Separation Sciences and Analytical Biotechnology Laboratory

Chemical characterization of Biologics by High-Performance Liquid Chromatography and Capillary Electrophoresis

At the Centre for Biologics Evaluation, we apply the latest technology in the development and application of analytical methods for the chemical characterisation of biomolecules based on high resolution separation techniques. We specialise in the application of High-Performance Liquid Chromatography (HPLC) and Capillary Electrophoresis (CE) for analysing Biologics.

Why we study biologics using separation sciences and analytical biotechnology

Biologics such as vaccines, blood products, and recombinant therapeutic protein preparations can contain simple or complex substances. As the amount, combination, and characteristics of these substances affect how the biotherapeutic product will act in the body, evaluating the quality of biologics is important. We use separation sciences and analytical biotechnology to analyse biologics for quality control. We also use them to identify and characterize the substances present and to detect contaminants. Furthermore, we use separation sciences and analytical biotechnology to investigate whether a formulation matches its description and whether active ingredients meet quality standards.

As we develop and improve analytical techniques, we acquire tools for evaluating biologics. New tools help us analyse complex mixtures and structures, detect contaminants, and examine new biomolecules and biologics.

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How we study biologics using separation sciences and analytical biotechnology

At the Centre for Biologics Evaluation, in the Separation Sciences and Analytical Biotechnology Laboratory, we develop and improve methods for the separation and analysis of substances in biologics. Analysis can be challenging because of the complexity of mixtures and structures. We need to separate small amounts of active ingredients, such as proteins, from samples that contain large amounts of added ingredients (excipients) such as diluents and carrier ingredients.

We use technically advanced instruments and separation methods to investigate and apply high-resolution analytical techniques. We use High-Performance Liquid Chromatography (HPLC) and Capillary Electrophoresis (CE) to study biomolecules and address concerns such as product quality, purity, contaminant detection, and batch consistency.

We apply our expertise and analytical abilities to

  • Develop and improve techniques in order to analyse sample characteristics such as product quality, purity, and batch consistency
  • Generate research information that will help establish criteria and standards for quality and purity testing of biologics as well as lot-release testing programs
  • Develop and improve techniques to study biosimilars
  • Analyse the quality of biologics to provide information, such as impurity profiles and contaminant detection, for assessing and responding to adverse reactions
  • Provide a Canadian perspective for setting international standards and methods for biological substances by participating in international collaborative studies with other regulatory agencies such as the U.S. Food and Drug Administration, World Health Organization, European Pharmacopoeia, and United States Pharmacopeia
  • Provide support to manufacturers for setting up and optimizing high-resolution separation techniques

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High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE)

High-performance liquid chromatography (HPLC) is a powerful tool that we use at the centre to separate, identify and measure the amount of a substance in a sample. In general, the mixture containing the Active Pharmaceutical Ingredient (API) is forced through a separation column under high pressure. The compounds from the sample move through the column at different speeds as a result of their affinity to the column material. Different compounds have different affinities and thus separate from each other. When a compound leaves the column, it goes through a detector (UV or Fluorescence detector) and is recorded. The collected and processed data allows for the characterisation of the Biologics and other compounds in the sample.

Capillary electrophoresis (CE) is a powerful approach that we use at the Centre for Biologics Evaluation to separate and characterize charged molecules, large or small, such as DNA and proteins. Samples are run through an electric field in a thin tube, of less than 100 micrometers, called capillary tubing. In the capillary electrophoresis instrument, light sources acting as detectors track and measure how molecules migrate or move. Since capillary electrophoresis uses small capillary tubing, it has advantages over standard electrophoresis methods, such as

  • enabling the use of high voltages without damaging samples
  • providing faster processing by using increased voltages, sensitivity or detection
  • using light detection measurements
  • analysing small amounts of sample
  • automating and analysing large sets of samples relatively quickly

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Research highlight 1: Development of new approaches for the fast separation and quantitative assessment of hemagglutinin content in influenza vaccines

Influenza is a major public health threat where each year the human influenza viruses cause seasonal influenza in about 10% of the world population with three to five million severe cases of illness and up to half a million deaths. Manufacturing of Influenza vaccines ahead of the targeted season is currently the most effective countermeasure against influenza.

Seasonal inactivated influenza vaccines are formulated to include antigens from strains of influenza A and B virus lineages. In such vaccines, each strain is composed of the immunogenic surface protein hemagglutinin (HA) and neuraminidase (NA) responsible for host protection against the Influenza virus. Currently, the Single Radial Immunodiffusion (SRID) test is the standard method for the determination of influenza vaccine potency by HA quantification. Although the SRID was successfully used for decades, one major disadvantage is the requirement for production of time-consuming calibrated reagents. This was evident in the 2009 H1N1 pandemic where fast production and release of vaccines was delayed because of the unavailability of SRID reagents. Furthermore, SRID has been found to be inadequate for the identity and potency testing of the B-influenza strains in QIV because of the cross-reactivity between the potency reagents. This lead to the conclusion that on top of improving the assessment of vaccine potency by SRID, the development and use of alternative approaches is necessary.

In our laboratory, we address this concern by developing HPLC- and CE-based approach for the analysis of HA and NA antigen content in Influenza vaccines. We have developed and continue to improve analytical methods for the fast evaluation of quality and HA strain identification in commercial Influenza vaccines.

We are currently working to

  • Develop new methods and approaches to quantify immunologically relevant hemagglutinin in influenza vaccine
  • Detect and characterize quality and quantity of Neuraminidase in finished product
  • Develop new tools for quality assessment of chemical key attributes of vaccine antigens such as glycosylation profile and consistency of production
  • Develop and adapt such methods to be applied to Influenza vaccine made by recombinant technology using cell-lines over egg for Influenza vaccine production

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Research highlight 2: Elaboration of analytical assays for the evaluation of Interferon-alpha and product safety

Interferons are proteins that are secreted by cells in response to foreign substance such as virus to stimulate the immune response of the infected cells. The name comes from their ability to interfere with production of new virus particles. For this reason, Interferon alpha-2 products have been widely used as antivirals for the treatment of diseases such as hepatitis B and C.

Recent advances in technology, such as recombinant DNA technology, have enabled the large-scale production of Interferon for therapeutic applications.

As biologics, Interferon preparations are analysed for quality using a wide range of specific standard tests to characterize the substances present in the sample to maximize their safety. Then, the biologics are compared against a standard to confirm specific biological activity based on protein structure and formulation.

In our laboratory, we use modern technology to characterize the structure of Interferon and develop better ways to analyse new and marketed products. We have used HPLC and CE combined with mass spectrometry to develop effective approaches to examine Interferon alpha-2 samples. In the course of this work, we have identified impurities that had been unreported previously.

We are working to:

  • Detect and characterize contaminants such as Interferon degradation forms and structural variants
  • Develop new methods and approaches to examine Interferon samples
  • Develop new tools for surveillance of marketed products and new formulations to prevent adverse drug reactions in patients

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