JAIMA – Recent Achievements and Current Interests in Research on the Characterization and Quality Control of Biopharmaceuticals

Presented: Thursday, May 19, 2022
Organizer: Kouhei Tsumoto, University of Tokyo

New analytical solutions to shed light on characterization research of next generation biologics, such as therapeutic antibodies themselves and their interaction with additives have become more and more important recently. Collaborative studies to develop new analytical solutions for biopharmaceutical characterization and quality control have been started in Japan leaded by National Institute of Health Science (NIHS) with the participation of the manufacturers of analytical instruments together with academia and pharma regulatory sector. The technical issues of biopharma and recent achievements using Raman spectroscopy, X-ray diffraction, Fluorescence A-TEEM, and separations methods will be discussed.

Recent Achievements and Current Interests in Research on the Characterization and Quality Control of Biopharmaceuticals
Akiko Ishii-Watabe, National Institute of Health Sciences, Japan
Biopharmaceuticals such as therapeutic antibodies comprise a very important category of drugs that have been developed to satisfy unmet medical needs. Their target disease area is expanding due to the advancements in the development of products with novel structures or novel modes of actions. In addition, in the field of CMC (Chemistry，Manufacturing and Control), novel manufacturing technologies such as continuous manufacturing and analytical method such as multi-attribute method are attracting interest. In order to ensure the safety and efficacy of these products, appropriate quality control strategy is necessary. For the characterization and quality control of these innovative products, the use of state-of-the-art technologies is required. Considering that the time frame of product development is getting shorter, and complexity of products become higher, the efficient use of innovative technologies will help to establish the appropriate establishment of control strategy. In this presentation, recent activities for characterization and quality control of biopharmaceuticals in Japan will be introduced. This includes the collaborative studies of National Institute of Health Sciences and pharmaceutical companies and academia in Japan (J Pharm Sci. 2020 May;109(5):1652-1661. doi: 10.1016/j.xphs.2020.01.001.; J Pharm Sci. 2017 Dec;106(12):3431-3437. doi: 10.1016/j.xphs.2017.07.024.). The plan and latest information of the new project started in July 2021 will also be introduced.

Absorbance-Transmission Excitation Emission Matrix Spectroscopy – Molecular Fingerprint for the Characterization of Low Concentration or Highly Similar Biopharmaceuticals
Linda Kidder, Horiba Instruments Inc.
The next generation of complex biopharmaceuticals products require fit-for-purpose analytical tools to provide robust characterization for QbD and manufacturing QA/QC. These exciting new therapeutics garner the headlines, but the right analytical tools are an absolute prerequisite for their successful commercialization. Molecular spectroscopy approaches like Raman, near-infrared (NIR) and Fourier-transform infrared spectroscopy (FT-IR), are well known and widely used, with attributes that make them a good fit: green, rapid, robust, and non-destructive. There is however a continuum of capability across these optical techniques, and some struggle to detect low concentration components, or to easily distinguish very similar components. The A-TEEM fluorescence method provides a complete and traceable optical fingerprint of liquid samples, overcoming limitations that previously hampered adoption of other fluorescence EEMs approaches. This technique is well suited to the characterization of vaccines and other biopharmaceutical samples, with not only the sensitivity and specificity comparable to chromatographic methods, but also the speed, cost savings, and reproducibility of vibrational spectroscopic approaches. We will show its ability to differentiate similar multi-component vaccine formulations, identify therapeutic samples with post-translational modifications, quantify the payload filling percent in AAV reference materials, and detect amino acid substitutions in vaccines. A-TEEM therefore has the potential to replace traditional instruments like HPLC, GC, LCMS, and GCMS as a simpler, and faster analytical tool, with significantly lower per-sample measurement costs.

Capturing the Physical Properties of Highly-Concentrated Antibody Solutions from Raman Spectroscopic Analysis
Satoru Nagatoishi, The Institute of Medical Science, University of Tokyo
A liquid formulation of antibodies is popular in the biopharmaceutical industry. The final concentrations of antibody solutions are almost high, over 50 mg/mL. However, a highly concentrated antibody solution has several troubles. At high concentrations, the antibody solution increases its viscosity, so it sometimes becomes difficult to prepare the antibody formulation. In addition, the increased concentration and viscosity are accompanied by an increased risk of protein aggregation or denaturation. These unfavorable changes may result in side effects, such as low drug efficacy and toxic reaction. Therefore the optimization of antibody formulations and the understanding of the behavior in a highly concentrated solution are important.
Since the short-range intermolecular interactions work in a highly-concentrated solution, we must study and discuss the protein based on a conformational approach. In other words, conformational knowledge is more important in a highly-concentrated solution. Which instrument / analytical method is a powerful tool to detect accurately and discuss clearly the conformational behavior of antibodies at high concentrations. Raman spectroscopy is one of the attractive methods to detect and discuss the conformational behavior of proteins at high concentrations. In this study, we perform Raman spectroscopy to analyze the therapeutic antibodies.

Evaluation of Protein Structure Using Small-Angle X-Ray Scattering (SAXS)
Angela Criswell, Rigaku American Corporation
Small angle X-ray scattering (SAXS) is a useful technique for extracting structural information from biological samples in solution. Most biological SAXS experiments are performed for the purpose of low resolution modeling of macromolecular structure. In such cases, the samples ae predominantly aqueous macromolecules present in low concentration. In other cases, it may be of interest to study solutions at high concentration, at high viscosity or at non-ambient conditions. In such cases, SAXS can provide useful information about the inherent structure and phase of solutions. In this study, we describe the applications using Rigaku’s SAXS system for biological SAXS and we also demonstrate the system is well suited for analysis of all types of solution samples, independent of concentration, viscosity and phase. In this presentation, we describe experiments with both typical and non-typical macromolecular solution samples.

Analysis of Monoclonal Antibody (mAb) and Related Biologics by LC and LC-MS Platforms
Mridul Mandal, Shimadzu
Monoclonal antibodies (mAbs) are a class of biotherapeutics that are rapidly gaining traction. The higher-order structures of mAbs play a crucial role in determining their efficacy and safety. It is crucial that mAbs are accurately characterized before they can become biotherapeutics. A better understanding of mAbs helps craft better bioprocesses, formulas, and dosages. In this study, a recombinant human IgG NIST mAb reference standard was analyzed using high-resolution quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LCMS). An innovative proteolysis method called nano-surface and molecular-orientation limited proteolysis (nSMOL) selectively cleaves Fab regions of mAbs. Regardless of the type of antibodies scientists use, nSMOL can be used to develop analytical methodologies. Triple quadrupole liquid chromatography-mass spectrometry (TQ-LCMS) permits multiple reaction monitoring (MRM) to determine the level of fragmentation in peptides derived from Fab. Furthermore, a protocol for selecting pharmacokinetically suitable signature peptides was developed using nSMOL and TQ-LCMS. Additionally, consistency of glycosylation during process validation is a critical quality attribute (CQA) as N-linked glycans can impact the stability, solubility, and recognition of glycoproteins by glycan-binding proteins. Proteins are attached to N-glycans by covalent bonds formed by N-glycosidic bonds forming at the asparagine (Asn) residues. Monoclonal antibodies and biosimilars can cause such an event at the crystallizable fragment (Fc). In this study, we measured the released glycans from mAbs using QTOF-LCMS. A variety of methodologies are available for the characterization of mAbs and related biologics. Any kind of analysis can be performed based on our tailored workflows, including whole protein analysis, subunit, and peptide fragment analysis, and released N-Glycan analysis.