E. Coli Cloned Bacteria Host Protein Residue Detection Kit (Enzyme-linked Immunosorbent Assay · One-step Method)

Escherichia coli (cloned strain) is the core prokaryotic expression host for biological therapeutic products such as mRNA vaccines, viral vector gene therapy and CAR-T products. Host cell protein (HCP) residues generated during the production and purification process are key process-related impurities. These trace contaminants may pose an immunogenic risk and compromise the purity and safety of the finished product. HCP residue detection is a core project for process characterization, process control and product release. The key raw materials such as antigens and antibodies in this kit are all independently produced by Zhuhai Baorui Biotechnology Co., LTD. The kit has undergone comprehensive methodological performance verification in accordance with regulations such as the United States Pharmacopeia, the Chinese Pharmacopoeia, and the European Pharmacopoeia, meeting the technical requirements for HCP detection set by domestic and international biopharmaceutical regulatory authorities. The detection limit (LOD) of this kit is approximately 0.249 ng/mL, and the limit of quantification (LOQ) is approximately 0.861 ng/mL.

Calibration curve (linear range)

Overview: Test the standard substances provided by the kit and one blank sample. Each concentration was repeatedly detected three times, and a total of three independent experiments were conducted. The calibration curve was fitted using a four-parameter logistic regression model.

Figure 1 Test results of the detection range

Conclusion: In the three repeated experiments, the calibration curve R² of the four-parameter model fitting was ≥ 0.99, and the RE and CV at all concentration points were ≤ 15% (the highest and lowest concentration points were ≤ 20%). The detection range of this kit is 1-243 ng/mL.

2. Accuracy

Overview: Prepare calibration curves within the verified range. Set five quality control (QC) levels - ULOQ, high (H), medium (M), low (L), and LLOQ, and repeat the testing of each level three times.

Figure 2 Accuracy test results

Conclusion: The CV and RE at each concentration of ULOQ, high (H), medium (M), low (L), and LLOQ were all ≤20%, which met the requirements.

3. Repeatability

Overview: Prepare calibration curves within the verified range. Prepare QC samples of high, medium and low levels, and conduct 8 parallel repeated tests for each level.

Table 1 Repeatability Test Results

Conclusion: The CV at each concentration point is ≤20%, which meets the requirements.

4. Precision among people, during the day, and between instruments

Overview: Simultaneously prepare calibration curves and HQC, MQC, and LQC samples to test the precision among different analysts, different dates, and different instruments.

Figure 3 Precision test results

Conclusion: Among the five QC concentration levels used for accuracy assessment, all RE values are within 20%. The CV values among different analysts, different dates and different instruments were all below 20%, which met the standards.

5. Specificity
Overview: Prepare calibration curves within the verified range while preparing potential interferers. Select the low (L) QC level as the final spiked concentration.

Table 2 Results of Specificity Tests

Conclusion: This kit has no cross-reaction with Pichia pastoris, Sf9, MDCK, and HEK293 HCP host cell proteins at a concentration of 1 µg/mL, and the spiked recovery rate is qualified.