Biopharmaceutical Analytical Testing – A Critical Step in Producing Cutting-Edge Therapies

Biopharmaceuticals – or biologics – are a rapidly growing segment of the global pharmaceutical market that has revolutionized the management of hard-to-treat diseases such as cancer and autoimmune disorders.

Biopharmaceuticals include therapeutic preparations such as proteins, antibodies and DNA preparations. Unlike chemically synthesized drugs, most biopharmaceuticals are highly complex molecules produced through a multistep manufacturing process in living systems such as microorganisms, animals or plants. Thus, extensive characterization and an arsenal of analytical techniques are required to support a biopharmaceutical product from discovery to market entry. Analytical testing is also one of the critical steps toward achieving a successful biosimilar (follow-on biologics) approval.

Analytical testing provides the information needed to produce a safe and effective biopharmaceutical product, and the efficacy or safety depends on a product’s critical quality attributes (CQA). The International Council for Harmonization (ICH) Q8 (R2) defines CQAs as “a physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range or distribution to ensure the desired product quality”. Such attributes can be assessed by multiple analytical procedures, each yielding different results.

What makes analytical testing of biopharmaceutics a challenging task?

Analytical testing is a pivotal step in the development and approval of biopharmaceuticals, but it comes with its own set of challenges. “There are two major factors,” explains Dr. Anurag Rathore, professor at the Department of Chemical Engineering, Indian Institute of Technology. “One is the complexity of this class of products. Compared to a small molecule pharmaceutical that is likely to have a handful of CQAs, a biopharma product can have as many as 15–30 CQAs. Not just that, but many of these (such as aggregation or glycosylation) often require two or three analytical tools for performing a thorough characterization. The second major factor would be the regulatory oversight that the industry operates under. All methods need to be validated and this means that they need to meet high thresholds of the various performance criteria: accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ), etc. Considering these factors, combined with the low levels at which some of the impurities may need to be quantified, analytical method development is non-trivial,” says Rathore.

Regulatory authorities such as the WHO and the USFDA suggest the use of orthogonal analytical platforms for qualitative and quantitative characterization of CQA. Orthogonal methods include the use of analytical tools differing in their principle of operation. So, one method may detect variants that another method does not.

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Analytical testing of biopharmaceutical – ICH Q6B Guidance

As per the ICH Q6B, characterization of a biopharmaceutical product should include an assessment of its physicochemical properties, biological activity, immunochemical properties, purity and impurities.

Physicochemical characterization

Physicochemical characterization includes the evaluation of the composition, physical properties and primary structure of the product. Details regarding the higher-order structure of the product may also be obtained using suitable methodologies. Analytical methods that can be used to elucidate physicochemical properties are outlined in Table 1.

Table 1: Analytical methods that are used to study physiochemical properties.

Mass spectroscopy is one of the most popular techniques. “Mass spectrometry in biopharmaceutical analysis is a powerful tool as it allows for the characterization of product quality attributes (PQAs) which can have an impact on the overall safety, stability and efficacy of the drug product,” says Craig Jakes, research scientist at National Institute for Bioprocessing Research and Training (NIBRT), Dublin, Ireland. Examples of PQAs that manufacturers would be interested in are post-translational modifications (e.g., deamidation and oxidation), charge variant profiling arising from C-terminal lysine clipping and glycan abundance. “Modern-day mass spectrometry allows for intact, subunit or peptide characterization of biopharmaceuticals with each level providing more site-specific information than the previous,” adds Jakes.

Mass spectrometry techniques have evolved dramatically over the past few decades and several technological advances have been made. “For me, the most important technological advance in mass spectrometry has been the development of the multi-attribute method (MAM). MAM is a powerful tool that allows for the simultaneous monitoring of product quality attributes (PQAs) and detection of product or process-induced impurities through new peak detection (NPD) algorithms. At its core, MAM is a high-resolution peptide mapping protocol that is intended to replace a number of traditional assays in QC environments with a single LC-MS run. In NIBRT, my colleagues and I showed just how powerful this technique is when we applied it to a 12-day cell culture experiment and used NPD to identify impurities such as host cell proteins,” says Jakes.

Elucidating the biological activity

The biological properties of a biopharma product help decipher the specific ability or capacity of a product to achieve a defined biological effect. In many complex molecules, the physicochemical information might not be able to confirm the higher order structure, but it can be elucidated from the biological activity. The biological activity can be measured using animal-based biological assays, cell culture-based biological assays and biochemical assays.

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Assessment of immunochemical properties

Evaluating the immunochemical properties is necessary when the therapeutic product is an antibody. Immunochemical properties are parameters used to describe the interactions and sensitive reaction between an antigen and an antibody. Immunochemical characterization can help detect the product’s affinity, avidity and immunoreactivity. The product’s immune response and the rate of anti-drug antibody production can also be assessed. ELISA and western blot are techniques that can be used for immunochemical characterization.

Evaluating the purity and impurities

Biopharmaceuticals are also monitored for product purity and the presence of impurities. Detecting the product’s purity highly depends on the analytical method used and can be challenging. Usually, multiple orthogonal analytical techniques are used, as outlined in Table 2.

Impurities can be produced during manufacturing (process-related impurities) or can be related to the drug product (product-related impurities). Impurities may interfere with the product’s biological activity and negatively impact the product’s safety, efficacy and stability. For example, some impurities may stimulate an immune response to the product, leading to the generation of antibodies or cell-mediated immunity. Hence, it is essential to characterize the impurities to the best extent and asses their biological activities wherever possible.

Table 2: A list of the analytical procedures that may be used to detect impurities.

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Recent advancements in biopharmaceutical analytical testing

Elucidating the recent advancement in analytical testing, Dr. Anurag explains, “If I were to look at major advances in analytical testing over the last five years, I see the following trends. First, is the increasing use of MAM which aims to maximize the outcomes from a single workflow. Second, the replacement of offline testing with online and atline testing to increase the efficiency of decision making and cost reduction. Third, increasing use of spectroscopy-based testing for a wide variety of applications, replacing gradually traditional and cumbersome analytical testing tools such as HPLC and MS.” 

The critical role that the analytical procedures play in the biopharmaceutical developmental process, highlights the importance of a well-designed analytical strategy and selection of the best analytical tool. Recent years have witnessed several innovations in analytical technology and processes. Using the right analytical tool at the correct stage can ensure the efficient development of a safe and stable biopharmaceutical product.