Innovations in Analytical Methodologies for Biopharmaceutical Characterization

By Marzia KhanReviewed by Lily Ramsey, LLM

Biopharmaceutical characterization is a significant step in developing biopharmaceutical products such as vaccines, blood products, and recombinant proteins (including monoclonal antibodies).¹

Image Credit: Pickadook/Shutterstock.com

Introduction

The characterization process includes exploring the structural features of a drug, which can have a great impact on variables such as stability, efficacy, and safety of the final drug product.² Additionally, characterization can aid in defining product-related impurities and in detecting and quantifying protein/DNA impurities derived from the host cell.¹

As a result of the complexity of biopharmaceuticals, the characterization analysis is completed using more than one assay for in-depth analysis and reduced biases.^2,3

New analytical innovations have been developed to address the complexity of biologics, such as monoclonal antibodies that are more complex than traditional small-molecule drugs.⁴

Key Innovations in Analytical Methodologies

High-Resolution Mass Spectrometry (HRMS)

High-resolution mass spectrometry (HRMS) is an analytical technique that is used to study biomolecules, especially in the biopharmaceutical industry. This technique consists of mass spectrometry that has a routine broadband resolving power that is higher than 10,000.⁵

HRMS is used to characterize covalent structures of protein therapeutics and has become significant in this area due to its improved performance and versatility; this further enhances the effect of MS in complex structural characterization, with improved accuracy, specificity, and higher throughput.⁵

HRMS has advanced the possibility of elucidating almost complete proteomes, enabling identification, quantification, and partial site-specific localization of an increasing number of post-translational modifications (PTMs).⁶

PTMs of proteins represent a significant mechanism for cellular control and also affect many areas of protein function, such as their activity, stability, and interactions, with many of these modifications leading to diseases that occur from their deregulation. This is why critical proteins involved in the regulation of PTMs are used as drug targets.⁶

The advantages of HRMS enable a wide range of applications for pharmaceutical development such as drug discovery, product characterizations of small molecules and novel drug modalities, as well as in vitro and in vivo metabolism studies, post-approval quality control, and pharmacovigilance, ensuring product consistency.⁷

Advanced Chromatography Techniques

Advanced chromatography techniques such as liquid chromatography-MS (LC-MS) enable easy detection of differences between a monoclonal antibody and a biosimilar.

An example of this includes detecting differences between an innovator monoclonal antibody, trastuzumab, and a biosimilar using LC-MS, which detected changes in glycosylation as well as amino acid mutations in the heavy chain.⁴

Researchers use multi-dimensional separation methods to characterize protein biopharmaceuticals such as monoclonal antibodies, which can have high heterogeneity with many variants that coexist and can impact drug safety and efficacy.⁸

Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) is ubiquitously used for impurity profiling of drugs, analysis of biomolecules, and characterization of biopharmaceuticals.⁹

A type of CE includes capillary isoelectric focusing (CIEF), which employs MS-incompatible ampholytes and buffers that can be combined with CE-MS to selectively detect charge variants of monoclonal antibodies.¹⁰

CE is currently a preferred method for analyzing biologics, food materials, and supplements as well as determining adulterants due to providing higher resolution, faster results, higher sensitivity, requiring a small volume of analytes, and consuming less reagents for drug analysis.⁹

Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy was a breakthrough technique that is highly compatible with analyzing O-linked glycans in proteins.⁴

While traditional NMR analysis of proteins was limited by the requirement of a large sample size for effective data collection, advancements in this technology, such as through flow NMR and microcoil NMR, have overcome this obstacle.⁴

Monitoring the higher-order structure of intact proteins is significant for characterizing biologics, which, when analyzed, can lead to awareness of differences in this structure and can provide information on any potential biological or immunological differences between proteins and variants.⁴

Analyzing the high-order structure of proteins can also be used to assess comparability between innovator product versions before and after process changes and for establishing a lack of comparability between an innovator product and a biosimilar version.⁴

With the development of smaller protein biopharmaceuticals, the use of a simple one-dimensional hydrogen-1 NMR can produce NMR fingerprints that provide comparability assessments that are useful for drug development.⁴

Next-Generation Sequencing

Next-generation sequencing (NGS) is a revolutionary tool that is used in genomic research, with the capacity to sequence millions of DNA fragments at one time. It provides comprehensive information about genome structure, gene activity, genetic variations, and alterations in gene behavior.¹¹

NGS's versatility has expanded the scope of genomics research, facilitating studies on cancer genomics, rare genetic diseases, and infectious diseases. It has also advanced the development of targeted therapies, approaches relating to precision medicine, and improved diagnostic strategies.¹¹

The power of NGS within translational medicine includes its advanced efficiency in multiplexing, as well as useful bioinformatic tools that are used for data curation and different reference databases, which aid drug manufacturers and designers in understanding the genetic basis of the disease.¹¹

Learn more about analytical chemistry techniques

Emerging Techniques for Biopharmaceutical Quality and Stability

Multi-Attribute Method

Multi-Attribute Method (MAM) consists of an LC-MS-based method that is used to directly characterize and monitor many product quality attributes (PQAs) of a biopharmaceutical product at the amino acid level.¹²

MAM can be used throughout the product life cycle, going from process development to upstream and downstream processes, as well as during quality control release or under current good manufacturing practices regulation, which regulatory agencies may enforce.¹²

Surface Plasmon Resonance

Surface Plasmon Resonance (SPR) is another example of an advanced technology that has been successful after its emergence within the past ten years. SPR can measure real-time interactions with a high level of sensitivity without requiring labels.¹³

SPR can be used for screening pharmacologically active molecules, enabling further understanding of molecular mechanisms involved in disease development, as well as for novel drug development and design.¹³

Artificial Intelligence

Artificial intelligence (AI) is also a novel emerging tool that has created advanced opportunities in drug discovery and formulation, with the use of AI algorithms that analyze biological data for researchers who can use this to detect disease-associated targets and predict interactions with potential drugs. This process enables a more targeted approach to drug discovery and leads to an increased success rate for drug approval.¹⁴

Discover more about how AI is transforming spectroscopy

Applications in Biopharmaceutical Development

Monoclonal Antibodies and Biosimilars

Advanced analytical methods ensure the quality and comparability of monoclonal antibodies and biosimilars, with the key step to achieve a successful biosimilar approval being to establish analytical and clinical similarity with the innovator.¹⁵

These methods aim to detect differences between monoclonal antibodies and biosimilar, characterizing protein biopharmaceuticals to produce more reliable therapeutic products, including drug safety and efficacy.^4,8

Gene and Cell Therapies

Cutting-edge methodologies such as NGS are essential for characterizing complex genetic material and cells that are involved in gene therapies.¹¹

Additionally, advancements in cell-based therapeutics, such as CRISPR and CRIPSR-Cas9 proteins, can be used to improve cell therapies, including chimeric antigen receptor T cell (CAR-T) therapy.¹⁶

Vaccine Development

NGS also has a significant impact on transcriptomics, which includes the study of the transcriptome, or the complete set of RNA molecules within an organism; profiling these can lead to comprehensive insight into gene expression as well as a range of biological processes and diseases.¹¹

mRNA sequencing can enable researchers to detect and quantify gene expression levels between various conditions, tissues, and cell types, which would be significant for developing mRNA vaccines.^11,17

Regulatory Considerations

Regulatory agencies such as the Food and Drug Administration (FDA) are responding to the increased complexity of novel therapeutic drugs by encouraging the use of more innovative approaches to clinical trials while also insisting these treatments should show evidence of having meaningful patient outcomes; this can include using a drug comparator that is not a placebo.¹⁸

Additionally, the European Medicines Agency (EMA) has many programmes that aim to accelerate the development of drugs for research that address significant unmet medical needs, including (i) Accelerated Review, (ii) Conditional Approval, (iii) Adaptive Pathways, and (iv) PRIME.¹⁸

ICH Q6B specifications consist of test procedures and acceptance criteria used as a scientific guideline for biopharmaceutical companies for biotechnological or biological products.

This uniform set of international specifications supports new marketing applications. It covers comprehensive standards that companies would be required to undertake, including extensive characterization of products in the development phase and when needed after significant process changes.¹⁹

With comprehensive quality checks and characterization undertaken by innovative analytical techniques, this improves compliance with these standards, which aim to provide opportunities to gain further knowledge of the product during the design process.^5,20

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Conclusion

Innovations in analytical characterization are significant for both the biopharmaceutical industry as well as public health, ensuring the safety, efficacy, and quality of biopharmaceuticals.^5,8

These advances aid in overcoming the complexity of biologics and aim to provide more reliable and effective therapeutic products in the future.

By undertaking extensive characterization of biologics and proteins, researchers can better comprehend drug targets and interactions with potential candidates, which leads to enhanced success rates and drug approval.^5,6,14

References

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9. Shah M, Patel N, Tripathi N, Vyas VK. Capillary electrophoresis methods for impurity profiling of drugs: A review of the past decade. Journal of Pharmaceutical Analysis. 2022;12(1):15-28. doi:10.1016/j.jpha.2021.06.009.
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11. Satam H, Joshi K, Mangrolia U, et al. Next-generation sequencing technology: Current trends and advancements. Biology. 2023;12(7):997. doi:10.3390/biology12070997.
12. Millán-Martín S, Jakes C, Carillo S, Rogers R, Ren D, Bones J. Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing. Nature Protocols. 2022;18(4):1056-1089. doi:10.1038/s41596-022-00785-5.
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15. Nupur N, Joshi S, Gulliarme D, Rathore AS. Analytical similarity assessment of Biosimilars: Global Regulatory Landscape, recent studies and major advancements in orthogonal platforms. Frontiers in Bioengineering and Biotechnology. 2022;10. doi:10.3389/fbioe.2022.832059.
16. Bashor CJ, Hilton IB, Bandukwala H, Smith DM, Veiseh O. Engineering the next generation of cell-based therapeutics. Nature Reviews Drug Discovery. 2022;21(9):655-675. doi:10.1038/s41573-022-00476-6.
17. Al Fayez N, Nassar MS, Alshehri AA, et al. Recent advancement in mrna vaccine development and applications. Pharmaceutics. 2023;15(7):1972. doi:10.3390/pharmaceutics15071972.
18. Lee M, Ly H, Möller CC, Ringel MS. Innovation in regulatory science is meeting evolution of clinical evidence generation. Clinical Pharmacology & Therapeutics. 2019;105(4):886-898. doi:10.1002/cpt.1354.
19. ICH Q6B specifications: Test procedures and acceptance criteria for biotechnological/biological products - scientific guideline. European Medicines Agency (EMA). https://www.ema.europa.eu/en/ich-q6b-specifications-test-procedures-acceptance-criteria-biotechnological-biological-products-scientific-guideline. Accessed November 8, 2024.
20. Guidance for Industry. Guidance for Industry Q8(R2) Pharmaceutical Development. https://www.fda.gov/media/71535/download. Accessed November 8, 2024.