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Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Time and sensitivity are essential for analytical technologies in all phases of biopharma development.
Although just a few decades old, the biopharmaceutical industry has evolved significantly since its inception. Many candidate biologics today-antibodies and antibody fragments, highly potent antibody-drug conjugates (ADCs), virus-like particles, cell- and gene-based therapies, etc.-are different from the first simple, recombinant proteins. Manufacturers have been continuously challenged to develop analytical methods for timely and accurate determination of the chemical, physical, and therapeutic properties of these different actives, as well as potential contaminants throughout the production process, from raw material selection to process analysis, formulation development, and release testing.
The introduction of biosimilars and the move toward continuous processing are creating the need for more rapid and sensitive analytical techniques. The advent of quality by design (QbD) has further increased the importance of analytical methods/technologies within a manufacturing environment, according to Fiona Greer, global director of biopharma services development at SGS Life Science Services.
Newer versions of traditional methodologies, such as capillary isoelectric focusing (cIEF) versus IEF gels, peptide mapping with liquid chromatography-tandem mass spectrometry (LC/MS/MS), and high-performance LC (HPLC) are available today. Notably, mass spectrometry-based methods and next-generation sequencing technologies are addressing the need for greater sensitivity in less time. Automation and high-throughput technologies are also having an impact. As the industry introduces more complex and increasingly potent molecular formats with novel, highly potent product-related impurities, however, ongoing advances will be required.
Many sensitive mass spec methods
For product characterization, the most appropriate techniques will depend on the class of molecule: protein, glycoprotein, pegylated, ADC, vaccine, etc. “Improvements in biopharmaceutical mass spectrometry in the past 10 years-in sensitivity, dynamic range, resolution, mass accuracy, and user-friendliness-have dramatically improved our ability to get detailed protein molecular information,” says Byron Kneller, director of analytical/formulation development with CMC Biologics.
The continued development and deployment of LC/MS-based applications are having a significant impact on both the characterization and quality testing of biopharmaceuticals, particularly for recombinant proteins and monoclonal antibodies, agrees Mike Garrett, senior director of global marketing for BioReliance. “While in the past these methods were reserved for early research into the structure of these molecules, today, methods are being developed that bring this technology closer to the quality control lab,” he observes. Access to more sensitive and detailed characterization data is allowing manufacturers to better understand and more carefully control the molecular structures of their products during the manufacturing process. Garrett also notes that LC/MS has enabled finer control of bioprocess optimization, allowing for correlation of process changes to both molecular structure and yield.
Some of the most recent advances in product characterization techniques have, according to Greer, been developed in response to challenges encountered with biotherapeutic products and their post-translational modifications (PTMs). Glycosylation analysis in particular has been advanced significantly with the advent of high-resolution mass spectrometry and the use of hydrophilic interaction liquid chromatography (HILIC) columns for glycans. “Introduction of the quadrupole orthogonal acceleration time-of-flight (Q-ToF) geometry and the increased resolving power of MS now allow the direct determination of the monoisotopic mass of antibody heavy chains including modifications such as deamidation,” Greer explains. Kneller adds that current-generation Q-ToF and Orbitrap instruments allow for high-resolution intact mass and peptide mapping measurements for both characterization and process-development support, and current software continues to make data processing easier and faster.
State-of-the-art MS instruments with markedly increased sensitivity are also providing profound insights into the impurity profiles of biotherapeutics and allowing the identification of previously unknown host-cell contaminants, according to Harald Wegele, head of analytical development and quality control in Europe for Roche. “Sensitive assessment of specific host-cell proteins (HCPs) and other contaminants provides crucial guidance for the development of impurity depleting process steps, which ultimately helps to warrant a maximum of product safety,” he states. Additional developments such as sequential window acquisition of all theoretical mass spectra (SWATH) and parallel reaction monitoring are improving the quantitative assessment of process-related impurities.
Greer also expects wider adoption of numerous other MS-based analytical techniques, including ion mobility-MS, capillary electrophoresis-MS (CE-MS), and hydrogen-deuterium exchange-MS (HDX-MS). Wegele adds that size-exclusion chromatography (SEC) coupled to native MS already provides-particularly for novel antibody formats like bispecifics-a fast and easy means for gaining unachieved levels of information on, for example, the size-variant distribution of biotherapeutic, as early as at the onset of clinical development. He also points to 2D-HPLC as providing a convenient and accurate method for characterizing single product peaks, side products, and excipients.
More rapid analyses
There is tremendous pressure on biopharmaceutical companies to get products to the market more quickly and at lower cost without compromising safety. Manufacturers are consequently looking for alternatives to conventional cell-based analytical methods. Newer personalized treatments such as cell-based therapies, in fact, require more rapid release testing because they do not have long-term stability and must be administered to patients soon after they are produced. Manufacturers are also moving to continuous processing, which requires process analytical technology (PAT) that provides real-time process monitoring data.
Several newer testing methods have been developed and are in the process of being implemented by the biopharmaceutical industry, largely in cooperation with regulatory agencies such as FDA. Improvements in real-time, quantitative polymerase chain reaction (qPCR)-based methods have allowed for broader detection of known potential contaminants with improved speed and accuracy, according to Garrett. Newer nucleic acid detection technologies, such as next-generation sequencing, are also being applied to the quality control testing lab. “Importantly, these technologies will allow manufacturers to test their biopharmaceutical products for both known and unknown adventitious contaminants,” Garrett says.
Advances in bioassays have also made potency testing easier, faster, and more reproducible, according to Kneller. “The broader availability of reporter-gene assays (e.g., for antibody-dependent cell-mediated cytotoxicity [ADCC]) testing has decreased the difficulty of implementing some potency assays, while access to soluble enzyme-linked immunosorbent assay (ELISA) formats and ready-to-use analytical cell banks has decreased both the time needed for potency assays and assay variability,” he explains. Wegele adds that novel LC-, cell-, and surface plasmon resonance (SPR)-based assay formats are facilitating the assessment of the impact of PTMs on antibody/bispecific antibody Fc (crystallizable fragment) effector functionality, including pharamacokinetic (PK) properties (e.g., via FcRn [neonatal Fc receptor] affinity chromatography). The Fc region of a therapeutic antibody interacts with receptors on various types of cells and is involved in immune-mediated effector functions, such as ADCC and complement-dependent cytotoxicity (CDC). It is therefore potentially important in determining drug safety and efficacy and must be fully characterized.
Advances in chromatography methods are also enabling more rapid analyses, according to Kneller. “The increased use of ultra high-pressure liquid chromatography (UHPLC) systems and sub-2 µm columns has enabled more rapid, higher-resolution chromatographic assays, which has decreased testing time for many release methods,” he comments.
Implementation of high-throughput (HTP) methods and expanding use of automation are additional avenues the biopharmaceutical industry is pursuing to achieve more rapid testing. The challenge has been to reduce testing times without loss of accuracy, precision, specificity, sensitivity, and robustness. Several successes have been achieved to date, however.
Microfluidic capillary electrophoresis (MCE) has, according to Wegele, become a central pillar for product quality analytics during clone selection and bioprocess development due to its ease of sample preparation, robustness, and unrivaled high-throughput capability. “This HTP method is indispensable for meeting the steadily growing demand for the shortest possible sample turnover time and enhanced time efficiency in present-day biologics development,” he says.
Automated high-throughput quantification of process-related impurities (e.g., HCPs and Protein A), titer, and fermentation broth supplements such as insulin, LongR3, etc., via electrochemiluminescence immunoassay (ECLIA) is also now used at Roche to support process development, process characterization/process validation studies, manufacturing, and in-process control/release testing, according to Wegele. “This technology is high-throughput-compatible and greatly reduces hands-on time. As a result, it enables novel insights for bioprocess development in near real-time and facilitates the assessment of process-related impurities depletion,” he says.
Higher-throughput screens for formulation development coupled with the use of design-of-experiment (DoE) tools have also enabled faster, more comprehensive screening of many formulation conditions and excipients and decreased the time required for formulation optimization, according to Kneller. Often, combinations of light-scattering, intrinsic and extrinsic fluorescence, and calorimetry are used to rapidly deliver information on protein stability in many excipient combinations.
Methods for emerging biologics
Recent years have seen growing interest in newer types of biologic actives. Significant numbers of antibody-based treatments have been commercialized, and many more, including those based on antibody fragments and ADCs, are in advanced stages of development. Successful initial studies with cell- and gene-based therapies are attracting interest in these therapies, many of which are now in clinical trials. While many of the analyses required to characterize these different classes of biologic drug substances are the same, their characterization does in many cases require different analytical techniques.
For newer antibody formats, both Garrett and Wegele note that LC/MS is a relatively fast method for gaining high levels of information on the size-variant distribution of biotherapeutics at early development stages. CE is also providing deeper insights into the structure of these molecules, according to Garrett. “Use of these techniques has led to numerous improvements in the manufacturing of antibodies and antibody fragments, particularly when considering the variables that can now be investigated and controlled as part of the manufacturing development process,” he asserts.
For cell-based therapies, Garrett notes that the development and adoption of rapid, molecular-based testing methods for both process and product safety will enable cell therapy products to be manufactured in the timeframes necessary to both manipulate patient-derived cells and then deliver them safely. The development of methods for assessing the safety of the viral backbones used to produce gene therapies has also kept pace with their advancement into the clinic. “Virology-based tests have been refined such that they now provide information on the specific properties and quality of vector backbones, which is crucial for ensuring the safety of these advanced therapeutics,” Garrett states. He also notes that molecular methods such as next-generation sequencing are being employed to investigate the identity, purity, and stability of virus-based gene therapies.
Full analytical characterization of branded biotherapeutics and potential biosimilar products is fundamental to the development of biosimilars, and the pathway for analytical method development for biosimilars is somewhat different from that of novel biotherapeutics, according to Jun Lu, director of analytical development for Catalent Pharma Solutions. “Both release and characterization methods are required at the very early stage of biosimilar development, because the reference product from multiple lots must be extensively characterized to establish the target product profile,” he says. More specifically, analytics are essential to defining the critical quality attributes (CQAs) that form the quality target product profile (QTPP).
Demonstration of similarities between the biosimilar and reference product through side-by-side comparison (i.e., physical, biological, and chemical characterization) is required before progressing into the clinic, according to Greer. Matching of the amino acid sequence and PTMs of the reference product determined by using LC/MS/MS and other protein characterization methods must be performed as a clone selection criterion, because upstream and downstream development has minimal impacts on changing these CQAs, adds Lu.
Use of orthogonal methods for biosimilar assessment is also emphasized by regulators, because subtle differences between a biosimilar and the reference product may be difficult to detect using only one analytical method. FDA in particular has introduced the concept of “fingerprint-like” analyses, according to Greer. “This approach entails the use of a carefully selected portfolio of characterization techniques for primary and higher-order structure, together with biological and potency assays producing data that, when combined, add up to more than the sum of the parts,” she says.
For instance, Lu notes that for analysis of high-molecular-weight (HMW) species, which present a high-risk safety concern, supplementing SEC with analytical ultracentrifugation (AUC) is strongly recommended. For the determination of higher-order structure, a combination of at least two techniques from a list including circular dichroism (CD), Fourier-transform infrared (FTIR), differential scanning calorimetry (DSC), and HDX-MS can be used to potentially elucidate any detailed structure differences. Garrett adds that advanced cell-based potency assays are important for determining whether the in-vitro effects of biosimilars are similar to the originator molecule. Greer observes, however, that the link between higher order structure and biological activity remains to be explored. She does note, though, that several techniques are emerging from research backgrounds to address these questions, such as HDX-MS and 2D-nuclear magnetic resonance (NMR) imaging.
Statistical analysis of analytical data for the determination of biosimilarity is also required by FDA to ensure confidence in the data. One consequence, according to Lu, has been the replacement of imaging methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IEF gels with CE-SDS and cIEF, respectively, which allows greater analysis of the data output.
More work to do
Despite the numerous advances in MS, CE, next-generation sequencing, and other rapid assays, further developments are still needed. Adoption of many new analytical technologies takes time given the need for extensive confirmation and validation of performance. Many of these newer methods are gaining acceptance further down the development pathway and closer to quality control, but are not yet widely used. Regulators are, however, starting to explore the potential advantages these technologies can provide, according to Garrett.
One specific issue for Kneller is HCP quantitation, which for early clinical work is typically performed using commercially-available ELISA kits, but then requires transition to costly custom assays later in development. “This transition can be difficult if kits do not provide adequate coverage of all HCPs potentially present in the product. Orthogonal approaches to HCP quantitation (e.g., mass spectrometry) are not yet feasible or widely-adopted, however,” he notes. Wegele points to the need for tools that enable the assessment of the criticality (e.g., safety, immunogenicity, PK, potency) of various product-related impurities/CQAs (e.g., HMW species, dimers, fragments, PTMs, charge variants, etc.) to identify control strategies that make sense and do not lead to excess testing burdens.
Reed Harris, senior staff scientist in Pharma Technical Development at Genentech, would like to see more effective methods for identifying the causes of excipient degradation, which may be due to trace-level impurities that are below current detection capabilities. He also points to the need for better resolution of higher-molecular-weight species using SEC. SEC aggregate resolution is needed because there is growing evidence that antibody aggregates are not as immunogenic as originally believed, and further work is necessary to establish the true patient risks for different aggregate types. “Current SEC columns resolve monomers from dimers, but do not resolve different dimer types or multimers such as trimers, tetramers, etc., very effectively,” he says. Furthermore, he notes that while CE-SDS is an advance over SDS-PAGE, further improvements are needed. The presence of SDS makes it difficult to analyze CE-SDS peaks with mass spectrometry, and therefore, most peak assignments are performed by spiking forms prepared using other methods into samples, which is time consuming.
Particle analysis is another issue for Wegele. He notes that currently available methods are mostly insufficient for precise and robust assessment of subvisible particles, particularly translucent proteinaceous particles and particles with diameters less than approximately 2 μm. Roche has developed a modified light-obscuration sensor that monitors the signal width rather than length, leading to improved detection of very small subvisible particles, reduction of artifacts during the analysis of low concentrations of translucent protein particles, and higher counting accuracy compared to flow imaging microscopy and standard light obscuration measurements.
Other ongoing needs, according to Wegele, include replacement of cell-based potency assays with novel, cell-free assay formats in the quality control environment; methods for the evaluation of the impact of combined administration of biotherapeutics; and more automated testing solutions to cope with the steadily increasing sample load of ever more complex biologics and next-generation biologics, which are often highly potent therapeutics with novel, highly potent product-related impurities. “It is important to address novel and critical product-related side products (e.g., immune-cell-activating side products acting at the crossroads of immunology and oncology) to ensure maximum patient safety and guarantee efficacy,” he asserts.
Finally, Harris notes that the industry is struggling to balance the needs for comprehensive testing, including testing to account for unexpected events, and more rapid product development. “Risk-based (i.e., QbD) test strategies will lead to a reduced set of tests, but it is also necessary to include tests that detect variation outside of process models. The two approaches present a fundamental conflict,” he states.
Indeed, biopharmaceutical manufacturers remain challenged to increase the speed and accuracy of product development while still ensuring safety in the face of more rigorous regulatory scrutiny, novel biologic molecules, and evolving manufacturing strategies. “All of these factors are adding complexity to analytical testing programs,” Garrett concludes.
Vol. 29, No. 2
When referring to this article, please cite it as C. Challener, “Fine Tuning the Focus on Biopharma Analytical Studies,” BioPharm International 29 (2) 2016.
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