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    Features

    Next-Gen Bioprocessing

    Overcoming the challenges of characterizing bioprocess residual impurities in biologics using HPLC/MS

    Next-Gen Bioprocessing
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    Fergus Hall, Section Manager, Pharmaceutical Chemistry, Eurofins BioPharma Product Testing05.09.17
    Effective profiling and removal of impurities is a critical step in the development of biopharmaceuticals. However, because residual impurities are typically present at low concentrations within difficult sample matrices, their detection and quantification can be challenging. This article discusses the need for improved techniques in process validation studies and looks at how high performance liquid chromatography mass spectrometry (HPLC/MS) may represent a powerful solution to overcome the challenges and meet regulatory expectations.

    Bioprocess residual impurities in biologics
    In recent years, biopharmaceuticals, or large molecule drugs, have surpassed their small molecule counterparts in terms of the quantity of new products coming through the drug development pipeline. Unlike small molecule drugs that are manufactured via chemical synthesis, biologics undergo multiple post-translational modifications when manufactured in living cells. Examples of biologic products include hormones, enzymes, monoclonal antibodies, vaccines and blood factors. Biologics are more complex than small molecule drugs and require more quality assurance testing during the manufacturing process to ensure potency, quality and purity.

    The manufacture of biopharmaceuticals involves the use of a variety of process additives that have the potential to become residual impurities and have a presence in the process stream. Tracking the clearance of these residual impurities is an essential element of process development and characterization, while being fundamental to achieving regulatory compliance and market approval for new products. Methods to detect, characterize and accurately quantify bioprocess residuals can be used to demonstrate removal of residual impurities in process stream samples.

    Biologic-related impurities can fall into several categories. Some may be introduced in the upstream steps as required components of a process, including proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Other impurities, such as antibiotics, media components and inducers, can result from various cell culture steps. There can also be residual impurities that are introduced downstream from resins, residual solvents and surfactants, while others can occur through contact with single-use bioprocess components. For example, extractable and leachable substances, where impurities can migrate from pharmaceutical container closure systems, process equipment and packaging.
    Tracking the clearance of these residuals is an essential part of process development and characterization. 

    Regulatory standpoint
    Regulatory authorities have established clear and rigorous guidelines, which dictate the identification of impurities. The development, validation and testing of biopharmaceuticals must be conducted in line with the International Conference on Harmonization (ICH) Technical Requirements for Registration of Pharmaceuticals for Human Use Q5A to Q5E (Quality of Biotechnological Products) and the Code of Federal Regulations (CFR) Title 21 parts 600, 601 and 610. However, since residuals are typically present at varying levels throughout the process, method development and optimization can be a challenging part of process characterization. For example, extraneous protein known to be capable of producing allergenic effects in human subjects shall not be added to a final virus medium of cell culture produced vaccines intended for injection. If serum is used at any stage, its calculated concentration in the final medium shall not exceed 1:1,000,000 (CFR 21 part 610.15).

    Detection methodologies
    Various analytical techniques are available for the identification and quantification of impurities in biologics. However, identification of trace levels remains challenging as conventional analytical approaches involve multiple instrument platforms and sample workup steps.

    Many of the methods used in a pharmaceutical quality control (QC) laboratory to characterize and classify residual impurities utilize high performance liquid chromatography (HPLC) or gas chromatography (GC) combined with a range of different detection methods. These include:
    • Charged aerosol detection (CAD)
    • Evaporative light scattering detector (ELSD)
    • Liquid chromatography mass spectrometry (LC-MS)
    • Gas chromatography with flame ionization detector (GC/FID)
    • Gas chromatography mass spectrometry (GC-MS)
    • Enzyme-linked immunosorbent assay; and
    • Quantitative polymerase chain reaction.
    The various methods are appropriate for different materials. For example, the detection of residual solvents and volatile molecules are most amenable to GC/FID, whereas qPCR works best for proteins. ELSD is commonly used for the analysis of compounds where UV detection might be a restriction, e.g. sugars, antivirals, antibiotics, lipids, phospholipids, terpenoids, and alcohols.

    The advantages of HPLC-MS
    Typically, the detection methods used for biologic products are HPLC/CAD and HPLC/MS. HPLC/CAD is a cost-effective universal detector, which is ideal for limit tests and screening. It is highly sensitive and has a broad dynamic range, which offers some real advantages to researchers and analysts in a pharmaceutical laboratory, particularly when analyzing compounds lacking UV chromophores. However, compounds of interest must be non-volatile and this technique has proven to be less robust than other methods.

    HPLC/MS has the advantage of being able to detect many impurities and is also an extremely sensitive and selective technique. It can therefore provide more information and a better indication of purity. The only requirement is that the residual impurity being analyzed must be ionizable.

    Unlike the CAD detector, MS can provide a wide-range of information on analyte identification. It yields both qualitative and quantitative information, making it one of the primary tools for monitoring and identifying residual impurities. It is also the only technique that provides the capability to both identify and quantify residual impurities, and offers sensitivity down to the picogram (10-12) level with high resolution and accurate mass.

    The need for a reliable outsourcing model
    Although there are multiple advantages in applying HPLC/MS for the detection of impurities, the technique is widely associated with high costs. When concurrently faced with ever-increasing pressure to cut development budgets, this means that it is simply not feasible for many manufacturers. The expense of procuring the instrumentation can reach the $600,000 mark, and this is without even beginning to count the cost of instrument installation and qualification, software validation, as well as maintaining an in-house team to operate the equipment and interpret the resulting data. Consequently, there is an increasing trend towards outsourcing residual impurities analysis using this technique.

    Final thought
    As biopharmaceutical production processes evolve, the detection and tracking of residual impurities is becoming increasingly difficult. The complexity of new process matrices coupled with requirements for increased sensitivity present multiple challenges. Combined with this, manufacturers of biologics are coming under increasing regulatory pressure to identify, quantify and monitor impurities in their products, meaning that they are faced with the choice of selecting the right technique to ensure compliance, as well as to determine the purity and safety of their final products.

    There is a growing need for methods that can quantitate and characterize impurities in increasingly complex matrices. In the manufacture of today’s biologics, HPLC/MS represents a powerful analytical tool with a high degree of sensitivity and selectivity. Recognized for its ability to provide manufactures with the most defensible data, HPLC/MS can assist the end user in complying with regulatory requirements by identifying and quantifying biopharmaceutical residuals with a high degree of precision, specificity and accuracy. 


    Fergus Hall is section manager for pharmaceutical chemistry at Eurofins BioPharma Product Testing in Dungarvan, Ireland. He is responsible for the management of five analytical teams, testing sample material on behalf of global biopharma and pharmaceutical clients. He has specialist expertise in contract testing, method development, transfer and validation. With a PhD in analytical chemistry, Fergus has spent more than 11 years working for contract research and contract testing organizations in managerial positions.
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