What are biologics?

Biologics are at the forefront of biomedical research and have been manufactured and developed using cutting-edge technologies, such as biotechnology methods, with the aim of treating a variety of medical conditions that previously had no other treatment available. Biologics have become known as the new therapeutic solution for many diseases.

What is a biological drug?

A biological drug can be made using complex combinations of substances, such as sugars, proteins, or nucleic acids, and may also be living entities such as cells and tissues, or come from a living organism. Biologic drugs, also known as a biologic or biological agent, are used in the prevention, diagnosis, or treatment stages of the medical process and include antibodies, interleukins, and vaccines for the treatment of cancer and other diseases. Some of the more well-known biological drugs include vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins.

Monitoring host cell proteins for downstream purification

During biologic manufacturing, it’s common to use host cells to express a target biological molecule as a final drug product, or a part of the final drug product. The host cell continues to produce its own biological molecules together with the drug product. These host cell-derived biological molecules are regarded as impurities and are subject to target substances to be removed during downstream purification. One of the main impurities are host cell-derived proteins, known as Host Cell Proteins (HCPs). The level of host cell proteins in biologics must be monitored carefully during development to optimize the downstream purification, as well as at final production to ensure the quality, efficacy and safety of the biological drugs.

Using ELISA tests to monitor host cell protein levels in biologics

The most widely used and accepted method to monitor host cell protein levels in biologics is an immunological assay, Enzyme-Linked Immunosorbent Assay, also known as ELISA. ELISA uses a set of polyclonal antibodies to detect as many host cell proteins as possible. These anti-host cell protein antibodies are critical reagents in the assay and should be evaluated to ensure compliance with industry standards.

The antibody coverage assay based on 2-dimensional (2D) electrophoresis and subsequent Western blot has been used to evaluate the performance of the anti-host cell protein antibodies. This method is written in both recent US and EU Pharmacopoeia, USP <1132> and Ph Eur 2.6.34, respectively. In this method, the host cell proteins, together with or without the final product, are separated by 2D electrophoresis and the anti-host cell protein antibodies are used at the subsequent Western blot to measure how many host cell protein spots have been detected by the antibodies. The result will be expressed as a percentage coverage of the antibodies to the total number of host cell protein spots separated by 2D electrophoresis.

However, this coverage assay comes with some known challenges:

  • Sensitivity of total host cell protein detection in the 2D electrophoresis
  • Gel-to-gel or blot-to-blot validations due to the electrophoresis and Western blotting
  • Spot matching between 2D electrophoresis detection and immuno-detection (Western blot)
  • Time consuming evaluation and image data analysis for those involved in process development or monitoring host cell protein impurities in biopharmaceutical products

An enhanced host cell protein analysis method

To address these challenges, we propose an enhanced anti-host cell protein antibody coverage analysis method, 2D Differential in Blot Electrophoresis (2D-DIBE). This solution includes:

  • Fluorescent multiplexed methodology based on CyDye pre-labeled and 2D separated proteins. The labeling is proven by a long history of 2D DIGE and provides high sensitivities in total host cell protein detection.
  • To minimize gel-to-gel and blot-to-blot variation, the labeled proteins separated by 2D electrophoresis are transferred to a membrane and immunological detection is carried out on those proteins with anti-host cell protein antibody (Western blot). The two images are directly visualized and compared to the proteins detected by CyDye pre-labeled host cell protein-antibodies on the same membrane.
  • Multiplex fluorescence image acquisition. Capture both host cell protein antigen and anti-host cell protein antibody images from a single membrane with laser based Amersham Typhoon™ by Cytiva. This avoids any mismatch issue due to the different detection methods.
  • Melanie™ Coverage Software entirely and solely focused on coverage analysis, with a specific workflow and dedicated tools developed in collaboration with Cytiva and major companies in the biopharmaceutical arena. This allows investigators to spend less time evaluating the data with more confidence.

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