This article from Ingenza explores the various analytical techniques used for assessing the properties of biologics.

The rapid evolution of bioprocessing in the pharmaceutical industry has revolutionised drug development by providing an efficient means to prepare biologics, which can offer more targeted treatments for complex diseases. However, unlike traditional small molecules – which are often chemically synthesised – biologics are derived from living systems, resulting in greater molecular complexity and variability in both mechanisms of action and means of production. As with all therapeutics, these bio-based medicines must also adhere to rigorous regulatory standards established by agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to ensure product quality, safety and efficacy. These factors present challenges for quality control and regulation of biotherapeutics, and manufacturers are therefore continuously seeking ways to improve the consistency and predictability of bioprocessing routes.¹ Succeeding in this endeavour hinges on use of robust analytical techniques, providing information about the characteristics, purity and functionality of bio-based products.

Overcoming challenges in biopharmaceutical production

Reliable analytical techniques allow manufacturers to closely track key variables and indicators during the production of biological materials, including host cell density and viability, metabolite levels, protein expression, oxygen and pH levels, nutrient consumption and contamination. The insights provided by these techniques enable bioscientists to monitor and optimise both upstream fermentation processes and downstream product purification to ensure the successful manufacture of biopharmaceuticals that meet necessary specifications for potency and efficacy, and satisfy regulatory requirements for safety and purity, easing the journey from lab to commercialisation.

Product complexity: Most bio-based medicines, such as monoclonal antibodies and other recombinant proteins, are characterised by their complex molecular structures. Biopharmaceuticals have sophisticated three-dimensional configurations and frequently undergo post-translational modifications (PTMs) that can significantly influence biological availability and therapeutic effectiveness, as well as their resistance to biological or chemical degradation.² The process of creating these molecules is therefore understandably complex, relying on highly delicate metabolic processes that are themselves sensitive to even minor changes in their environment. Fluctuations in fermentation conditions, variations in the quality of raw materials or slight deviations in process parameters can therefore significantly impact cell growth and the quality of the purified final product. In- and post-process analytical testing is essential to identify inefficiencies in the production process and detect any inconsistencies in the final drug substances and drug products. For example, PTMs such as glycosylation, oxidation and phosphorylation can introduce variability, so analytical testing methodologies must be able to detect and characterise these.

Impurity profiling: Analytical testing can also be used to identify harmful impurities and contaminants that can form or be introduced at various stages of production. For instance, residual host cell proteins or DNA fragments from the expression host may persist after purification if downstream processing is not fully effective in removing them. These contaminants can pose immunogenic or safety risks to patients if they exceed predefined levels in the final therapeutic product.³ In addition, protein aggregates and process-related contaminants must be carefully monitored, as they can affect the safety, efficacy and purity of biopharmaceuticals.⁴ Rigorous testing is crucial for accurately identifying and quantifying these impurities, ensuring that the target protein meets the necessary purity and safety standards to comply with regulatory requirements.

Choosing an appropriate test

The intricacy of biopharmaceuticals requires a combination of orthogonal techniques for effective characterisation and quality control. The methods used for these processes must align with relevant regulatory guidelines and standards,¹ and be conducted in accordance with Good Manufacturing Practices (GMP). The chosen combination of solutions should also offer rapid results – to enable proactive decision-making and real-time process adjustments^5,6 – while still being highly sensitive and specific to detect trace impurities and contaminants.¹ This often requires the use of advanced analytical techniques – such as liquid chromatography (LC) and mass spectrometry (MS) – that are capable of achieving high resolution separation and detection. These various analytical techniques are broadly divided across four key applications: physicochemical characterisation, biological activity measurement, immunochemical analysis and purity testing.

Physiochemical properties: Physicochemical characterisation involves analysing a drug’s composition, physical properties and primary structure, all of which can influence pharmacokinetics.⁷ For instance, lipophilicity is a key characteristic for permeating cell membranes and interacting with receptors.⁸ Additionally, a compound’s size, shape, hydrogen bonding and charge distribution can impact its absorption and distribution.⁹ Various techniques can be used to evaluate these attributes (see Table 1) and provide insights into the higher order structure of a compound.²

Table 1. Analytical methods used to study the physiochemical properties of biopharmaceuticals²

Physiochemical property	Analytical techniques
Molecular weight or size	·      Size exclusion chromatography ·      SDS polyacrylamide gel electrophoresis ·      Mass spectrometry
Isoform pattern	·      Isoelectric focusing
Extinction coefficient	·      UV/visible spectrophotometry
Electrophoretic patterns	·      Polyacrylamide gel electrophoresis ·      Isoelectric focusing ·      SDS polyacrylamide gel electrophoresis ·      Western blot ·      Capillary electrophoresis
Liquid chromatographic patterns	·      Size exclusion chromatography ·      Reverse-phase liquid chromatography ·      Ion-exchange liquid chromatography ·      Affinity chromatography
Spectroscopic profiles	·      Circular dichroism ·      Nuclear magnetic resonance

 

Biological activity: Some physicochemical analyses may be unable to confirm the higher order structures of complex drug products. However, these structures can often be inferred from the product’s biological activity.² Understanding biological properties of a biopharmaceutical product is also important to demonstrate its capacity to induce the desired effect. This activity is typically evaluated using both animal-based in vivo assays – to measure the overall biological response in an organism – and cell culture-based assays, which focus on biochemical or physiological responses at the cellular level. Biochemical assays can also be used to assess activities like enzymatic reaction rates, immunological responses or ligand or receptor binding to help further elucidate the product’s biological interactions.

Immunochemical properties: Evaluating the immunochemical properties of antibody-based therapeutic products is crucial to assess their therapeutic potential, safety and quality. This characterisation helps to identify the product’s affinity, avidity and immunoreactivity, as well as to assess likely immune responses and anti-drug antibody production rates.³ Gaining an understanding of these factors enables the optimisation of treatment strategies, minimises adverse effects and ensures adherence to regulatory standards. Common techniques for immunochemical characterisation include enzyme-linked immunosorbent assays (ELISAs) and western blot assays.³

Detecting impurities: Biopharmaceuticals must be monitored for impurities that could disrupt their efficacy or compromise product safety. Contaminants arising during the manufacturing process – referred to as process-related impurities – are typically identified using hybridisation techniques, immunoassays or clearance studies.³ Conversely, product-related impurities – those originating from the drug product – are analysed using various methods, including high performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism, mass spectrometry and size exclusion chromatography.³

Q-TOF LC–MS: a powerful tool

Quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS) is a more recently developed advanced analytical technique that can be used to separate, identify and measure the components of complex pharmaceuticals. LC can separate drug components by their polarity, size and unique structural characteristics, which are then ionised to form charged particles. These ions are passed through a quadrupole mass filter in the mass spectrometer, selecting ions with specific mass-to-charge ratios (m/z) for further analysis. The selected ions are accelerated through a high voltage flight tube, where their time-of-flight is measured to determine their molecular weight with high accuracy.

Q-TOF LC-MS offers unparalleled biopharmaceutical characterisation due to its high sensitivity, allowing the detection of molecules at very low concentrations, making it ideal for analysing complex biopharmaceuticals with trace amounts of target molecules. It provides accurate mass measurements to verify the composition and integrity of products, and enables the differentiation of molecules with similar masses, aiding in the identification of closely related species, like protein variants with differing PTMs. The technique also supports comprehensive analysis across a broad range of biomolecules – from small peptides to large proteins – in a single test, providing structural information and offering insights into the sequences, modifications and interactions of biopharmaceuticals to better understand drug activity.

Analytical aid

The information provided by analytical testing is vital for the successful development and manufacture of biopharmaceuticals. Manufacturers need to either maintain their own in-house analytical capabilities and expertise, or work with a contract research, development and manufacturing organisation (CRDMO) that is proficient in a variety of analytical methods. CRDMOs can provide a cost-effective helping hand, offering the specialised support, knowledge and infrastructure crucial to accelerate the drug development process and get new biologics to the market sooner, with minimal risks.

Ingenza offers a suite of instrumentation and analytical techniques to provide both qualitative and quantitative options for biopharmaceutical characterisation. By employing advanced techniques such as Q-TOF LC-MS, the company empowers bioscientists to evaluate the quality and efficacy of the pharmaceutical products that they are creating, detect and measure any impurities, and assess the overall effectiveness of their bioprocesses. These analytical capabilities aid in both process monitoring and optimisation efforts, ensuring consistent production of high quality pharmaceuticals. Each analytical approach is rigorously qualified to confirm its suitability for specific tests and the streamlined in-house workflows ensure rapid analysis. This quick feedback loop enables timely adjustments to bioprocesses, improving efficiency and reducing costs.

This expertise proved crucial in engineering a 60-protein nanoparticle vaccine that displays SARS-like betacoronavirus spike protein receptor binding domains (RBDs) from eight different human and animal coronaviruses.⁹ Using Pichia pastoris as a host, the challenge was to ensure consistent and reproducible distribution of all eight RBDs to provide vaccine efficacy against multiple coronavirus isolates. This project required detailed characterisation of the nanoparticle structure, so the team developed over ten assays to assess various physicochemical properties, biological activity, immunochemical properties and purity of both the individual vaccine components and the final product. By using unique markers for each RBD, our approach allowed their relative ratios and distribution to be determined, ensuring consistency in every batch.

Summary

The complexity of biopharmaceuticals demands advanced analytical techniques for accurate characterisation and quality control. These methods are crucial for assessing physicochemical, biological and immunochemical properties, and ensuring compliance with stringent regulatory standards. Ultimately, they support the reliable production and consistent quality of biopharmaceuticals, including complex products like nanoparticle vaccines. As the field of biopharmaceuticals continues to evolve, both new and existing analytical approaches will be fundamental to advance therapeutic innovation and ensure patient safety.

References

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2. Ratanghayra, N. 2022. Biopharmaceutical analytical testing – a critical step in producing cutting-edge therapies. Technology Networks Biopharma. Accessed: 2^nd May 2024. Available at: https://www.technologynetworks.com/biopharma/articles/biopharmaceutical-analytical-testing-a-critical-step-in-producing-cutting-edge-therapies-365496.
3. Wolter, T. and Richter, A. 2005. Assays for Controlling Host-Cell Impurities in Biopharmaceuticals. BioProcess International. Accessed: 7^th May 2024. Available at: https://eu-assets.contentstack.com/v3/assets/blt0a48a1f3edca9eb0/bltd0dbeefc34014a41/65c4be5b08ab13040aab54b2/0302ar06_77171a.pdf.
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8. Davis, A.M. and Leeson, P.D. 2023. Physicochemical properties. The Handbook of Medicinal Chemistry, 1-39. doi:10.1039/9781788018982-00001.