Since the 1982 approval of Eli Lilly’s recombinant human insulin, Humulin, biotherapeutic drug development has steadily grown into a global market valued at $140 billion in 2013, increased from $25 billion in 2001¹ (Table 1).

Biologics, or biotherapeutics, make up a class of drugs including proteins and peptides, which are generally produced in living organisms and used for the treatment of a wide variety of diseases including cancer, diabetes, and rheumatoid arthritis. These products also provide genuinely new strategies against infectious agents and orphan diseases alike. There are currently over 200 approved biotherapeutic drugs on the market² (in the US and EU) and hundreds more potential products in clinical trials or awaiting regulatory approval, a strong indication of their continuing value. The biologics pipeline is comprised of an extensive array of products with monoclonal antibodies (mAbs) representing the most broadly developed and profitable class (Figure 1).² Currently, mAb-based biotherapies account for 50% of the top 100 drugs3 and are projected to maintain a dominant position in the pharmaceutical market over the coming years.

The continuing growth of biopharmaceuticals can be attributed to their many advantages over small-molecule therapies including minimal safety/toxicity issues, well-understood mechanisms of action, and high target specificity. The challenge of developing a biotherapeutic, such as a biologic protein, stems from the structural complexity of these large molecules and their production processes. Because biologics are manufactured in living cellular systems, they often undergo modification as a result of growth/media conditions, the bioprocessing environment, or purification and/or formulation. Some of these modifications are primary sequence variants, while others are post-translational modifications (PTMs) to the protein sequences (Figure 2). This structural heterogeneity is dynamic and can influence the protein’s overall conformation with the potential to change its clinical efficacy. Advances in recombinant DNA technology, as well as in fermentation and purification processes have provided robustness in generating large amounts of protein-based therapies. Typically, biologics are produced in mammalian cell systems like Chinese hamster ovary (CHO) cells using carefully controlled growth and environmental conditions. However, cell culture can be unpredictable, and even minor adjustments to the manufacturing process, like changes in the culture media or even in the purification procedure, can alter a molecule’s structure or stability, and therefore its safety and efficacy. Consequently, implementation of chemical manufacturing controls (CMC) and analytical characterization processes at each stage of manufacturing is necessary to ensure product biocomparability.

As the biologics market continues to grow, there is a significant need for established analytical and bioanalytical methods that can help improve discovery and development. SCIEX offers technology including capillary electrophoresis (CE) and Mass spectrometry (MS) to help facilitate these improvements.

Capillary electrophoresis (CE) is a technique commonly used to assess purity and heterogeneity of biologics. In principle, CE separates molecules based on differences in their charge by generating an electric field across a capillary into which sample has been introduced. The voltage applied facilitates differential migration of analytes through a capillary and across a detection window where their absorbance or fluorescence can be measured. In practice, conventional gel electrophoresis techniques like SDS-PAGE and isoelectric focusing (IEF) have been replaced with capillary-based methods (CE-SDS and CIEF) using the PA 800 Plus platform because of its ability to generate highly reproducible results in an automated, quantitative fashion. In addition, oligosaccharide characterization can similarly be performed allowing for ultra-high resolution separation of glycoforms associated with protein biologics. Coupling high-resolution separations with validated methodology and powerful software analysis on the PA 800 Plus has been providing critical information across development and quality control processes in the biopharmaceutical sector. CESI technology couples the power of CE with the detection capability of electrospray ionization mass spectrometry (MS). Utilizing flow rates as low as 10 nl/min, CESI minimizes ion suppression, thereby enhancing overall sensitivity at the MS when performing key assays, including peptide mapping and characterization of intact biologics.

MS technology is being widely used to characterize proteins due to exceptional sensitivity, selectivity, and specificity, positioning itself as a superior method for profiling protein sequence, PTMs and other structural attributes of biotherapeutics.

Recent innovations in source electrospray ionization (ESI) has improved detection sensitivities.⁴ Expanded mass ranges can now accommodate very large proteins, and improved quantitation as a result of state-of-the-art detector design has enabled increasingly smaller detection limits approaching the femtomole range.¹ Additionally, powerful software analysis has streamlined the management of overwhelmingly large data sets generated in unbiased, comprehensive sample analysis. Together, these analytical advances have triggered an emerging dominance in the use of CE, and MS by the biopharmaceutical industry.

Download and read the Biologics Analytical Characterization Compendium to help you in navigating the growing array of options for comprehensive product characterization to in-depth analysis. Several SCIEX technologies and applications are highlighted in the compendium that emphasize efficiency, while still providing the precision and accuracy necessary for rapid decision-making regarding a product’s quality. Each section identifies solutions that accelerate biotherapeutic characterization through the use of automated sample preparation and analysis workflows and optimized separation methods. These sections focus on analytical areas that are critical for product development, and are segmented in a similar manner as the basic Biologics Workflow:

• Biologics Research
• Development and Quality Control
• Bioprocessing and Biosimilar Comparability

In the Biologics Research section, applications in four areas— molecular weight determination, peptide mapping, disulfide bond assignment, and antibody-drug conjugate analysis—explore next-generation analytical technologies that create a deeper view of protein structure. The key to accelerating these processes lies in automating data processing and decreasing data complexity. To streamline the evaluation of large data sets, BioPharmaView™ Software automatically computes protein structural features, such as intact molecular weight, antibody-drug ratios, glycoforms, and terminal modifications, generating a global overview of any lot-to-lot changes. The software’s smart-filtering capacity efficiently deciphers peptide-mapping data, shrinking analysis time to just a few hours. By enhancing selectivity, the orthogonal chromatography techniques of differential mobility spectroscopy (DMS) and capillary electrophoresis electrospray ionization (CESI) can reduce spectral complexity resulting from background interferences, simplifying the resulting data and its processing—to give an all-inclusive structural overview.

For more information on:

• Development and Quality Control
• Bioprocessing Biosimilar Comparability
• Biosimilar Comparability

Prepared by Laura Baker, Ph.D., Science and Technical Writer 

References:

1. Chen, G., Warrack, B.M., Goodenough, A.K., Wei, H., Wang-Iverson, D.B., and Tymiak, A.A. “Characterization of protein therapeutics by mass spectrometery: recent developments and future directions.” Drug Discovery Today. January 2011; 16(1/2): 58-64. 
2. Walsh, G. “Biopharmaceutical benchmarks 2014.” Nature Biotechnology. October 2014; 32(10): 992-1000. 
3. Van Arnum, P. “Moving to the Next Level in Biomanufacturing.” Pharmaceutical Technology. April 02, 2010; 34(4). Retrieved at: http://www.pharmtech.com/moving-next-levelbiomanufacturing. 
4. Cotte-Rodriguez, I., et al. “Introduction to Protein Mass Spectrometry.” In Guodong Chen (Ed.) Characterization of Protein Therapeutics using Mass Spectrometry. 2013; Springer, New York.