The drug development landscape has undergone a massive transformation in recent years, driven by advancements in bioprocessing and the rise of biologics. Compared to traditional small-molecule drugs, biologics differ significantly in terms of their production methods, molecular complexities, and mechanisms of action.1 Their large size and elaborate structures enable these bio-based medicines to target specific biological pathways, offering efficient and tailored treatments for a variety of diseases with minimal off-target effects. However, this complexity also creates significant challenges when it comes to manufacturing, quality control, and regulation.2 As a result, manufacturers are continually working to improve the consistency and predictability of bioprocessing.
Successful production of biologics hinges on the use of robust analytical techniques that provide crucial information about the characteristics, purity, and functionality of these bio-based products. These insights enable precise process management and quality control, allowing any deviations in biological formulation or production processes to be promptly addressed. Fortunately, bioscientists do not have to navigate the intricacies of biopharmaceutical analysis alone; contract research, development, and manufacturing organizations (CRDMOs) can offer specialized support and novel analytical approaches to help ensure compliance with safety and regulatory standards, as well as to enhance the overall efficiency of biopharmaceutical production.
An Introduction to Biomanufacturing
While traditional small-molecule drugs can be chemically synthesized, biologically derived molecules are produced using living organisms or biological systems, including bacteria, yeasts, plants, or mammalian cells. This fundamental difference results in distinct development and manufacturing processes. Small molecule pharmaceuticals are created through well-defined – and often long-established – chemical reactions, yielding products that are generally smaller and structurally simpler, which makes their development more straightforward and their behaviors more predictable. In contrast, biologics – such as vaccines, blood products, growth factors, monoclonal antibodies, and other recombinant proteins – are generated through intricate biological processes.
Biomanufacturing involves a series of carefully orchestrated steps, each designed to ensure the safety, efficacy, and consistency of the final biological drug. The process begins with upstream processing – which includes cell line development and optimization of fermentation or cell culture conditions – laying the groundwork for producing therapeutic proteins. During these early stages, bioscientists may genetically modify the genome of host cells to produce the target proteins and optimize growth conditions to maximize protein yields. This optimized process can then be scaled up to manufacture the product. Once the desired protein has been produced within the cell or secreted to the extracellular culture medium, downstream processing steps are conducted to recover, purify, and isolate the target protein for formulation into the final drug product.
Analytical Approaches to Tackle Bioprocessing Challenges
The unique characteristics of biologics mean that they are generally more difficult to characterize than small molecule drugs, making the use of advanced analytical techniques crucial to assessing the consistency of production, as well as product integrity and purity.3 Manufacturers use a variety of analytical approaches to closely track key variables throughout biological production, including host cell density and viability, metabolite levels, protein expression, oxygen and pH levels, nutrient consumption, and any potential contamination of the culture.3 These insights are vital for optimizing both fermentation processes and downstream product purification, helping to ensure that the resulting biopharmaceuticals meet safety, efficacy, and regulatory standards.
Product Variability
The inherent complexity of bio-based compounds underscores the need for comprehensive analytical testing. Most biologics have sophisticated three-dimensional architectures and often undergo post-translational modifications (PTMs) that can influence biological availability and therapeutic effectiveness, as well as their resistance to biological or chemical degradation.4 For example, PTMs such as glycosylation, oxidation, and phosphorylation can introduce variability in biopharmaceuticals, so analytical testing methodologies must be able to detect and characterize these.2
The biomanufacturing process is also influenced by a range of factors, relying on dynamic and highly sensitive metabolic processes that are affected by even minor environmental changes. Fluctuations in fermentation conditions, variations in raw material quality, and even slight deviations in process parameters can significantly affect cell growth and metabolism, as well as the quality and efficacy of the final purified drug product. In- and post-process analytical testing is therefore essential to identify bottlenecks and inefficiencies in both upstream and downstream processes and to detect any variations in the final drug products. Testing methods should provide accurate and reliable data in real-time, so that bioscientists can make proactive decisions and adjustments during drug production, ensuring optimal product yield, quality, and productivity.5,6
Susceptibility to Impurities
Analytical testing is also essential to identify potentially harmful impurities and contaminants that can be introduced at various stages of production. For instance, residual host cell proteins or DNA fragments may remain in the drug formulation if downstream purification steps are not entirely effective at eliminating them. These contaminants can present immunogenic or safety risks to patients if their levels exceed predefined limits.7 Protein aggregates and process-related contaminants in biopharmaceuticals must also be closely monitored, as they can impact their safety, efficacy, and purity.8 Analytical testing methods need to be highly sensitive and specific to detect even trace levels of these impurities.1
Ensuring Regulatory Compliance
Extensive analysis of both bioprocesses and the final pharmaceutical products is necessary to ensure that they meet required quality control specifications. As with all therapeutics, bio-based medicines must meet stringent regulatory standards established by agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA).8 Various tests – including batch release, stability checks, and product quality investigations – should be carried out at several stages of drug development, and the assay methods chosen need to meet regulatory guidelines and standards.9 Most importantly, analytical tests should be properly validated and must be conducted by Good Manufacturing Practices (GMP) to ensure the reliability of data and the quality, safety, and efficacy of commercialized biologics.
Choosing the Right Method for Analysis
Analyzing complex biopharmaceuticals demands a combination of orthogonal techniques for effective characterization and quality control. A variety of advanced analytics – such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) – are capable of achieving high-resolution separation and detection, enabling sensitive assessment of product quality and purity. Testing methods can be broadly divided across four key applications: physicochemical characterization, biological activity measurement, immunochemical assessment,t and purity testing.
Assessing Physicochemical Properties
Physicochemical characterization is the process of examining a drug’s composition, physical properties, and primary structure, all of which can affect how it is absorbed and distributed in the body.3 For example, lipophilicity – a drug’s ability to dissolve in fats – is a crucial property that enables it to pass through cell membranes and interact with target receptors.10 Additional factors like a compound’s size, shape, hydrogen bonding, and charge distribution can also influence its pharmacokinetics.11 Specific techniques can be used to assess these characteristics and gain insights into the higher-order structures of biologics (as shown in Table 1).3
Measuring Biological Activity
While some physicochemical analyses might not be able to directly confirm the higher-order structures of complex drug products, this can often be inferred from the product’s biological activity.3 Understanding a biopharmaceutical product’s biological properties is also crucial for determining how effectively it can achieve its desired effects. Several assay types can be used to measure a drug’s effectiveness and interactions with biological systems, including cell culture-based and in vivo assays.12 Cell culture-based assessments focus on biochemical or physiological responses at the cellular level, while animal-based in vivo assessments allow the measurement of the overall biological response in an organism. Additional assays – such as biochemical, ligand, or receptor binding assays – can examine specific biological activities, like enzymatic reaction rates or immunological responses, as well as interactions between the drug and target molecules in the body.
Assessing Immunochemical Properties
The immunochemical properties of antibody-based therapeutic products – such as their affinity, avidity, and immunoreactivity – can also provide valuable information about their effectiveness, safety, and overall quality.3 Factors such as the elicited immune response and rate of anti-drug antibody production can be assessed to help determine how well the product binds to its target, and how strong this binding is. Gaining an understanding of these factors enables improved treatment strategies, minimizing side effects, and ensuring that the product meets regulatory requirements. Common methods for immunochemical characterization include enzyme-linked immunosorbent assays (ELISAs) and western blot assays.3
Detecting Impurities
Biopharmaceuticals must be carefully monitored for any impurities that could affect their biological activity, safety, and efficacy. Contaminants produced during the manufacturing process – known as process-related impurities – can interfere with a drug’s intended function, so they should be detected using immunoassays, hybridization techniques, or clearance studies.3 In contrast, impurities related to the drug product itself require different analytical techniques. A variety of methods can be used to identify these product-related impurities, including HPLC, MS, SDS-PAGE, circular dichroism, and size exclusion chromatography.3
Q-TOF LC-MS: Revolutionizing Analytical Testing
Analytical testing methods are constantly evolving to enhance sensitivity and accuracy and to keep pace with increasingly sophisticated biologics. Quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS) is a modern analytical method used to separate, identify,y and measure the components of complex biopharmaceuticals. This advanced approach combines liquid chromatography (LC) and MS to give detailed information about the composition, integrity, and biological activity of these medicines.
How Q-TOF LC-MS Works
The process begins by using LC to separate drug components based on their polarities, sizes, and unique structural characteristics. Once separated, the individual components are ionized to create charged particles. These ions are introduced into the mass spectrometer, where they are passed through a quadrupole mass filter that selects ions with a specific mass-to-charge ratio (m/z) for further analysis. The selected ions are then accelerated through a high-voltage flight tube, and their time-of-flight – the amount of time it takes to travel from the ion source to the detector – is measured. This value allows precise determination of the molecular weight of the ions with exceptional accuracy.
Benefits of the Technique
Q-TOF LC-MS offers several key benefits for biopharmaceutical characterization. It provides accurate mass measurements to help confirm the composition and integrity of biopharmaceutical products, as well as to distinguish between closely related species, such as protein variants and differing PTMs. Its sensitive detection capabilities also allow the identification of molecules at very low concentrations, which is essential for analyzing complex biologics that may contain only trace amounts of target molecules or contaminants. Crucially, the technique supports thorough analysis of a wide range of molecules – from small peptides to large proteins – in a single test, providing structural details and valuable insights into biopharmaceutical sequences, modifications, and interactions to better understand drug mechanisms.
Leveraging CRDMO Expertise for Advanced Biopharmaceutical Analyses
State-of-the-art analytical technologies like Q-TOF LC-MS offer extensive insights into biological drugs, providing critical information that is vital for the successful development and manufacture of these complex pharmaceutical products. While these advanced technologies are invaluable, setting up and maintaining in-house analytical capabilities can be time-consuming and costly. Fortunately, manufacturers have a viable alternative to managing biopharmaceutical analyses independently: partnering with a CRDMO that excels in a range of analytical methods. This type of collaboration provides access to cutting-edge technology and expertise, without the burden of establishing and managing costly in-house facilities.
CRDMOs offer a strategic advantage to biomanufacturers by providing specialized support, in-depth knowledge, and robust infrastructure. A reputable CRDMO will be proficient in a variety of analytical methods and should be equipped to handle complex testing requirements with efficiency and precision. CRDMO partnerships give manufacturers access to advanced, cost-effective,e and GMP-compliant analytical testing solutions, allowing them to streamline the drug development process and redeploy resources to other areas of the business. By providing efficient and less resource-intensive analyses, CRDMOs help to accelerate the necessary analytical testing throughout drug development and production, enabling the rapid optimization of biomanufacturing routes and, ultimately, more efficient manufacturing. This not only speeds up the time-to-market for new biopharmaceutical products but also ensures that they meet stringent regulatory standards. As a result, CRDMOs play an important role in helping manufacturers achieve high levels of quality and reliability in their products.
A New Era of Biopharmaceutical Production
The biopharmaceutical field is rapidly evolving, propelled by major technological innovations and growing demand for biologics. Novel technologies are revolutionizing bioprocessing to significantly accelerate the commercialization of new therapies, where cutting-edge analytical techniques – such as Q-TOF LC-MS – offer unparalleled insights into the intricate structures and functions of biologics. At the same time, the increased use of CRDMO partnerships is reshaping the industry, providing specialized expertise and state-of-the-art infrastructures that many companies would struggle to develop in-house. Outsourcing analytics can enhance biomanufacturing efficiency, drive innovation, and provide a significant competitive advantage to biopharmaceutical companies.
References
- Jacoby, R, Heim, M, Pernenkil, L, Sabad, A, Harutunian, S. Advanced Biopharmaceutical Manufacturing: An Evolution Underway. Deloitte Life Sciences. Available at: https:// www2.deloitte.com/content/dam/Deloitte/us/Documents/life-sciences-health-care/ us-lshc-advanced-biopharmaceutical-manufacturing-white-paper-051515.pdf. Accessed August 6, 2024.
- Berkowitz, SA, Engen, JR, Masseo, JR, Jones, GB. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nature Reviews Drug Discovery. 2012;11(7):527-540.
- Ratanghayra, N. Biopharmaceutical analytical testing – a critical step in producing cutting-edge therapies. Technology Networks Biopharma. Available at: https://www. technologynetworks.com/biopharma/articles/biopharmaceutical-analytical-testing-a-critical-step-in-producing-cutting-edge-therapies-365496. Accessed August 6, 2024.
- Walsh, G. Post-translational modifications of protein biopharmaceuticals. Drug Discovery Today. 2010;15(17-18):773-780.
- Rathore, AS, Mishra, S, Nikita, S, Priyanka, P. Bioprocess Control: Current progress and future perspectives. Life (Basel). 2021;11(6):557.
- Mitra, S, Murthy, GS. Bioreactor control systems in the biopharmaceutical industry: A critical perspective. Systems Microbiology and Biomanufacturing. 2021;2(1):91-112.
- Wolter, T, Richter, A. Assays for Controlling Host-Cell Impurities in Biopharmaceuticals. BioProcess International. 2005;40-46.
- Geiger, J. Complex Process-Related Impurity Profiles. In: The Challenge of CMC Regulatory Compliance for Biopharmaceuticals. Springer, Cham. 2019.
- Ritter, N, Russel, R, Schofield, T et al. Bridging Analytical Methods for Release and Stability Testing: Technical, Quality and Regulatory Considerations. BioProcess International. Available at: https://www.bioprocessintl.com/product-characterization/bridging-analytical-methods-for-release-and-stability-testing-technical-quality-and-regulatory-considerations. Accessed: August 6, 2024
- Karlgren, M, Bergström, CA. How physicochemical properties of drugs affect their metabolism and clearance. New Horizons in Predictive Drug Metabolism and Pharmacokinetics. 2015;1-26.
- Davis, AM, Leeson, PD. Physicochemical properties. In: Ward, SE, Davis, A, eds. The Handbook of Medicinal Chemistry. Royal Society of Chemistry. 2023:1-39.
- ICH Q6B Specifications: Test procedures and acceptance criteria for biotechnological/ biological products - scientific guideline. European Medicines Agency. Available at: https:// www.ema.europa.eu/en/ich-q6b-specifications-test-procedures-acceptance-criteria-biotechnological-biological-products-scientific-guideline. Accessed: August 6, 2024.
Author Details
Fraser Brown, PhD, Head of Chemistry- Ingenza
Dr Fraser Brown joined Ingenza in 2006, after completing his PhD in Medicinal Chemistry at the University of Edinburgh. He has extensive experience in bioprocess design, development, and optimization for a range of small molecule, protein,n, and biopharmaceutical products. This expertise led to his promotion to Head of Chemistry in 2015. In this role, he leads a team of scientists involved in various aspects of bioprocess evaluation, assay development, product isolation, and analytical characterization.
Publication Details
This article appeared in American Pharmaceutical Review: Vol. 27, No. 6Sept/Oct 2024Pages: 60-64
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