-
E-mail
gptchina@gyrosproteintech.com
-
Phone
13764581292
-
Address
Room 1609-1613, Building A, 380 Fenglin Road, Xuhui District, Shanghai
Mesa Labs
How to make the analysis of host cell protein (HCP) residues more efficient and reliable
Date: 2024-12-10Read: 0
Host cell protein (HCP) is a non target protein produced or encoded by host cells (CHO, HEK293, E. coli, Sf9, etc.), which is the main component of process related impurities in biopharmaceuticals. Although the vast majority of host cell proteins (greater than 99%) are removed in downstream processes, there may still be a small amount of HCP residue co purified with drug products. Some residual proteins are considered high-risk HCPs, which may have immunogenicity, biological activity or enzymatic activity, or may degrade product molecules or excipients, and have important impacts on drug quality and safety. Therefore, these high-risk HCPs are the focus of attention for pharmaceutical companies and regulatory agencies.
The Chinese hamster ovary cell CHO expression system has always been one of the commonly used mammalian expression systems in the production of monoclonal antibodies and other biotechnological drugs, as it can produce complex post-translational modified proteins similar to those produced by humans. The downstream purification of CHO cell culture medium typically involves protein A affinity chromatography, followed by a purification step to further remove aggregates, charge variants, HCP, and host cell DNA. During the purification process, the vast majority of HCPs are cleared, but there may be some HCPs that "escape" clearance due to the following two ways:
1
HCP has the ability to bind specifically or non specifically to products and is carried during the manufacturing process;
2
HCP has the ability to interact with protein A chromatographic packing, resulting in its co elution with the product.
HCP hazards and classification
The potential risks of HCPs are influenced by multiple factors, including drug indications, route of administration, frequency of administration, number of HCPs per dose, patient population, homology of HCPs with their human counterparts, and previous non clinical and/or clinical experience.
Potential impact of HCP on product quality, safety, and effectiveness:
Immunogenicity:Drug resistant antibodies, induction of cytokine production, etc
Potency:Protein aggregation or degradation
stabilityLipase degradation of excipients
Security:Interacting with endogenous proteins and acting as an immune adjuvant
The BPDG HCP working group categorizes the impact of high-risk HCPs into four main types based on their ability to co purify individual HCPs with products, frequency of occurrence in downstream processes, ability to alter or degrade drugs/excipients, and potential immunogenicity:
1) Product quality, 2) formulation, 3) direct biological function on the human body, and 4) immunogenicity.
There are currently multiple methods to identify and quantify the levels of all and individual specific HCPs present in the drug development process. Each method has its own advantages and disadvantages. According to the following comprehensive analysis strategy, known HCP impurities can be accurately measured. If identified as high-risk HCPs, control strategies can be developed to monitor and/or eliminate these impurities.
01 Measuring total HCP through immunoassays such as ELISA is the main means of determining residual HCP levels to guide process development and support GMP batch release testing. The key reagents required for the development of the overall HCP immunoassay method should undergo detailed characterization.
02 Additional orthogonal methods, such as 2D gel electrophoresis/Western, and/or mass spectrometry after antibody affinity extraction (AAE) should be used to evaluate the coverage of key antibody reagents to process specific HCPs.
03 The total HCP immunoassay should be routinely performed to determine the level of HCP in the process sample.
04 It is recommended to use orthogonal methods such as mass spectrometry and/or additional single HCP detection methods to ensure product purity, especially when an increase in total HCP levels is detected by immunoassays, dilution nonlinearity is observed, or product quality impact is observed.
05 If individual HCPs are identified and considered potentially high-risk:
a
It may be necessary to develop a specific immunoassay to monitor known impurities, or to develop a targeted mass spectrometry analysis method to better understand the levels and potential effects of HCP.
b
If the identified impurities have enzymatic activity, an activity assay can be developed to guide process and product development, monitor, remove, and/or inactivate the protein.
c
Immunogenicity assessment can be performed using the in silica method. If the prediction indicates a high risk of immunogenicity, in vitro comparative immunogenicity assessment (IVCIA) can be performed.
d
Toxicological studies assess clinical risks.
The main method used for qualitative and quantitative analysis of total HCP is immunoassay, usually using one or more ELISA methods for detection. However, traditional ELISA has the following limitations:
✦
•
Multiple incubation and washing steps require a lot of manual operation and analysis time, and the sample detection flux is very limited;
✦
•
The dynamic range is very narrow (± 100 ng/mL), requiring a large amount of repeated testing during downstream process research;
✦
•
The high consumption of samples and reagents poses a challenge for the detection of specific low-level HCPs, as it is very difficult to generate sufficient HCPs from drug substances. This has created bottlenecks for ELISA in meeting the increasing speed, flux, and sample consumption of downstream process development teams.
So, more and more pharmaceutical companies have turned to high-throughput immunoassay platforms that help improve throughput and accelerate turnover, such as Gyrolab from Gyros®The fully automated immunoassay platform utilizes Gyrolab technology to capture biotinylated capture reagents using streptavidin coated microspheres. The bound samples are detected using fluorescently labeled detection reagents after binding with the capture reagents. This immunoassay performed in flow through mode on an affinity column is very fast and does not require incubation time, greatly reducing the time required to complete the analysis and the consumption of samples and reagents compared to traditional ELISA. The fully automated control operation enables excellent repeatability in Gyrolab testing. Therefore, the high-throughput analysis method of Gyrolab automation can provide effective support for HCP analysis in process development and QC release testing:
✓
Quickly analyze turnover efficiency;
✓
Excellent detection throughput;
✓
The data quality is reliable and the results have high repeatability.
Overall, this method can improve the efficiency of the biological analysis department, greatly reduce manual operation time, eliminate retesting requirements caused by dilution errors or human operation errors, and significantly improve the yield, quality, and reproducibility of analysis results.
