods: The ThermoHypersil GOLD column (250 mm×4.6 mm, 5 μm) was selected for analysis with a gradient program of mobile phase A[0.1 mol·L-1 sodium dihydrogen phosphate solution(adjusted to pH 1.8 with phosphoric acid)]and B[acetonitrile-0.1 mol·L-1 sodium dihydrogen phosphate solution(adjusted to pH 1.8 with phosphoric acid) (6.5∶93.5, v/v)].The column temperature was 55 ℃. The UV detection wavelength was 214 nm and the flow rate was 1.0 mL·min-1. The injection volume was 100 μL. The LC-MS method adopted distillate collection method to extract and enrich each peptide, Waters ACQUITY UPLC Peptide BEH C18 column (15 cm×0.21 cm, 1.7 μm) was selected for liquid-mass spectrometry method, flow rate was 0.2 mL·min-1, the mobile phase A was 0.1% formic acid solution, mobile phase B was 5% acetonitrile-0.1% formic acid solution, gradient elution. Thermo Q-Exactive Plus, ESI ion source, cation detection mode, atomization voltage was 3 800 V, ion transfer tube temperature was 300 ℃, scanning range of m/z 200-2 000. Results: The HPLC method established in this paper could identify the protamine sulfate from salmon and herring provided by the two companies. The content of protamine sulfate was 94.4%-96.2% from salmon, and 88.4%-92.3% from herring, protamine sulfate purity determination result was 96% from salmon, and 66%-69% from herring. The amino acid sequence of the target peptide of protamine was retrieved and obtained from Uniprot protein database, and analyzed by ThermoBioPharmaFinder v3.0 software to map fragments ion peaks were matched with theoretical fragment ions of the target sequence, and the amino acid sequence identification results of 4 main peptides from salmon and 3 main peptides from herring were obtained. Conclusion: The method established in this paper can be used for the identification, content determination and purity analysis of protamine sulfate. The LC-MS method can be used for the identification of protamine sulfatepeptide sequences from salmon and herring.
  • Special Column for Quality Analysis of Biochemical Drugs
    Application of MFI in the detection of particulate matters in urokinase for injection
    XU Ming-ming, YAN Cui-xia, ZHOU Qin, CHEN Gang, ZHENG Lu-xia, SHAO Hong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(1): 29-36. https://doi.org/10.16155/j.0254-1793.2023.01.04
    Abstract (18) PDF (5)   Knowledge map   Save
    Objective: To evaluate the application of micro-flow imaging(MFI) in the detection of particulate matters in biochemical protein drugs(urokinase for injection), and to investigate the status of particulate matters in urokinase samples for injection from various manufactures, and the differences of particulate matters after resolution with two different solvents, and find out the main origin of particulate matters. Methods: The urokinase for injection was re-dissolved in either sterilized water for injection or sterilized normal saline for injection respectively, and the particulate matters were analyzed by MFI. The MFI methodology was validated, comparing with light obscuration method. Results: For 5 μm and 15 μm particulate standard detected by MFI, the counting accuracy(deviations) of particle count were 11.6% and 8.5%, precision(RSDs) were 1.6% and 3.8%, the accuracy (deviations) were 2.5% and 0.8%, precision(RSDs) were 0.6% and 0.1%. In the results of determination of particulate matters detected by MFI with the particle size of 2-10 μm were mainly proteinaceous, and the partide size of more than 10 μm were mainly non-proteinaceous. The result of non-proteinaceous particulate matters detected by MFI was similar to light obscuration method. The particulate matters in urokinase for injection produced by different manufactures were quite different, and the particulate matters for manufacturer A was more. There was no significant difference in the results of particulate matters after dissolved with two different solvents for samples from manufacturer A and C, and significant difference for samples from manufacture B and D. Particulate matters were less for samples re-dissolved with sterilized normal saline. Conclusion: MFI was first applied to detect particulate matters in biochemical protein drugs (urokinase for injection). It provides new ideas for exploring the source of particulate matters in biochemical protein drugs. The results of particulate matters in individual manufactures were close to the limit, suggesting that the manufactures need to strengthen research and development to improve product quality control. And manufactures can analyze the main sources of particulate matters from endogenous and exogenous cause according to the result of 2-10 μm range and particle size more than 10 μm.Reconstitution with different two solvents affected the results of particulate matters for some samples, and particulate matters are less for samples dissolved with sterilized normal saline. But the cause of the above results need further study.
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