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The Flow Chart of ACROBiosystems Product Quality Control

Comprehensive Quality Inspection

Well-established Stability

Biological product stability is a critical part throughout the entire drug research and development, clinical trials, marketing and post-marketing quality study. This stability is the basis for the manufacturing, process, drug product formulation, packaging materials, storage, transportation conditions of the biological product. Real-time stability studies are the most reliable method to investigate product stability and determine product shelf life. However, this method is time-consuming, which greatly limits its use. Therefore, it is particularly important to find a more efficient method. The accelerated stability testing method based on Arrhenius equation is one of the recognized strategies. In recent years, more and more studies have proved its applicability and predictive accuracy.


ACROBiosystems products are validated for stability in four aspects: accelerated, freeze-thaw, real-time, and simulated transportation.

  • Accelerated stability:
    Accelerated stability

    The Cell based assay shows that ActiveMax Human IL-7 （Cat. No. IL7-H4219） is stable at 37°C for 48 hours.

  • Freeze-thaw stability:
    Freeze-thaw stability

    The Cell based assay shows that ActiveMax Human IL-7 （Cat. No. IL7-H4219） is stable at freezing and thawing 3 times.

  • Real-time storage stability:
    Real-time storage stability

    The Cell based assay shows that Monoclonal Anti-Human CD3 Antibody （Cat. No. CDE-M120a） is stable at 4°C for 1 year.

  • Simulated transportation stability:
    Simulated transportation stability

    The Cell based assay shows that Human IL-15 （Cat. No. GMP-L15H13） is stable at 37°C for 14 days.

Accurate Quantification

The biopharmaceutical development process often requires accurate concentrations of protein reagents used in experiments to avoid errors. As such, the loading quantity between batches should be kept strictly consistent, which puts forward higher requirements for the accurate quantification of protein reagents. Meanwhile, strict method validation is also important.

According to ICH Q2 and Chinese Pharmacopoeia standards for different proteins, we use the relevant standards for traceability as well as a comprehensive method validation. The results of repeatability show that the CV% of the same method performed by different operators on different days was less than 3%, and the recovery rates ranged from 90% to 108%, ensuring quantitative accuracy.


Traceability data for analytical procedures:
Validation item Test method Traceability method
Validation criteria Validation result Validation criteria Validation result
Repeatability RSD≤3% 0% RSD≤3% 1%
Different analysts on the same day RSD≤3% 0% RSD≤3% 3%
Same analyst on different days RSD≤3% 0% RSD≤3% 3%
Accuracy 90%-108% 100%-106% 90%-108% 97%-103%
Robustness RSD≤3% 0% RSD≤10% ＜10%
Linearity and range R2>0.999 0.9996 R2>0.999 0.9999
Specificity / / 90-110% 96%

There are many methods for protein quantification, such as UV spectrophotometry (UV 280), Folin-phenol method (Lowry method), BCA method, Bradford method, etc. These methods are based on different principles, and have their own advantages and limitations. For example, as long as the molar extinction coefficient is calculated according to the amino acid sequence, the UV absorption method can be used for fast protein quantification. It is a simple method which does not require special instruments, equipment, or training, making it the most used method in most laboratories. However, UV absorption method is susceptible to the interference of UV-absorbing impurities, such as residual trace nucleic acids, resulting in false high protein concentrations and batch differences caused by inconsistent quantification.

ACROBiosystems addresses these issues by adopting various quantification methods including UV absorption and BCA (modified Folin-phenol method) during the quantification of each batch of protein drug substance and finished product. Separate quantitative standards and standard batches for each protein product are also established. This fundamentally avoids the error of protein quantification caused by the limitations of any single method, and ensures the consistency of quantification among different batches of the same product, providing customers with the minimal batch-to-batch differences.

Purity/Molecular Weight

To meet the needs of biopharmaceutical customers for high-purity protein, ACROBiosystems uses various methods including SDS-PAGE, SEC-HPLC, SEC-MALS, DLS to test the purity and homogeneity of products.

  • SDS-PAGE
    SDS-PAGE

    ActiveMax® Human VEGF165， Tag Free （MALS verified） on SDS-PAGE under reducing （R） and non-reducing （NR） conditions. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 98%.

  • SDS-PAGE
    SDS-PAGE

    Human IL-7 on SDS-PAGE under reducing （R） condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 95%.

  • SEC-HPLC&SEC-MALS数据
    SEC-HPLC&SEC-MALS数据

    The purity of ActiveMax® Human VEGF165， Tag Free （MALS verified） （Cat. No. VE5-H4210） is more than 95% in HP-SEC， and the molecular weight of this protein is around 40-55 kDa verified by SEC-MALS.

  • DLS数据
    DLS数据

    The mean peak Radius of VLP is 60-80 nm with more than 95% intensity as determined by dynamic light scattering （DLS）.

Click to read: Sharp-sighted SEC-MALS: Accurately determine the molecular weight and confirm the aggregation state of the protein

Multiplatform Bioactivity Testing

The bioactivity testing of drugs is performed throughout the R&D and production of biopharmaceuticals. In order to better help customers in the biopharmaceutical field, ACROBiosystems has established multiple bioactivity testing platforms, including ELISA, SPR, flow cytometry and cell-based assays, to provide customers in the biopharmaceutical field with better products of more consistent quality and referable bioactivity analytical protocols.

  • ELISA
    ELISA

    Immobilized Human IL-17 RE （155-454）， Fc Tag （Cat. No. ILE-H5256） at 2 μg/mL （100 μL/well） can bind Biotinylated Mouse IL-17C， His，Avitag with a linear range of 0.2-20 ng/mL （QC tested）.

  • ELISA
    ELISA

    Immobilized Human IL-15， Tag Free （Cat. No. IL5-H4117） at 5 μg/mL （100 μL/well） can bind Human IL-2 R beta， Fc Tag （Cat. No. ILB-H5253） with a linear range of 5-156 ng/mL （Routinely tested）.

  • FACS
    FACS

    FACS assay shows that Biotinylated Human SIRP alpha， Fc，Avitag （Cat. No. CDA-H82F2） can bind to Jurkat cell expressing CD47. The concentration of SIRP alpha used is 3 μg/mL （QC tested）.

  • FACS
    FACS

    FACS analysis shows that the binding of Biotinylated Human SIRP alpha， Fc，Avitag （Cat. No. CDA-H82F2） to Jurkat expressing CD47 was inhibited by increasing concentration of neutralizing Anti- Human CD47 antibody. The concentration of SIRP alpha used is 1 μg/mL. IC50=0.1303 μg/mL （Routinely tested）.

  • Cell based assay
    Cell based assay

    ActiveMax Human IL-7， Tag Free （Cat. No. IL7-H4219） stimulates proliferation of PHA-P-activated human peripheral blood mononuclear cell （PBMC）. The EC50 for this effect is 1.565 ng/mL， corresponding to a specific activity of > 1.0 ⅹ10^8 IU/mg， which is calibrated against human IL-7 WHO International Standard （NIBSC code: 90/530） （QC tested）.

  • SPR
    SPR

    Human IL-2 R beta Protein, His Tag (Cat. No. CD2-H5221) captured on CM5 chip via anti-His antibody, can bind Human IL-15, premium grade (Cat. No. IL5-H4117) with an affinity constant of 21.7 nM as determined in a SPR assay (Biacore T200) (Routinely tested).

Batch Release

In order to ensure product quality and high batch-to-batch consistency, ACROBiosystems adopts the batch inspection and release strategy. Each new batch is compared to the corresponding reference standard, and only the batch of the equivalent quality level to the standard will be approved for release. Moreover, each batch of products is supplied with its own quality control report.

  • Batch Release

    In the above ELISA analysis， three different lots of biotinylated hTNF-alpha （Cat. No. TNA-H82E3） were used detect immobilized Adalimumab （5ug/ml）. The result showed that the batch variation among the tested samples is negligible.

  • Batch Release

    Recombinant Human TNF-alpha （Cat. No.TNA-H4211） induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The ED50 for this effect is 0.007-0.014ng/ml. The result shows that the batch variation among the tested samples is negligible.

  • Batch Release

    The binding affinity between Herceptin and different batches of FcRn / FCGRT & B2M Heterodimer Protein （Cat. No. FCM-H5286） were determined by SPR assay. The result shows that the batch variation among the tested different lots is negligible.

  • Batch Release

    Bioactivity of three different lots of GMP Human IL-15 （GMP-L15H13） verified by cell-based assay， and the result shows very high batch-to-batch consistency.

Validation of Analytical Method

The use of accurate and credible analytical methods for testing is the basis for strict quality control of the product. For GMP grade proteins, we have completed sufficient analytical method validation according to ICH Q2 (R1) and Guidelines for Validation of Analytical Procedures in Chinese Pharmacopoeia to ensure the accuracy of the test results.

FcRn & Hercepetin
Analytical Procedure Test items Source of procedures Validation items
Lowry Protein content ICH Q2 (R1) and 9101 Guidelines for Validation of Analytical Procedures in Volume IV of ChP 2020 specificity, accuracy, precision, linearity, range and robustness
SDA-PAGE Identity ICH Q2 (R1) and 9101 Guidelines for Validation of Analytical Procedures in Volume IV of ChP 2020 specificity, accuracy, limit of detection (LOD), robustness
Endotoxins Biological assay methods Kinetic Chromogenic Assay in General Chapter 1143 Bacterial Endotoxins Test in Volume IV of ChP 2020 reliability test of standard curve, and interference test of test sample
Sterility Biological assay methods Sterility Test in General Chapter 1101 Microbiological Examination in Volume IV of ChP 2020 sensitivity test of culture medium, and method suitability test
Mycoplasma Biological assay methods ICH Q2 (R1) and 9101 Guidelines for Validation of Analytical Procedures in Volume IV of ChP 2020 specificity, accuracy, precision, limit of quantitation (LOQ), linearity, range and robustness
DNA residues Impurity determination/ Quantification ICH Q2 (R1) and 9101 Guidelines for Validation of Analytical Procedures in Volume IV of ChP 2020 specificity, accuracy, precision, limit of quantitation (LOQ), linearity, range and robustness
Cell Viability Biological assay methods 9401 Guidelines for Biological Activity/Potency Assay Validation of Biological Products in Volume IV of ChP 2020 specificity, relative accuracy, intermediate precision, linearity, range
HCP Residues Impurity determination/ Quantification ICH Q2 (R1) and 9101Guidelines for Validation of Analytical Procedures in Volume IV of ChP 2020 specificity, accuracy, precision, limit of quantitation (LOQ), linearity, range and robustness
Kanamycin Residues Impurity determination/ Quantification ICH Q2 (R1) and 9101 Guidelines for Validation of Analytical Procedures in Volume IV of ChP 2020 specificity, accuracy, precision, limit of quantitation (LOQ), linearity, range and robustness
Table 1. Example of the validation of cell viability analytical procedure
Validation item Validation criteria Validation result Validation conclusion

Specificity

Negative reaction, interference ≤ 20%.

average RSD = 5%
maximum RSD = 12%

Conformed

Relative accuracy

The relative bias should be within ± 12%.

The maximum bias was 6%.

Conformed

The slope of regression equation should range from 0.80 to 1.25.

The slope was 1.11.

Intermediate precision

The geometric coefficient of variation (GCV, %) of relative potency of each potency level measured by different analysts on different dates should not be greater than 20%.

a±GCV15%

Conformed

Linearity

The correlation coefficient of linear regression equation should not be less than 0.95.

0.96

Conformed

Range

It should cover the range of potency quality standard of the product.

This method covered 64%–156% potency levels.

Conformed

Table 2. Example of precision validation of the enzyme activity testing
Test date Number of detections Measured value Different analysts on the same day
RSD 		Employee A, same analyst on different days
RSD 		Employee B, same analyst on different days
RSD 		Employee A, same-day repeatability
RSD 		Employee B, same-day repeatability
RSD 		Different analysts on different days
RSD
EmployeeA

EmployeeB

DAY 1

1

1.49

1.52

1%

5%

4%

2%

3%

6%

2

1.46

1.48

1%

3

1.52

1.52

0%

4

1.49

1.47

1%

5

1.45

1.40

3%

6

1.51

1.51

0%

DAY 2

1

1.36

1.56

9%

2%

3%

2

1.33

1.53

10%

3

1.39

1.60

10%

4

1.39

1.58

9%

5

1.32

1.52

10%

6

1.39

1.61

11%

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