Special column on the quality analysis of recombinant hormone drugs

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Editor: Invited Column Editor-in-chief: LIANG Cheng-gang

Recombinant hormones are pioneers among biotechnology products for clinical treatment, and their total global sales volume second only to monoclonal antibodies in current biotherapeutic proteins market. They played an irreplaceable role in clinical treatment of various chronic diseases and degenerative diseases, such as diabetes mellitus, reproductive diseases, and osteoporosis. Therefore, they are of great significance to national medical insurance, public health and other livelihood projects. Since the design basis of recombinant hormone drugs mainly comes from the active molecules of proteins and polypeptide naturally existing in human and animals bodies, after more than 100 years of research, human beings have established a comprehensive understanding of their molecular category, pharmacology and efficacy. Therefore, it is very difficult to develop innovative drugs based on the source of new targets and new molecular entities presently. International research and development hotspots mainly focus on long-acting, protein engineering modification and compound preparation products with great clinical value, such as insulin glargine, insulin lispro, insulin degludec, liraglutide and dulaglutide are all "blockbuster" biological drugs. Due to the large population base and gradually emerged aging problem in our country, the clinical demand for recombinant hormone drugs is still in the stage of rapid growth. Numbers of approved domestic products and relative manufacturers have increased significantly in recent years. Some clinical urgently needed new recombinant hormones, which still rely on imports, have already been submitted to the process of license.

With the development of biotechnology industry and the persistent support of the national drug regulatory agency, National Institutes for Food and Drug Control (NIFDC) has accumulated rich experience in the research of quality control analytic methods and quality standards of recombinant hormone drugs. Consequently, such monographs recorded in the national pharmacopoeia continue to maintain the international level. In addition, based on a number of creative studies carried out by NIFDC in polyethylene glycol modified recombinant hormone drugs, the first general monograph for “pegylated recombinant protein and polypeptide products for human use” in the world was successfully recorded in Volume Ⅲ of Chinese Pharmacopoeia 2020.

Due to the needs of development, the monographs of recombinant hormones were transferred to Volume Ⅲ since 2020. To achieve the goals, NIFDC undertook a series of standard improvement projects of the National Pharmacopoeia Committee. As a result, 10 monographs, including human insulin and its preparations, insulin glargine and its preparations, insulin lispro and its preparations, and human growth hormone for injection, were deliberately drafted or revised.

This special column includes 10 papers which written by relevant professionals of NIFDC. The content involves the strategy and technical considerations of the national standard research of recombinant hormone drugs, targeted research works carried out in the process of standard improvement, exploratory studies on in vitro bioassay methods aimed to method replacement, and the development of national reference substance. The purpose is to provide reference for how to make the national standard of recombinant hormone drugs further meet the strategic needs of supporting regulation, promoting industry development and international mutual recognition. Meanwhile, we hope that this special column may provide some inspiring ideas for the quality design and R & D decision-making in the development of recombinant hormone products and may help native listed products to enhance the global value.

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Chinese Journal of Pharmaceutical Analysis (10)

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2022 (10)

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Special column on the quality analysis of recombinant hormone drugs (10)

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Special column on the quality analysis of recombinant hormone drugs
Study on modification sites of novel site-specific modification of pegylated recombinant human growth hormone^*

LI Jing, SHAO Zheng-kang, HU Xin-yue, LI Yi, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 86-93. https://doi.org/10.16155/j.0254-1793.2022.01.10

Abstract (10) PDF (2) Knowledge map Save

Objective: To establish a new site-specific modified pegylated recombinant human growth hormone(PEG-rhGH) modification site research method, and provide a basis for the quality evaluation of the products of this new type of modification technology. Methods: MALDI-TOF mass spectrometry was used to analyze the number of modified PEGs and the mass of modified peptides. LC-Q-TOF mass spectrometry was used to analyze the modification sites and the analysis column was UPLC column(ACQUITY UPLC Protein BEH C₄, 150 mm×2.1 mm, 1.7 μm, 300Å). Gradient elution was performed using 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution. The column temperature was set to 40 ℃. The conditions of mass spectrum were a　below: MS^E mode with a primary mass spectrum energy of 4 eV and a secondary fragmentation voltage of 30-55 eV. Results: The average relative molecular mass of PEG in the modified protein was 30 000, and only single PEG molecule was found in PEG-rhGH molecule; the arginine at position 134 in the recombinant human growth hormone of the prototype protein was replaced with lysine, and the ε-amino group of the lysine was covalently linked to the carboxyl end of the linker HOOC-O-CH₂-CH₂-N₃, and the azide group at the other end of the linker was coupled with the activated PEG to generate a modified product. Conclusion: Combined with a variety of mass spectrometry techniques, by comparing the national reference substance of human growth hormone, the cleavage peptide map of the prototype protein and the modified protein, and the molecular weight analysis of the primary and secondary fragments of specific peptides, it is capable of determining the new site-specific modified poly modification sites of PEG-rhGH.
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Special column on the quality analysis of recombinant hormone drugs
Development of the first national reference standard of beinaglutide^*

DING Xiao-li, LÜ Ping, CAI Yong-qing, DUAN Xu-hua, WANG Lü-yin, CHEN Ying, XU Ke-zheng, LI Jing, ZHANG Wei, HU Xin-yue, LI Yi, SUN Yue, ZHANG Hui, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 78-85. https://doi.org/10.16155/j.0254-1793.2022.01.09

Abstract (15) PDF (7) Knowledge map Save

Objective: To establish the first national reference standard of beinaglutide. Methods: TOF-MS, N-terminal sequence determination, ratio coefficient of amino acid, peptide mapping were used to confirm the structure of beinaglutide. The purity was determined by HPLC. The content of active pharmaceutical ingredients A(API,sample of large size) was determined by mass balance method. Using API A as reference substance, the content of API B(candidate standard) was determined by HPLC external standard method. The potency of API B　was determined by cell culture in vitro. Andthe stability and uniformity of API B(the candidate preparation) were investigated. Results: The structure of the beinaglutide was confirmed, the content of the first beinaglutide natio-nal reference standard was assigned as 0.932 mg per ampoule and the potency was 1.0×10⁴ U per ampoule. The purity of API B(candidate preparation) was not significantly decreased when it was placed at 4 ℃ or 25 ℃, RH 80% for 10 d, and the purity was significantly decreased when it was exposed to light for 10 d. RSD of 15 API B(candidate standard) was 0.86%, showing good homogeneity. Conclusion: The first batch of national standard beinaglutide could be used for HPLC identification, content determination and biological activity determination of benalutide and related preparations of beinaglutide.
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Special column on the quality analysis of recombinant hormone drugs
Preparation and collaborative calibration of the 1st national standard of recombinant human chorionic gonadotrophin for bioassay^*

LI Zhan-jun, LIANG Yu-ling, LI Yi, HU Xin-yue, HU Yu-chi, CAO Chun-ran, WEI Xia, ZHU Qing-fen, ZHANG Juan, FENG Run-dong, WANG Li-fang, HE Li-xiu, LI Jing, ZHANG Hui, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 68-77. https://doi.org/10.16155/j.0254-1793.2022.01.08

Abstract (16) PDF (6) Knowledge map Save

Objective: To prepare and calibrate the first batch national standard(NS) of recombinant human chorionic gonadotrophin (rHCG) for bioassay. Methods: The bioactivity of the first batch of the candidate preparation of rHCG was collaboratively calibratedby five laboratories taking the fifth batch of rHCG of WHO international standard 07/364 with a titer of 162 IU per vial as the standard, the 1209 bioassay method of rHCG in Volume Ⅳof the Chinese Pharmacopoeia was adopted. The structure of candidate preparation concentrate was confirmed by imaging capillary isoelectric focusing(icIEF), peptide mapping and determination of relative molecular mass. The key physicochemical properties were measured, such as the protein content by ultraviolet spectrophotometry, purity and polymers by SEC-HPLC, oxidation of subunits by RP-HPLC. The stability tests for the candidate preparation were carried out. Results:The biopotency of the candidate preparation of rHCG was finally determined to be 6 400 IU per vial after collaborative calibration. The content of rHCG was about 290 μg per vial, and the purity by SEC-HPLC was about 100%. Structure confirmation results meet the requirements; Physical and chemical indicators such as polymer, dissociated subunit, and oxidized subunits were conformed to the standard; the stability test results meet the requirements. Conclusion: The candidate preparation is recognized as rHCG to sever as the 1st NS rHCG for bioassay of rHCG and related preparations, and its biopotency is 6 400 IU per vial.
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Special column on the quality analysis of recombinant hormone drugs
Study on biological activity assay of fusion protein of rhFSH-Fc based on the reporter gene^*

SUN Shuang, WANG Lü-yin, LI Jing, LÜ Ping, XU Ke-zheng, LIANG Yu-ling, ZHANG Hui, LI Zhan-jun, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 60-67. https://doi.org/10.16155/j.0254-1793.2022.01.07

Abstract (12) PDF (3) Knowledge map Save

Objective: To establish a method to detect the biological activity of recombinant human follicle stimulating hormone Fc fusion protein(rhFSH-Fc) by transgenic cell method. Methods: Using CHO-K1-FSHR-CRE-Luc transgenic cells to seed the plate at a certain cell density. After the strain was cultured for 16-20 h, the medium was abandoned, and rhFSH-Fc was diluted in a multiple and added to the cell plate. After the drug acted for a period of time, the lucifase detection reagent was added, and the biological activity of rhFSH-Fc was detected through the lucifase detection system, and the parameters of each test condition were optimized and the methodology was verified. Results: rhFSH-Fc has a dose-effect relationship in this method and conforms to the four-parameter curve equation, R² was greater than 0.99; after the method was optimized, the cell seed plate density was determined to be 5×10⁵ cells·mL^-1, and the initial dilution concentration of rhFSH-Fc was 750 ng·mL^-1, at 1:5 times dilution factor, induction time was 4 h, sample dilution was DMEM/F12K+10% FBS. The method has good specificity. The RSD of the precision test within the day was less than 5% in 9 independent days. The recovery samples of 5 different dilution groups were measured 6 times, and the relative titers were (51±2.00)%, (78±2.05)%, (94±2.23)%, (124±3.46)% and (141±4.45)%. The corresponding average recoveries were(101±3.94)%, (104±3.00)%, (94±2.24)%, (99±2.68)%and (94±3.03)%, RSD were all less than 4%. Conclusion: The biological activity assay of rhFSH-Fc was successfully established by transgenic cells.The method is simple, reproducible and accurate. It could be used for routine detection of rhFSH-Fc products.
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Special column on the quality analysis of recombinant hormone drugs
Time-resolved fluorescence immunoassay for the determination of the biological activity of human insulin^*

YANG Yan-feng, WANG Lü-yin, LIANG Yu-ling, LI Zhang-jun, ZHANG Hui, LI Jing, GAO Xiang-dong, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 51-59. https://doi.org/10.16155/j.0254-1793.2022.01.06

Abstract (11) PDF (2) Knowledge map Save

Objective: To establish a method for detecting the biological activity of human insulin against transgenic cells using a time-resolved fluorescence immunoassay system. Methods: CHO INSRB 1284 transgenic cells with 2.5×10⁵ cells·mL^-1were used as target cells. The initial concentration of 400 pmol·mL^-1was used to dilute human insulin in a series of 3 times. Then the drug was treated with the target cells for 20 min, biological activity was detected by time-resolved fluorescence immunoassay system. Key parameters of the method were optimized and verified according to the 2020 edition of the Chinese Pharmacopoeia, the four general rules 9401 and the ICH guidelines Q2(R1)、Q6B for validation. Results: The method shows good dose-response relationship, which conforms to the four-parameter equation: y=(AD)/[1+(x/C)B]+D. The method had a strong specificity. The geometric mean of the relative biological activity of the 5 potency levels(64%, 80%, 100%, 125% and 156%)(n=8) was respectively (55.8±2.06)%, (82.1±5.52)%, (94.4±5.46), (121.4±5.94)% and (154.4±8.37)%, the relative bias and its 90% confidence interval were respectively -12.8% (-16.3%, -9.9%), 2.6% (-4.4%, 9.5%), -5.6% (-11.2%, -0.3%), -2.9% (-7.7%, -1.8%) and -1.0% (-7.1%, 3.9%). The absolute value of relative bias and confidence interval did not exceed 20%, indicating that the method was relatively accurate. The geometric coefficient of variation(GCV) and the upper limit of its confidence interval for the five potency levels were all less than 20%. The results showed that the proposed method has good precision. The linear range was 64%-156%, the slope was 1.031, and the R² was greater than 0.98. Conclusion: The time-resolved fluorescence immunoassay system is used to successfully establish the biological activity detection method of human insulin based on transgenic cells, which can be used for efficacy evaluation in vitro and quality control.
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Special column on the quality analysis of recombinant hormone drugs
Establishment of a UPLC method for the determination of related substances in recombinant human growth hormone bulk and injection^*

LU Jun-jie, LI Jing, CHEN Ying, LI Yi, ZHANG Wei, ZHANG Hui, LÜ Ping, QIN Xi-yue, GAO Xiang-dong, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 41-50. https://doi.org/10.16155/j.0254-1793.2022.01.05

Abstract (10) PDF (5) Knowledge map Save

Objective: To establish a UPLC method for determination of related substances in recombinant human growth hormone bulk and injection. Methods: The analysis was conducted on Waters BEH C₁₈ 300Å column (150 mm×2.1 mm, 1.7 μm). The mobile phases were 12.5 mmol·L^-1 dipotassium hydrogen phosphate buffer solution and acetonitrile with gradient elution, the flow rate was 0.3 mL·min^-1, the column temperature was 45 ℃, and the detection wavelength was 220 nm. Results: Under the selected chromatographic conditions, more related substances could be separated, and the main peak and each peaks of related substances could be well separated. The linear relationship was good in the concentration range of 0.25-5 mg·mL^-1. Precision, and durability was good. Stability within 72 h was good. According to the qualitative results of mass spectrometry. The method was explored that preparing the main related substances such as deamination impurities, oxidation impurities and dehydration impurities. For recombinant human growth hormone injection,compared with the existing standard, this method could compensate for the shortcomings of the registration standard such as the small number of substances and the low degree of separation between each other. and overcome the problem thatthe European Pharmacopoeia requires two independent experiments with different mechanisms to complete the control of related substances. Conclusion: This method is suitable for the determination of related substances in recombinant human growth hormone bulk and injection.
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Special column on the quality analysis of recombinant hormone drugs
Study on analysis method of recombinant human follicle stimulating hormone oxidized subunits^*

YANG Hui-min, ZHANG Wei, SUN Yue, LÜ Ping, WANG Lü-yin, LI Yi, ZHANG Hui, LI Jing, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 33-40. https://doi.org/10.16155/j.0254-1793.2022.01.04

Abstract (9) PDF (2) Knowledge map Save

Objective: To study and establish a method for analyzing the oxidized subunits of recombinant human follicle stimulating hormone(rhFSH), and prepare a stable system suitability solution for the analysis of oxidized subunits. Methods: Hydrogen peroxide was added to destroy rhFSH, and then methionine was added to stop oxidating for preparing the system suitability solution. The RP-HPLC method was used to separate and analyze the oxidized subunits of rhFSH. The C₄ chromatographic column(250 mm×4.6 mm, 5 μm) was used, mobile phase A was 0.2 mol·L^-1 potassium dihydrogen phosphate buffer (pH 2.5), mobile phase B was 80% acetonitrile. Flow rate was 1 mL·min^-1, column temperature was 30 ℃, detection wavelength was 210 nm, gradient elution (0-10 min, 82%A→74%A; 10-25 min, 74%A→60%A; 25-26 min, 60%A→20%A; 26-56 min, 20%A; 56-56.1 min, 20%A→82%A; 56.1-75 min, 82%A). Results: The addition of methionine could effectively terminate the oxidation and a stable system suitability solution was prepared. The optimized analysis method of oxidized subunits had good specificity, repeatability(RSD=0.22%), column robustness(RSD=3.21%), and solution stability within 36 h(RSD=0.65%). The contents of oxidized subunits in 9 batches of samples ranged from 0.687% to 1.001%. Conclusion: The optimized method of oxidized subunits analysis could accurately quantify oxidized subunits in rhFSH, and could be used for quality research and release inspection of rhFSH. The system suitability solution could accurately locate the peaks of oxidized subunits.
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Special column on the quality analysis of recombinant hormone drugs
Study on analytical methods of related substances in insulin glargine injection^*

DING Xiao-li, CHEN Ying, HU Xin-yue, LI Jing, ZHANG Hui, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 23-32. https://doi.org/10.16155/j.0254-1793.2022.01.03

Abstract (11) PDF (2) Knowledge map Save

Objective: To optimize the analysis method of related substances of insulin glargine injection, demonstrate the scientific limits for the current standards and identify the main related substances. Methods: The separation was performed on Thermo scientific Bio-Basic-C₁₈ column (250 mm×4.6 mm, 5 μm). Sodium chloride, phosphate buffer and acetonitrile were mixed in a 18.4 g:250 mL:250 mL ratio, and added with water to 1 000 mL to be used as mobile phase A; sodium chloride, phosphate buffer, acetonitrile were mixed in a 3.2 g:250 mL:650 mL ratio,and added with water to 1 000 mL to be used as mobile phase B; gradient elution program was used at the flow rate of 1.0 mL·min^-1. The detection wavelength was 214 nm and the column temperature was 35 ℃. The maximum content of related substances was collected and identified by LC-MS. The chromatographic separation was carried out on a column ACQUITY UPLC BEH C₁₈ column(100 mm×2.1 mm, 1.7 μm, 300Å) with the mobile phase consisting of 0.1% formic acid aqueous solution (A)-0.1% formic acid acetonitrile solution (B) in a gradient mode. The column temperature was set to 50 ℃. The mass spectra was acquired with MS^E mode. The primary mass spectrum energy was 40 V. Results: The RP-HPLC method for the analysis of related substances has good methodological verification results, and could effectively separate and accurately determine the related substances in the samples.It was identified that the main related substance of insulin glargine injection was the 3^B-succinimide-insulin glargine,which was deaminated degradation product of asparagine at the third position of chain B. Conclusion: The optimized RP-HPLC method could be effectively used for the analysis of related substances in insulin glargine injection. The current national standards for related substances limits are scientific and reasonable, which could meet the quality control of insulin glargine injection during the shelf life.
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Special column on the quality analysis of recombinant hormone drugs
Analysis and study on the complete sequence of insulin and its analogues by UPLC-MS/MS^*

HU Xin-yue, DING Xiao-li, CHEN Ying, ZHANG Hui, LI Jing, LIANG Cheng-gang

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 13-22. https://doi.org/10.16155/j.0254-1793.2022.01.02

Abstract (17) PDF (5) Knowledge map Save

Objective: To establish a UPLC-MS/MS method to identify the complete sequence of insulin and its analogues. Methods: This method took human insulin as the main research object.It was first pre-treated by reductive alkylation and then analyzed by UPLC-MS/MS. ACQUITY UPLC peptide BEH C₁₈(100 mm×2.1 mm, 1.7 μm, 300Å) column was adopted, 0.1% formic acid/water for solvent A and 0.1% formic acid/acetonitrile for solvent B as mobile phase. The gradient mode was 3%-60% solvent B over 10 min, 10-10.5 min, 60%B→95%B; 10.5-14 min, 95%B; 14-14.5 min, 95%B→3%B; 14.5-20 min, 3%B at a flow rate of 0.3 mL·min^-1, with column temperature 50 ℃. The top-town complete sequencing analysis of human insulin and its analogues was carried out by ESI⁺ scanning and optimized mass spectrometry parameters. The sequencing method of human insulin was studied in depth,and the applicability of the method to insulin aspart and insulin lispro was also investigated. Results: The method could cover the whole sequence of A and B chains of human insulin well. The RSD of ion strength of five high strength b/y ions was within 20% by b/y ions statistical analysis, The first-order mass number of chain A and B was accurate, with good precision and accuracy; the method could also distinguish the sequence differences from other insulin analogues with good specificity and has good applicability to insulin aspartic and insulin lypro. Conclusion: The method is simple, accurate and specific,and provides data support for the complete sequence determination of insulin and its analogues.
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Special column on the quality analysis of recombinant hormone drugs
Considerations on research of national standards for recombinant hormones^*

LIANG Cheng-gang, LI Jing, ZHANG Hui, LÜ Ping, LI Zhan-jun

Chinese Journal of Pharmaceutical Analysis. 2022, 42(1): 3-12. https://doi.org/10.16155/j.0254-1793.2022.01.01

Abstract (19) PDF (6) Knowledge map Save

Recombinant hormones drugs are pioneers among therapeutic biotechnology products for clinical use, and their total global sales volume are the second class of recombinant protein drugs next to monoclonal antibodies. Such drugs usually have “counterarts” of listed natural drugs extracted from tissues,with mature studies on the correlations between their structures and functions and clear efficacy. It plays an irreplaceable role in the treatment of various chronic diseases and degenerative diseases. The technical points, issues and solutions, the basis of the research and the evaluation of the results which were generally followed and paid attention to in the process of the standard study of the pharmacopoeia of recombinant hormones drugs were discussed. Some research strategies for future activities on national standard drafting of recombinant hormones were also prospected and discussed.

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