Key considerations and cases of mesenchymal stem Cell bioanalysis Share | Hongren Pharma Report of the 2nd Greater Bay Area Cell Gene Therapy Development Forum

Dr. Langshiwei was invited to attend the conference and gave a speech on "Key considerations and cas

2024-11-25 13:25 Hongren Biopharmaceutical
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From November 21 to 22, 2024, InnoXBio 2024 2nd Greater Bay Area Cell Gene Therapy Development Forum was held as scheduled in Guangzhou Knowledge City International Convention and Exhibition Center. 70+ speakers gather for an industry event to share the latest advances and achievements in cell therapy, gene therapy, RNA therapy and stem cell therapy.

Dr. Lang Shiwei, Director of Biological Products BioAnalysis Department of Wuhan Hongren Biomedicine Co., LTD., was invited to attend the conference and gave a speech on "Key Considerations and Case Sharing of mesenchymal stem cell bioanalysis" in the special session of "Technological Exploration under the support of Regenerative Medicine and Stem Cell Technology "!


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Dr. Lang Shiwei, Director of Bioanalysis Department of Wuhan Hongren Biopharmaceutical Inc. Pharmaceutical

Hongren pharmaceutical report sharing


《Key considerations and case studies for the biological analysis of mesenchymal stem cells》


Introduction to the speaker


Lang Shiwei received his bachelor's degree from Beijing Normal University and his master's degree from Chinese Academy of Sciences. More than 10 years of experience in biological products/macromolecular bioanalysis. As the main person in charge, he participated in the establishment of the preclinical and clinical macromolecular analysis platform of a listed company and served as the department head. Familiar with the development and validation of various biological drug analysis methods. We have successfully completed the analysis of more than 100 biological drugs in accordance with the GLP specifications of NMPA, FDA and OECD, including polypeptides, fusion proteins, monoclonal antibodies, double antibodies, AAV, oncolytic viruses, CAR-T, stem cell drugs, nucleic acid drugs, vaccines, etc


Report highlights



Biological analysis of stem cells mainly includes: 1, PK and distribution. Among them, clinical distribution detection is difficult due to the limitation of sample sampling. 2. Immunogenicity assessment. This includes overall immunogenicity, anti-drug antibodies, human leukocyte antigen (HLA), and donor-specific antibody analysis (DSA). The immunogenicity of exogenously expressed proteins (if any) should also be evaluated clinically. 3. Biomarker detection. Including a variety of cytokines that reflect the biomarkers of cell viability and disease status.


PK and distribution analysis of stem cells


Unlike traditional small molecule/large molecule therapeutic drugs, cellular drugs are characterized by "living", and their fate in vivo, such as distribution, migration, homing and duration, is the most important factor affecting their effectiveness and safety. At present, the dynamic process of the fate of stem cells in the body and its correlation with drug efficacy are not clear. By studying the biological distribution process, influencing factors and rules of stem cells entering the body, it is helpful to understand the mechanism and principle of stem cells, and provide basis and guarantee for patent drugs. The guidelines on stem cells also suggest the importance of analyzing stem cell PK and distribution in clinical and non-clinical studies.

Detection methods: 1. In vivo tracer, including radioluminescence imaging (BLI), fluorescence imaging (FLI), magnetic resonance imaging (MRI), etc. 2. In vitro testing, including qPCR, bDNA, immunohistochemistry, etc. The advantage of these methods is that they can dynamically observe the real-time state of stem cells in vivo without damaging the animal. But this approach requires labeling of cells and has limited resolution, which makes it impossible to use in clinical human trials.
The qPCR method requires samples for in vitro analysis, but is highly sensitive and can be used in clinical and non-clinical studies. It is still the main means of analyzing PK and distribution of stem cells.


qPCR cell product analysis:
  1. Preparation of standards: PCR for cell product standards rarely uses the stem cell itself as a standard, usually using plasmids. The concentration of the plasmid is mostly mass concentration, which needs to be converted to copy number according to molecular weight.
  2. Primers/probes: need to have specific properties to distinguish between foreign stem cells and somatic cells; RNA enzyme is added to extract DNA, and the quality of DNA extraction is identified. In this way, the final concentration measurement results can be accurate.
  3. qPCR reaction: High quality reaction Mix and TaqMan method were selected for PCR experiment. The stem cell concentration was calculated back according to the standard curve qPCR.

Important validation items for the qPCR analysis method:
  1. Standard curve and quantitative range: the curve established by at least 6 calibration standard samples should meet R2≥0.98; The amplification efficiency was 90~110%
  2. Accuracy and precision: The accuracy of the PCR method is often not good, often related to the quality of the primer itself and the parallelism of the operation, a good method can achieve accuracy within 25%
  3. LLOQ: The sensitivity must be 50 copies/μg DNA
  4. Extraction recovery rate: A certain amount of cells were added to the matrix to calculate the recovery rate



Flow cytometry was used to analyze the anti-drug antibodies of stem cell products


In stem cell therapy, the strength of immune response is related to the matching degree of HLA human leukocyte antigen between donor and recipient. The lower the matching degree, the stronger the immunogenicity, including natural immunity and acquired immunity. Natural immunity is mainly activated by NK cells. Acquired immunity activates T cells and B cells, of which B cells produce anti-drug antibodies.

ADA detection is the content of clinical and non-clinical analysis of stem cells. The ADA assay method for stem cell products is different from that for protein drugs. ADA detection of protein drugs is usually based on LBA experiments (such as ELISA, MSD, etc.). For stem cell products, LBA methods often do not meet the needs. Macro Pharma has developed an analytical technology based on the flow platform to capture ADA using stem cells and then analyze it through flow.



  1. During the test, the sample to be tested is incubated with an excess of stem cells so that the antibodies in the sample bind to the stem cells and are captured.
  2. Fluorescein labeled antibodies are added to detect antibodies, and if there are anti-stem cell antibodies in the sample, the fluorescein antibodies will bind further, thus forming a drug resistant antibody-fluorescein labeled antibody complex on the cell surface.
  3. The presence of antibodies in the original sample can be detected by flow analysis of the numerical strength of fluorescein-labeled antibodies on the cell surface.

It can be seen from a typical result in the figure above that the fluorescence intensity of PE gradually increases when PC is not added, low concentration PC and high concentration PC are added, indicating that this method can detect specific anti-stem cell antibodies.


Clinical HLA (human leukocyte antigen) analysis


In clinical trials, HLA analysis is also very important in cell therapy. HLA is an expression product of human histocompatibility complex (MHC) and an important antigenic substance constituting transplant rejection. The products encoded by the HLA gene complex are located on the short arm of chromosome 6 and are classified into class I antigens, Class II antigens and Class III antigens. The higher the HLA compatibility between donor and recipient, the lower the rejection rate and the higher the transplant success rate.


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In hematopoietic stem cell transplantation, it is very important to judge the HLA compatibility between donor and recipient before transplantation, and certain criteria must be met before hematopoietic stem cell transplantation.

For the treatment of MSC (mesenchymal stem cells), although HLA gene level matching is not required before administration, HLA differences may still affect cell distribution and survival after MSC perfusion.

HLA compatibility analysis requires two levels of analysis:
  1. Gene matching analysis: At present, second-generation sequencing (NGS) has become the gold standard for HLA matching analysis.
  2. Serum antibody analysis: that is, to detect whether the recipient has specific antibodies against the donor HLA, this test is currently the most widely used Luminex test.
In addition, Dr. Lang Shiwei shared further technical details with some cases of the Hongren experimental platform. Such as stem cells non-clinical mouse tissue distribution analysis, qPCR analysis of clinical stem cell PK, flow cytometry detection of human umbilical cord mesenchymal stem cells non-clinical ADA, flow cytometry analysis of human umbilical cord mesenchymal stem cells clinical ADA, clinical DSA Detection - analysis methods and typical results, clinical biomarker detection - stem cells for the treatment of liver failure, clinical biomarker detection - stem cells for the treatment of idiopathic pulmonary fibrosis, etc. Welcome interested experts and teachers to visit Hongren Pharmaceutical exchange and discussion.

In summary, compared with small molecule therapies and biopharmaceuticals, cell products have higher complexity, which brings new challenges for bioanalytical detection.

  1. Traditional drugs have a single component, and the cell itself is highly complex, a cell contains tens of thousands of substances such as proteins or nucleic acids, and the biological analysis of cell products is more complex.
  2. The PK and distribution of stem cells can be traced in vivo and detected in vitro. qPCR is a widely used detection method in clinical and non-clinical studies. Due to the limitations of analytical methods and clinical sampling, the results of tissue distribution were mainly derived from non-clinical studies. Detection of PK and distribution in clinical studies is more difficult than in non-clinical studies.
  3. Due to species differences, the immunogenicity of stem cell products is usually stronger in non-clinical than clinical. Clinical immunogenicity testing requires the analysis of anti-drug antibodies and HLA.
  4. Stem cell biomarker detection needs to combine the characteristics of stem cells and clinical indications to select suitable analytes.




Clinical Bioanalysis




Rich project experience：Up to now, the platform project experience covers about 200 biologic drug clinical bioanalysis projects. According to the number of projects, the market share of biological drugs in the past three years from 2021 to 2023 has increased year by year. Developed more than 300 biological analysis and detection methods for drugs, and the analysis and detection projects involved more than 100 innovative drugs. A number of new biological drugs were approved for market, such as liraglutide, tiricizumab and so on.

Difficult bioanalysis solutions：Rich experience and service capability in multi-type and challenging bioanalysis projects, covering all types of drugs: polypeptide, insulin, fusion protein, enzyme, monoclonal antibody, double antibody, triple antibody, PDC, ADC, AAV, oncolytic virus, immune cell drug, stem cell drug, nucleic acid drug, vaccine, etc.

Laboratory multi-area coverage：Bioanalysis laboratories in Wuhan, Shanghai and Guangzhou can flexibly deploy equipment and personnel according to needs in project undertaking and execution, and solve some special project sample transfer and preservation problems. P2/P2+ biosafety laboratory over 400 m2.

Led by a bioanalyst：About 100 mature talent echelons, the core backbone has more than 9 years of domestic and foreign top CRO bioanalysis project experience, the number of 3-year boss in the team accounted for about 70%.

Configuration full/high throughput：The platform has more than 50 advanced analysis and detection instruments (including multiple flow cytometry, MSD, Simoa, PCR, enzyme spectrometer, ELISPOT, etc.), and the throughput of biological sample analysis and detection can reach 100,000 / month, and customers usually do not need to wait for the project schedule.

With the ability to report both China and the United States：The completed project can meet the NMPA and FDA reporting requirements.
声明：此篇为武汉宏韧生物医药科技有限公司原创文章，转载请标明出处链接： https://www.hrbiopharm.com.cn/en/h-nd-77.html
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