LC-MS/MS (DIA) analysis of biopharmaceutical product HCP

Biomolecules obtained by eukaryotic or prokaryotic expression and gradual purification. The heterologous protein (Host Cell Protein, hereinafter referred to as HCP) in the product is a potential immune response to the human body and interferes with macromolecular products. Stability and other risks, research on HCP has become one of the hot spots in the quality of biopharmaceutical products.

HCP content research

The type and content of HCP are very important for the quality research of biomacromolecule drugs. First of all, the application of LC-MS/MS technology in the detection of biological drugs enables the qualitative analysis of HCP. For example, PLBL2, which has attracted much attention in the CMC process, has been shown to be related to the human immune response.

Secondly, in the detection and analysis of HCP content, ELISA method and LC-MS/MS method respectively analyzed the total content of HCP and the content of target HCP. The ELISA method is more suitable for the establishment of release standards, and the LC-MS/MS method as a complementary method is suitable for the study of the analysis of the CMC process.

LC/MS/MS Method Development & Validation

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Chemical Analytical Center of Medicilon provides general analysis and purification services for the clients. Our experts have rich experience and proficient skills to solve problems, which are highly appreciated by clients.

General Analytical Chemistry Services

• NMR 1D、2D（H-NMR、C-NMR、P-NMR、F-NMR、HSQC、HMBC、COSY、NOESY, et al）

• LC-MS Analysis

• HPLC Analysis (including ELSD)

• Chiral HPLC Analysis

• General LCMS Testing (ROI、LOD、Cl-、SO42-、mp、HM、Specific Rotation、Water Content, et al）

HCP detection method

The HCP detection and analysis methods of biological drugs are mainly: ELISA (LBA) method and LC-MS/MS method.

ELISA method

1. Use commercial antibodies and ELISA kit for analysis;

2. After obtaining the HCP antigen, self-made antibodies;

Advantages and disadvantages:

1. Quantify HCP in the stock solution

2. Simple equipment and high throughput

3. Difficult to monitor the target HCP

4. What you get is cumulative quantification

5. The affinity of HCP molecules is different

LC-MS/MS method

1. DDA method

2. DIA method

3. MRM/PRM verification method

4. Affinity mass spectrometry (affinity based MS)

Advantages and disadvantages:

1. High specificity

2. High sensitivity (ppm level)

3. High accuracy

4. Short development cycle

DIA basic principles

DIA (data independent acquisition) data independent acquisition technology is one of the latest developments in the qualitative and quantitative application of LC-MS/MS. Compared with DDA (data dependent acquisition) data dependent acquisition technology, DIA technology has the advantages of fast speed, strong qualitative ability and wide quantitative range.

Based on the DIA technology in HCP detection and analysis, there have been many applications (BMS, Lilly, etc.) in recent years. This technology can achieve a shorter method development time and complete the detection of trace amounts (down to ppm) in biomacromolecule drug products. Qualitative and quantitative analysis of HCP molecules.
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Basic schematic diagram of DIA technology

DIA-HCP

For better quantitative analysis, in the DIA-HCP detection and analysis, five internal standard protein molecules were added in the range of 1 ppm to 100 ppm. In addition, because the protein composition in different purification steps is very different, APT tests the products in the entire process. It can be seen from the above figure that the stability of the internal standard protein is good in the entire purification process, and the linear performance is good within the quantitative range of the internal standard.

PRM verification

When the DIA method is used for the HCP detection and analysis of biopharmaceutical macromolecules, the HCP concentration is calculated from the internal standard protein standard curve. Zhongke New Life provides further MS or ELISA verification. Using MRM (triple quadrupole analysis) or PRM (QE-plus) analysis, peptide molecules labeled with stable isotopes can be used to accurately quantify the concentration of target HCP molecules. PRM verification results of two HCPs with a concentration of about 1 ppm.

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