The Race for Better Biotherapeutics: Why a Comprehensive Analysis is Key
Medicinal products derived from a biological source are commonly referred to as biotherapeutics or biologicals. They are typically proteins or polypeptides of >40 amino acids whose active components are constructed using biotechnology. A range of structural modifications are made to optimize the desirable functional characteristics of the target proteins and fine-tune their activity to meet a specific therapeutic need. Similarly, modifications may also be needed to be reduce side effects, not least immunogenicity, and improve the safety profile of the treatment.
This broad drug class includes a wide variety of therapeutic agents, including recombinant proteins and hormones, monoclonal antibodies (mAbs), cytokines, growth factors, hormones, vaccines, cell-based products, gene-modifying therapies, tissue-engineered products, and stem cell therapies. Monoclonal antibodies with their high specificity are a particularly valuable tool for providing targeted treatments. Of the 46 drugs approved by the FDA in 2017, 22 were biotherapeutics, of which 10 were mAbs. This reflects their application across a wide spectrum of indications, such as oncology, anti-immunity, and chronic inflammatory diseases. They may serve to deliver cytotoxic compounds to tumor cells, block binding sites to prevent ligand activation of targeted molecular pathways, or act as an agonist to promote specific molecular functions.
The potential for manipulating a diverse range of systems and disease processes, has led to a rapid expansion in the number of therapeutic recombinant proteins. Amongst the highest revenue drugs, 10% were biopharmaceuticals in 2004 and this had risen to 70% in 2012. However, the therapeutic benefits come at a cost. The manufacture of biotherapeutics is significantly more complex than that of small-molecule drugs, typically involving more than 5,000 critical process steps. There is consequently considerable opportunity for deviation, such as inaccurate or failed modification or conjugation, and contamination, such as virus used for transfection or unconjugated components. A host of complex purification and quality control processes are thus required to ensure that the final drug product is of the intended structure and free from impurities. Furthermore, these must be conducted without interfering with the intricate secondary and tertiary structures of the therapeutic proteins that are critical for achieving the desired efficacy.
Indeed, such capabilities must be demonstrated for a biotherapeutic to be granted marketing approval. Before a biotherapeutic can be licensed, the product, the manufacturing process, and the manufacturing facilities must all be shown to ensure the continued safety, purity and potency of the product.
This article will give an overview of the field of biotherapeutics, and the ways in which biotherapeutic proteins can be characterized to ensure their safety in vivo.
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Chapter 1 – The Importance of Quality Control in Biotherapeutics
In the development of biotherapeutics, a key concern is immunogenicity. It is highly undesirable for the introduced therapeutic protein to elicit a response from the patient’s immune system. The production of anti-drug antibodies can lead to a range of unwanted effects, such as reduced efficacy, destruction of the endogenous counterpart, alterations in drug pharmacokinetics, and hypersensitivity reactions.
Although tested in animals prior to clinical use, the level of immunogenicity observed in animal models is not predictive of immunogenicity in humans. Regular antibody assessment, through binding assays, confirmatory assays and neutralizing assays, is thus required to ensure the safety and efficacy of biotherapeutics. The introduced therapeutic protein could potentially activate both cellular and humoral (antibody) immunogenic responses, but immunogenicity is usually assessed through the measurement of anti-drug antibody levels. These may include IgM, IgG, IgE, and/or IgA immunoglobulins.
The level of immunogenicity of therapeutic proteins is to a large extent dependent on the production process. Structural variability arises due to differences in post-translational modifications in different cell lines. The favorable properties of CHO cells have made them the preferred mammalian cell line for the production of recombinant protein therapeutics for many years. However, with the ever-increasing demand for biotherapeutics, there is an ongoing search for new production cell lines with high yield. This involves identifying cell clones that are stable and grow well in suspension culture, are of low risk, eg, few human viruses are able to propagate in them, and are amenable to bioengineering methodologies. The selected clones are then evaluated in controlled bioreactors and stored for future use.
Due to the complex production process, protein products are often a mixture of product, product variants that can arise during protein synthesis, and degradation products. In addition, the target protein may differ in terms of the position of post-translational glycosylation, molecular structure, size, charge, degree of oxidation, and even sequence variation. Consequently, there are numerous opportunities for structural heterogeneity in recombinant proteins. Such differences can affect product safety and efficacy. For example, glycation in the complementary-determining region of mAb1 was associated with the loss of antigen binding. The detection and characterization of the structural, biophysical, and molecular information of a biotherapeutic as well as any anti-drug antibodies elicited in vivo are thus essential.
Intact liquid chromatography-mass spectrometry (LC-MS) techniques are valuable tools for assessing structural modifications, such as glycation, of proteins. In his talk at Pittcon 2019 entitled “Mass Spectrometry Imaging of Bacterially Infected Human Skin Tissue on Silicon Nanopost Arrays”, Jarod Fincher of George Washington University highlights the ability of mass spectrometry imaging (MSI) to not only detect a wide range of biomolecules but also simultaneously map out their spatial distributions. MSI analysis was applied in conjunction with silicon nanopost arrays (NAPA) and a matrix-free laser desorption ionization (LDI) platform. MSI plus the ability of NAPA-LDI to efficiently ionize certain interesting species, such as glycosylated ceramides, provided detailed chemical and sub-mm spatial information.
A range of companies providing mass spectrometry technologies will be onsite at Pittcon 2019 to discuss their capabilities. Advion will highlight the benefits of their compact mass spectrometry plus UHPLC gradient system for the analysis and identification of peptides and proteins. Extrel will be presenting their quadrupole mass spectrometers that provide more detailed identification and separation of peptides. Waters Corporation will also be on-hand to explain the combinations of advanced mass spectrometry imaging technologies they offer, including matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI).
Detailed characterization of biopharmaceuticals and the identification of impurities, including by-products of their manufacture, in quality control processes often utilizes ultra-high-performance liquid chromatography (UHPLC). This technique offers greatly enhanced resolution and separation and faster analyses compared with HPLC. In UHPLC, the sample is passed through a column packed with sub-2µm particles under pressures of up to 15000 psi. UHPLC has tremendous separation power, although complicated methological process development may be needed for more complex proteins, such as those with chiral centers. Techniques for tailoring UHPLC methodologies for the analysis of biologicals will be detailed by Michael Dong in his talk at Pittcon 2019 entitled “UHPLC in Method Development of Stability-Indicating Methods”.
Prior to the development of UHPLC, reversed-phase chromatography of proteins was plagued with problems of carryover, multiple peak formation, and peak diffusion. Broad peaks reduced resolution necessitating longer gradients, which increased run times and decreased throughput. UHPLC helped overcome these difficulties by speeding up intra-pore diffusion and the development of novel materials for the stationary phase reduced secondary interactions. At Pittcon 2019, Hitachi will be presenting their LaChromUltra II column, which is the longest UHPLC column available producing exceptional high-separation performance with a carryover of only 0.001%. SilcoTek will also be at Pittcon 2019, where you can learn more about using their bioinert coatings to reduce interactions between the stationary phase and proteins and ensure consistent, accurate UHPLC test results.
The importance of UHPLC is highlighted by the fact that it will be the focus of the 30th James L Waters Symposium at Pittcon 2019.
UHPLC can be adapted, eg, reversed‐phase chromatography, ion exchange chromatography, and coupled with other technologies, such as mass spectrometry, to tailor it to the particular separation and identification required. For example, hydrophilic interaction chromatography (HILIC) is particularly useful for glycan profiling and light-scattering detection combined with size-exclusion UHPLC is very effective for the characterization of proteins and antibodies. Representatives of Wyatt Technology will be present at Pittcon 2019 to show how light-scattering can be a useful tool for characterizing proteins and antibodies. Also at Pittcon 2019, Bruker will available to show how their Elute™ HPLC systems, which minimize gradient delay volume to enable faster run times, can be used in a variety of configurations to suit a range of analytical requirements.
UHPLC in its various guises thus plays an important role in high‐resolution and high‐throughput characterization of protein therapeutics. This will be explored in more detail at Pittcon 2019 by Jennifer Rea of Genentech in her presentation “Characterization and Quality Control of Recombinant Protein Therapeutics Using UHPLC”.
The higher pressure limits of UHPLC enabling faster analysis has made it feasible for host cell metabolites and glycosylation levels to be monitored during production. In this way, correlations between process parameters and product quality attributes can be identified to help advance process understanding, improve product quality, and increase production efficiency. These concepts will be presented in more detail at Pittcon 2019 by Michael Dong in his presentation “Implementing Ultra-High-Pressure LC (UHPLC) in Pharmaceutical Analysis”.
Michael Dong has also been researching methodologies for the analytical characterization of mAbs. He has shown that a peak capacity of 700 can be achieved with UHPLC.
- Advion website 2019. Available at https://advion.com/applications/compact-mass-spectrometry-applications/proteins-and-peptides-analysis/
- Bruker website 2019. Available at https://www.bruker.com/products/mass-spectrometry-and-separations/lc-ms/liquid-chromatography/elute-lc-series/overview.html
- CEM website 2019. Available at http://cem.com/uk/exploration-of-phospholipids-with-the-oracle
Ultra-high-performance liquid chromatography UHPLC is the standard LC platform
- Doneanu CE, et al. Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry. MAbs. 2012;4(1):24-44. https://www.ncbi.nlm.nih.gov/pubmed/22327428
- Extrel website 2019. Available at http://www.extrel.com/Module/Catalog/ProductsCategory/default/Quadrupole_Mass_Spectrometers_and_Residual_Gas_Analyzers/MS_MS_Systems?id=33
- Fekete A, et al. Current and future trends in UHPLC. TrAC Trends in Analytical Chemistry 2014;63:2 13
- Fekete S, et al. High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography column coupling. Journal of Pharmaceutical and Biomedical Analysis 2013;83:273 278. https://www.sciencedirect.com/science/article/pii/S0731708513002239
- Fincher JA, et al. Matrix‐free mass spectrometry imaging of mouse brain tissue sections on silicon nanopost arrays. JCN 2018. https://doi.org/10.1002/cne.24566
- Hitachi website 2019. Available at https://www.hitachi-hightech.com/global/product_detail/?pn=ana-chromasterultra
- Krishna M and Nadler SG. immunogenicity to Biotherapeutics –The Role of Anti-drug immune Complexes. Frontiers in Immunology 2016; 7:2. doi: 10.3389/fimmu.2016.00021
- Lai T, et al. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production. Pharmaceuticals (Basel). 2013;6(5):579–603. . doi: 10.3390/ph6050579
- Mo J, et al. Quantitative analysis of glycation and its impact on antigen binding. MAbs 2018;10:404 415. https://doi.org/10.1080/19420862.2018.1438796
- Rathmore A, et al. The Use of Light-Scattering Detection with SEC and HPLC for Protein and Antibody Studies, Part I: Background, Theory, and Potential Uses. LCGC North America 2012;30(9):842–849
- Rea J, et al. UHPLC for Characterization of Protein Therapeutics. In Ultra‐High Performance Liquid Chromatography and its Applications. Ed Xu QA. https://doi.org/10.1002/9781118533956.ch8
- Rowe L and Burkhart G. Analyzing protein glycosylation using UHPLC: a review, Bioanalysis 2018;20:1691-1703
- SilcoTek website 2019. Available at https://www.silcotek.com/blog/tips-for-improved-bioinert-hplc-surfaces
- U.S. Food and Drug Administration. The immunogenicity of therapeutic proteins- what you don’t know can hurt YOU and the patient. SBIA REdI Spring 2014. Available at https://www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/SmallBusinessAssistance/UCM408709.pdf
- U.S. Food and Drug Administration. Guidance for Industry Assay Development for Immunogenicity Testing of Therapeutic Proteins. 2009. Available at https://www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/SmallBusinessAssistance/UCM408709.pdf
- Waters Corporation website 2019. Available at http://www.waters.com/waters/en_GB/Mass-Spectrometry-Imaging-Technologies-including-MALDI%2C-DESI-and-Ion-Mobility/nav.htm?cid=134833481&locale=en_GB
Chapter 2 – Chromatographic and Mass Spectrometric Approaches for the Characterization of Biotherapeutic Proteins
The massive growth in the marketing of biotherapeutic mAbs discussed in Chapter 1 has necessitated the development of techniques to facilitate full characterization of these complex proteins. Liquid chromatography has become the most commonly used technique in the quality control of biotherapeutic proteins. It can be used in combination with a variety of detection technologies to allow absolute quantification and qualitative characterization of protein biotherapeutics. An analytical capability commonly used to evaluate elutes of liquid chromatography is mass spectrometry. Indeed, liquid chromatography and mass spectrometry (LC/MS) is a powerful analytical technique for ensuring the purity of biotherapeutic proteins.
Historically, ligand-binding assay was the only platform available for protein bioanalysis. As new LC/MS technologies became available, these were used to augment ligand-binding assays, providing a truly orthogonal detection principle that is less prone to interferences from other proteins present in the sample. This is particularly important for the clinical evaluation of mAbs when there is the possibility of anti-drug antibodies being present.
Further benefits of LC/MS, such as high reproducibility and quicker analyses, have resulted in it being increasingly used in the pharmaceutical industry for the evaluation of biotherapeutics. Indeed, LC/MS is now regarded as the gold standard for purifying and characterizing biotherapeutics. It is used across the development process from discovery to manufacture. There are strict regulatory requirements to ensure the safety and effectiveness of biotherapeutics, and so bioanalytical assays must be validated to meet strict global regulatory requirements.
At Pittcon 2019, Hu Ping of Janssen R&D, will be giving a presentation entitled “High-Throughput LC-MS Analysis for Cell Culture Metabolites”. A high-throughput LC-MS approach using Waters Xevo-G2-XS-QTof™ quadrupole time-of-flight mass spectrometer will be described that that is capable of simultaneously monitoring 93 CHO cell culture metabolites, including amino acids, nucleic acids, vitamins, and sugars, within a 17-minute cycle.
There are a range of liquid chromatography separation methods available for the characterization of biotherapeutics, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), and hydrophilic interaction liquid chromatography (HILIC). The method most applicable to the separation required must be selected according to the type of protein moieties to be characterized. These may be peptides, protein aggregates, isoforms, or polypetides with varying post-translational modifications, eg, glycosylation. Detection of the various elutes is usually performed using UV, fluorescence or light scattering and additional structural information is acquired using mass spectrometry. Such a multidimensional analytical approach serves to increase the potential sensitivity. In particular, it enables the characterization of more complex proteins, which would be difficult to achieve with a single technology. In addition, a multimodal approach in which several different types of chromatography, eg, SEC, IEC, are used in series may be adopted to improve the separation of biotherapeutics and achieve a higher level of purity. In fact, it is common for the a biotherapeutic purification process to include three to five different chromatography steps.
Atis Chakrabarti of Tosoh Bioscience LLC will be giving two presentations on the analysis of antibodies at Pittcon 2019. The first,” Fast and Robust Separation of Immunoglobulin G (IgGs) from Various Species and Subtypes Using an Analytical Recombinant Protein A Affinity Column”, will show that the separation of different types of antibody is readily possible with chromatography techniques. The second will explore the use of different chromatographic modalities in the characterization of mAbs, “Different Modes of Analytical Chromatography Techniques for the Characterization of Biomolecules”.
Chromatographical approaches can also be preceded by the fragmentation of mAbs to provide a full analysis. Antibody fragmentation is achieved by exposing them to reducing agents and proteases that digest or cleave certain portions of the immunoglobulin protein structure. The use of such fragments can help with studies of the function of particular areas of an immunoglobulin without interference from the rest of the molecule. This is important in the construction of novel mAbs as antibody fragments are preferred due to their lower propensity for immunogenicity.
The antibody fragments of primary interest are the antigen-binding fragments, such as Fab, and the class-defining fragments, such as Fc, that do not bind antigen. Fab fragments and Fc fragments can be obtained using proteases that cleave the protein hinge. The protease used depends on the fragment that is required. For example, papain is primarily used to generate Fab fragments. Backbone cleavage can also be catalyzed by non-enzymatic means using metals or radicals. Specific cleavage patterns can be obtained by adjusting the pH. Fragmentation rates are at a minimum in the pH range 5–6.
A many companies producing a range of specialist chromatographic systems will be onsite at Pittcon 2019 to discuss their capabilities and help you choose the right technologies to meet your antibody characterization needs. Amongst those present will be Merck, ThermoFisher, and Tosoh Bioscience LLC who will be able to provide more information about their comprehensive chromatography portfolios, which include immobilized proteases for antibody fragmentation, the EcoSEC™ separation systems and the High-Performance Liquid Chromatograph Chromaster™. In addition, Bruker will be available to discuss their range of mass spectrometry systems that can be integrated with a variety of third-party chromatographic instrumentation to facilitate powerful LC/MS analyses.
- Bruker. LC-MS. https://www.bruker.com/products/mass-spectrometry-and-separations/lc-ms.html
- Chakrabarti, Atis. Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography. 201810.5772/intechopen.73321 https://www.researchgate.net/publication/323346305_Separation_of_Monoclonal_Antibodies_by_Analytical_Size_Exclusion_Chromatography
- Dong J, et al. A High-Throughput, Automated Protein A Purification Platform with Multi-Attribute LC-MS Analysis for Advanced Cell Culture Process Monitoring. Analytical Chemistry 2016;88(17). https://www.researchgate.net/publication/305826242_A_High-Throughput_Automated_Protein_A_Purification_Platform_with_Multi-Attribute_LC-MS_Analysis_for_Advanced_Cell_Culture_Process_Monitoring
- Hitachi. High-Performance Liquid Chromatograph Chromaster.https://www.hitachi-hightech.com/global/product_detail/?pn=ana-chromaster
- Jenkins R, et al. Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics. AAPS J. 2015;17(1):1–16. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287296/
- Merck. Chromatography solutions for biopharma http://www.merckmillipore.com/GB/en/products/biopharmaceutical-manufacturing/downstream-processing/chromatography/oPGb.qB.fJIAAAFAZ8hkiQpx,nav
- Qu M, et al. Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry. Mass Spect Rev 2017;36(6):734 754. https://onlinelibrary.wiley.com/doi/abs/10.1002/mas.21500
- Thermo Fisher. Antibody fragmentation. https://www.thermofisher.com/uk/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/antibody-fragmentation.html#ab-frag-adv
- Tosoh Bioscience. Characterizing Biotherapeutics with HPLC Smart Solutions for Large Molecules. https://www.separations.eu.tosohbioscience.com/OpenPDF.aspx?path=/File%20Library/TBG/Products%20Download//Application%20Note/a15l49a.pdf
- Vivek H, et al. Multimodal Chromatography for Purification of Biotherapeutics – A Review. Ingenta Connect. https://www.ingentaconnect.com/contentone/ben/cpps/2019/00000020/00000001/art00005
Vlasak J and Ionescu R. Fragmentation of monoclonal antibodies. MAbs 2011;3(3):253–263. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149706/
- Waters. Quadrupole-Time-of-Flight-Mass-Spectrometer. http://www.waters.com/waters/en_GB/Xevo-G2-XS-QTof-Quadrupole-Time-of-Flight-Mass-Spectrometer/nav.htm?cid=134798222&locale=en_GB
Chapter 3 – Validating New Techniques
Little more than a decade ago ligand-binding assay (LBA) was the sole technique used to measure protein concentrations and assess immunogenicity in biological samples. Although a series of developments led to the creation of new variations of LBA to reduce variability and obviate the need for analyses to be conducted in triplicate, there was essentially just a single technique available. Having since had an unprecedented run of technological advances, there are now myriad techniques providing accurate bioanalysis of biopharmaceuticals. Furthermore, new techniques are being devised all the time.
With the number of biopharmaceutical products rapidly increasing, novel analytical strategies are needed to balance quality, speed and cost. Before a new technique or machine can be implemented into a biopharma procedure, it must be validated against current methods. This is called analytical method validation (AMV). This chapter will outline the importance of such validation and the use of critical quality attributes to identify appropriate methodologies.
AMV is required in the biopharmaceutical industry to provide evidence of suitability for all methods used to test final containers (release and stability testing), raw materials, in-process materials, and excipients. This regulation is designed to ensure that bioanalytical methods enable the timely delivery of data that adequately meet scientifically justified and fit-for-purpose criteria. The increasing complexity of biopharmaceuticals raised greater challenges to bioanalytical analyses for accurately evaluating efficacy, safety and immunogenicity. Regulators needed to know that appropriate techniques were being used to obtain these data in order to put their confidence in the results reported by pharmaceutical companies.
In addition to satisfying regulatory requirements, AMV enables product specifications to be defined and helps reduce process variability. AMV does not improve a test method but, by ensuring the quality of the development work, it drives the quality of the production process and consequently the final product.
Method validation is becoming more critical than ever as labs are increasingly choosing to outsource bioanalytical testing. Such outsourcing frees up the internal labs to focus on product research and development. However, it also involves significant initial time input to evaluate possible contract laboratories and select the one that is most appropriate, transfer methods and ensure that the outsourced results are consistent with the originating lab. It also requires ongoing monitoring of the method and the performance of the contract lab. The decision to outsource analyses is thus not a simple one. In his presentation at Pittcon 2019, “Outsourcing Antibody-Drug Conjugate Testing for Commercial Products: Contract Lab Selection, Method Transfer, and Contract Lab Monitoring”, Gregg Monten of Seattle Genetics will be evaluating the pros and cons for each approach.
Every analytical process is associated with specific properties or characteristics, be they physical, chemical, biological, or microbiological, that should be within an appropriate limit, range, or distribution to ensure the desired product quality. These are known as a critical quality attributes (CQA). The CQA may be method attributes or method parameters. For example, in HPLC the CQA are the mobile phase buffer, pH, diluent, column selection, organic modifier, and elution method. Furthermore, the CQA depend on the nature of the drug and potential impurities, so will need to be determined for every analysis.
Identifying CQA is the starting point for the implementation of systematic scientific approaches to the development of product development and manufacturing processes, such as QbD (Quality by Design) and PAT (Process Analytical Technology). The use of such tools minimizes the risk of product development as it ensures that it is based on product and process understanding and sound scientific knowledge, thereby increasing productivity and ensuring quality.
Determining CQA for antibody-drug conjugates (ADCs) is critical yet it is a major undertaking. ADCs form a valuable subset of biotherapeutics as they enable treatment to be limited to the target area. For example, they can provide targeted delivery of a cytotoxic agent to a tumor. An ADC comprises a potent small molecule conjugated to an antibody. ADCs are thus highly complex and structurally heterogeneous, typically containing numerous product-related species. The identification of CQA is an important aspect of the development of ADCs because such an identification process results in a thorough understanding of quality attributes and the potential impact on safety and efficacy.
At Pittcon 2019, Li Tao of Bristol Myers Squibb will be discussing analytical development centered around critical quality attributes and how this relates to the characterizing of ADCs in her presentation entitled “Current Status of Analytical Development During Process Development for Biologics”.
Malvern Panalytical will also be at Pittcon 2019 presenting their portfolio of low-volume, low-concentration stability screening techniques that can provide early indications of adverse molecular interactions. The measurement of such attributes can provide important information to inform decisions on the identification and progression of viable drug candidates.
Mass spectrometry (MS) is used during development for in-depth characterization of ADCs to determine the level and sites of drug conjugation and heterogeneities present due to the conjugation chemistry. It is also used during manufacture to confirm the sequence fidelity of the ADC as well as the extent and integrity of the conjugation. Although liquid chromatography with mass spectroscopy has more broad-reaching capabilities than LBA, there are still significant challenges in isolating, concentrating, detecting and quantifying specific proteins by LC–MS in the presence of an overwhelming abundance of endogenous proteins. In some cases, this can be facilitated by separating out the bulk of the unwanted proteins, for example using electrophoresis, before detection or analysis.
It is important to ensure that the techniques used to improve sensitivity and resolution of LC–MS must not alter the precise structure of the target protein that is essential for its efficacy. For this reason, electrospray ionization (ESI) MS is commonly used as it enables proteins to be ionized without denaturation; non-covalent bonds and protein-ligand complexes remain intact. However, ESI MS analysis of many intact ADCs remains challenging due to the presence of extensive chemically unstable and hydrophobic linkers. Recent developments to optimize the mobile phases during LC separation and the ESI source have enabled reliable MS-based assays for determining drug-to-antibody ratio.
Bioanalytical technologies are continually evolving to provide innovative methodologies that are capable of characterizing and purifying increasingly complex drug modalities. The implementation of regulatory guidance on best practices and criteria to ensure that the techniques are scientifically sound will guarantee that ongoing developments continue to provide efficient bioanalysis of biopharmaceuticals.
Numerous companies producing equipment to facilitate the separation and characterization of biotherapeutics will be attending Pittcon 2019 to discuss their portfolios, including Bio-Rad, Bruker, Wyatt. ThermoFisher will be on site at Pittcon 2019 to discuss their electrophoresis range.
- Bio-Rad – http://www.bio-rad.com/en-uk/product/mass-spectral-databases?ID=NH261UE8Z
Bruker – https://www.bruker.com/products/mass-spectrometry-and-separations/ms-software/biopharma-compass-30/overview.html
- FDA 2015. How to Identify Critical Quality Attributes and Critical Process Parameters. http://pqri.org/wp-content/uploads/2015/10/01-How-to-identify-CQA-CPP-CMA-Final.pdf
- Friese O, et al. Heightened Characterization of ADCs: Overcoming Challenges to Support Process and Product Understanding. Abstract at 15th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry 2018
- Malvern Panalytical. Stability profiling. https://www.malvernpanalytical.com/en/industries/bioscience/stability-profiling
- Raman NVVSS, et al. Analytical Quality by Design Approach to Test Method Development and Validation in Drug Substance Manufacturing. Journal of Chemistry 2015; Article ID 435129, 8 pages. http://dx.doi.org/10.1155/2015/435129
- ThermoFisher. Protein Gel Electrophoresis. https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-gel-electrophoresis.html and https://www.thermofisher.com/uk/en/home/industrial/pharma-biopharma/biopharmaceutical-analytical-testing/mass-spectrometry-biopharma-qc.html
- Wagh A, Challenges and new frontiers in analytical characterization of antibody-drug conjugates. MAbs. 2018;10(2): 222–243. doi: 10.1080/19420862.2017.1412025
- Wyatt – https://www.wyatt.com/solutions/sectors-served/biopharmaceutical.html
- Zhang YJ and Hyun JA. Technologies and strategies for bioanalysis of biopharmaceuticals. Bioanalysis 2017;9(18). https://www.future-science.com/doi/full/10.4155/bio-2017-4981
Chapter 4 – The Hidden World of Biosimilars
A biosimilar is a highly similar version of a biological medicinal product that is already on the market for which the patent has expired. Biosimilars are the biotherapeutic equivalent of generics for small molecule drugs. Biosimilars (and generics) must undergo a comprehensive comparison with the original product in order to demonstrate that they exhibit comparable physicochemical characteristics, efficacy and safety. The introduction of biosimilars, with their complex structures, has thus increased the demand for highly efficient analysis methods.
In 2009, the US government made it easier for biosimilars to be approved based on the approval of the original biotherapeutic. New versions of a biotherapeutic already on the market can now be licensed as long as they are proven to be comparable to the product they copy. This abbreviated licensure pathway for biosimilars was introduced in order to provide more treatment options, increase access to life-saving medications, and potentially lower healthcare costs through competition. However, such potential benefits were overshadowed by widespread concerns over patient safety.
These concerns arose due the complex nature of protein-based therapeutics and the high potential for heterogeneity. This makes it difficult to ensure their precise structure and composition. Even when a recombinant product is manufactured using a single process under controlled conditions, there can be variations between batches. Many well-characterized, highly purified proteins thus exhibit microheterogeneity. Biosimilars produced by a different manufacturing process may thus differ from the original biotherapeutic despite showing a high level of similarity. Since the full characterization of such products is a challenge to available analytical techniques, biosimilars could have undetected differences from the parent product and even minute differences have the potential to significantly impact safety and efficacy.
For a small molecule drug, it is relatively straightforward to demonstrate that the generic drug is pharmaceutically equivalent (that is, it contains the same active ingredient in the same purity, strength, dosage form and route of administration) and bioequivalent (that is, it is absorbed into the body at a similar rate and extent) to the original drug. However, the complexity of some biotherapeutics, such as mAbs, mean it is not possible to demonstrate that the two products are absolutely identical due to the limits of analytical technologies. In particular, full characterization of biotherapeutics in terms of post-translational modifications, three-dimensional structures and protein aggregation remains a challenge. The acceptable level of comparability required to confirm that two biotherapeutics must therefore be arbitrarily defined. This is achieved using the biosimilarity index, a disaggregated, probability-based, scaled, and weighted equation for determining biosimilarity.
In addition, when a biosimilar is being manufactured by a company other than that which produces the original product, it is likely that it will not be produced using the same methodologies that were proven to be scientifically sound and accepted by the regulatory agencies for the analysis of the original biotherapeutic. Only limited information regarding the manufacturing procedures of the originator molecule is made publically available, and so the biosimilar may use a different cell line, or more recently developed advanced cell culture conditions and purification processes. CQAs (discussed in Chapter 3) must therefore also be identified when developing a biosimilar protein product.
In a similar manner used for the original biotherapeutic, a comprehensive analytical biosimilarity approach is designed based on the identified CQAs. The CQAs are determined using various risk assessment approaches and are continuously monitored during manufacturing process development to provide feedback for process parameter changes.
Akos Szekrenyes of Glenmark Pharmaceuticals will be giving an overview of the current analytical techniques used to assess the similarity of biosimilars at Pittcon 2019 in his presentation “Comprehensive Analysis of Biosimilar Monoclonal Antibodies: Current Trends based on the Lately Approved Follow-On Products”.
Akos Szekrenyes was also involved in the recent development of a rapid, capillary gel electrophoresis based method to quantitatively assess the glycosimilarity between a biotherapeutic and a biosimilar. The technique highlighted differences in their N-linked carbohydrate profiles. Only mannosylation was deemed to be a CQA for this particular biosimilar. The study used reagents produced by Millipore Sigma, who will be exhibiting at Pittcon 2019.
Numerous additional companies supplying products and equipment needed in biosimilarity analyses will be attending Pittcon 2019 and be available to discuss ways to optimize outcomes. These include Bruker, who developed the integrated search engine for glycans, GlycoQuest; LGC who provide bioanalytical method development and validation services; and ThermoFisher with their high-performance, silica-based high-HPLC column, GlycanPac™ , for the analysis of glycans present in biological molecules.
Berkowitz SA, et al. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov. 2012;11(7):527–540. doi: 10.1038/nrd3746
Borza B, et al. Glycosimilarity assessment of biotherapeutics 1: Quantitative comparison of the N -glycosylation of the innovator and a biosimilar version of etanercept. Journal of Pharmaceutical and Biomedical Analysis 2018;153. https://www.researchgate.net/publication/323213370_Glycosimilarity_assessment_of_biotherapeutics_1_Quantitative_comparison_of_the_N_-glycosylation_of_the_innovator_and_a_biosimilar_version_of_etanercept
Bruker. GlycoQuest https://www.bruker.com/products/mass-spectrometry-and-separations/ms-software/proteinscape/glycoquest.html
FDA 2018. Biosimilars. https://www.fda.gov/drugs/developmentapprovalprocess/howdrugsaredevelopedandapproved/approvalapplications/therapeuticbiologicapplications/biosimilars/default.htm
Hajba L, et al. On the glycosylation aspects of biosimilarity. Drug Discovery Today 2018;23(3):616-625
Weise M, et al. Biosimilars—why terminology matters. Nature Biotechnology 2011;29:690–693. https://www.nature.com/articles/nbt.1936
LGC – https://www.lgcgroup.com/services/drug-development-solutions/bioanalytical-sciences/#.XEs7Zvn7SUk
Thermo Fisher. GlycanPac – https://www.thermofisher.com/order/catalog/product/082468
Woodcock J, et al. Opinion – The FDA’s assessment of follow-on protein products: a historical perspective. Nature reviews. Drug discovery 2007;6:437 42. 10.1038/nrd2307 https://www.researchgate.net/publication/6204172_Opinion_-_The_FDA’s_assessment_of_follow-on_protein_products_a_historical_perspective)
Advances in biotechnology capabilities during the last two decades have made it possible to engineer biological molecules for medical purposes. Such biotherapeutics now account for a significant proportion of new drug approvals. Monoclonal antibodies (mAbs) are the fastest growing subset of biotherapeutics. Their increasing popularity stems from the ability to provide specific targeted treatments for a range of life-threatening diseases that proved difficult to manage with traditional small molecule medications.
As is the case for small molecule drugs, biotherapeutics must be accurately characterized in terms of safety, efficacy and purity to protect the patients they are designed to help. The complexity of biological therapies results in a huge scope for structural and functional variations and so it is especially important that structure-function relationships are fully understood and well defined. The need to gain such in-depth knowledge of biopharmaceuticals in order to reap the therapeutic rewards has placed huge demands on bioanalytical capabilities. Indeed, it has catalyzed the development and enhancement of numerous techniques for analyzing proteins and determining immunogenicity.
Recently, the pressure increased further as the patents for licensed biotherapeutics started to expire and biosimilar products could be produced. Biosimilars can gain marketing approval more quickly via the abbreviated licensure pathway by demonstrating that they are structurally similar to the parent product they copy.
Thus, bioanalytical techniques are required that can accurately and quickly provide in-depth characterization and quantitation of both originator biotherapeutics and their biosimilars. This is made particularly challenging by the complex heterogeneous nature of these biological products. The high propensity for post-translational modifications, such as glycosylations, further complicates such analyses. The positon of these modifications differ according to the cell line used to produce the biotherapeutic and can also occur also during purification, formulation and storage processes as well as in vivo after administration. Slight differences in these structural modifications have the potential to impact on PK and pharmacodynamics and also to give rise to immunogenicity.
Manufacturers must demonstrate to regulatory agencies that the methodologies used to define a biotherapeutic or assess the comparability of a biosimilar with its originator molecule are scientifically sound. A thorough understanding of critical quality attributes (CQA) is essential to inform method development and analytical method validation ensures that the methods used are fit for purpose and provide robust data.
With the increasing volume of biotherapeutics, such analytical processes must also be easy to perform and cost-effective to incorporate into development and manufacturing procedures. The viability of bioanalytical techniques for the characterization of biotherapeutics with diverse structural complexities is thus a balance of quality, speed and cost.
Despite tremendous advances in bioanalytical techniques suitable for the analysis of biotherapeutics, significant analytical challenges still remain. For example, in isolating, concentrating, detecting and quantifying specific proteins from a sample containing a myriad of other proteins, often including the endogenous form of the biotherapeutic. In addition, strategies for evaluating post-translational modifications are still in their infancy. Several techniques for the determination of N-glycoyslation have recently been described, but there is still a need for improved evaluation of O-glycosylation. Recent technological advances have also made substantial contributions to quantifying and characterizing mAbs, but further improvements in immunogenicity assays are desirable.
With the high therapeutic potential of biological products, the flurry of novel biotherapeutics is set to continue. Accordingly, the pressure to develop more and more sensitive bioanalytical techniques can only increase. The next decade will inevitably bring more powerful bioanalytical techniques and stringent method validation criteria will evolve in parallel.
The presentations and exhibits at Pittcon 2019 will provide a taste of the analytical innovation and strategy evolution we are likely to see. Visit the Pittcon 2019 guide to learn more about the symposia, oral presentations and short courses that will be taking place.
In addition, numerous market-leading providers of analytical equipment, products and services will be on-site at Pittcon 2019 to discuss the latest additions to their capabilities and address your analytical requirements.
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