Higher Order Structure Consortium for biopharmaceutical proteins
Case studies on technical decision making with biophysical data
During the development of proteins for use as biopharmaceuticals, the primary structure (amino acid sequence) is important in defining protein activity.
Due to the complex nature of protein drugs, compared to small molecule drugs, biophysical techniques are important tools in the characterization of the protein’s higher-order structure (HOS) to understand the protein’s stability, folding, structure, and functional activity. HOS is characterized variety of biophysical methods including:
- Mass spectrometry (MS)
- Circular dichroism (CD)
- Fourier transform infrared spectroscopy (FTIR)
- Raman spectroscopy
- X-ray crystallography
- Nuclear magnetic resonance (NMR)
- Near-UV CD
- Size exclusion HPLC
- Static and dynamic light scattering (SLS and DLS)
- Differential scanning calorimetry (DSC)
- Analytical ultracentrifugation (AUC)
Functional assays include Isothermal Titration Calorimetry (ITC), surface plasmon resonance (SPR) and fluorescent and spectroscopic potency assays. With complementary and orthogonal techniques, HOS data are used to make decisions which drugs to move forward in development, how to formulate the drugs, and as quality control and biocomparability studies.
Scientists in the biopharmaceutical industry, academic researchers, and government agencies are increasingly aware of the critical role that HOS plays in the stability and intended biological function of biopharmaceutical products. Biophysical HOS characterization is included in regulatory submissions.
At the Well Characterized Biologicals conference in 2014, a group of scientists and researchers from industry and academic institutions organized the HOS Consortium, to address how HOS data can be used most effectively to drive decisions during product development. Scientific advances in HOS analysis, as well as continued dialogue among academia, industry, and regulatory agencies will ensure that appropriate methodologies are used to inform technical decision-making during biopharmaceutical development. The commentary introduces the HOS Consortium and its purpose:
Gabrielson, J. P. and Weiss, W. F. (2015), Technical Decision-Making with Higher Order Structure Data: Starting a New Dialogue. J. Pharm. Sci., 104: 1240–1245.
As of July 2015, there have been four case studies published from the HOS Consortium. In these case studies several technologies from the Malvern portfolio were used for the HOS characterization.
Case study 1:
Jiang, Y., Li, C., Li, J., Gabrielson, J. P. and Wen, J. (2015), Technical Decision Making with Higher Order Structure Data: Higher Order Structure Characterization During Protein Therapeutic Candidate Screening. J. Pharm. Sci., 104: 1533–1538.
During biopharmaceutical protein development, a potential drug product will go through a variety of different purification, production and storage conditions (i.e. pH extremes, temperature changes) that can stress the drug product. Any stresses can potentially lead to changes in the protein structure resulting in aggregation. For a commercially viable product, it is important to choose a candidate that is most stable, and least prone to aggregation during production, formulations, and storage.
This case study from a group at Amgen reports on the use of selected HOS characterization methods for protein drug candidate selection at early stages of development. Amgen scientists looked a two IgG2 monoclonal antibodies (X and Y) that exhibited similar biological activities. HOS and the relative stabilities of the two antibodies were assessed for manufacturability and overall product quality. The antibodies were placed in pH 3, 5 or pH 7 buffer, then analyzed with differential scanning calorimetry (using MicroCal VP-Capillary DSC), dynamic light scattering (using Malvern Zetasizer Nano ZS), near UV CD, FTIR, fluorescence spectroscopy, and size-exclusion HPLC.
Evaluation of the results showed that both antibody A and B had properties that could lead either of them to be moved forward in development. HOS data, used in conjunction with effectiveness of purification processes, led to the choice of the drug product with the best long-term stability and ease of manufacturing. Use of HOS data also helped design process and manufacturing methods.
Case study 2:
Gruia, F., Du, J., Santacroce, P. V., Remmele, R. L. and Bee, J. S. (2015), Technical Decision Making with Higher Order Structure Data: Impact of a Formulation Change on the Higher Order Structure and Stability of a mAb. J. Pharm. Sci., 104: 1539–1542.
Changes in formulation may be required during the development of protein biopharmaceuticals. Some of the changes may alter the protein HOS. In this case study, scientists from Medimmune used DSC (MicroCal VP-Capillary DSC), CD, and FTIR to show how the change from a trehalose-based formulation to an arginine-based formulation impacted the tertiary structure and the thermal stability of a monoclonal antibody. The secondary structure was not disrupted by the formulation change. The destabilization of the tertiary structure did not affect the long-term stability or the bioactivity of mAb1. This indicates that loss of conformational stability was likely compensated by improvements in the colloidal stability of mAb1 in the arginine-based formulation. The formulation-induced changes in HOS were reversible as proven by measurements after dilution in phosphate-buffered saline. For aggregation driven by assembly of aggregates, small changes in conformational structure and stability as measured by HOS methods may not necessarily be predictive of long-term stability.
Case study 3:
Budyak, I. L., Doyle, B. L. and Weiss, W. F. (2015), Technical Decision-Making with Higher Order Structure Data: Specific Binding of a Nonionic Detergent Perturbs Higher Order Structure of a Therapeutic Monoclonal Antibody. J. Pharm. Sci., 104: 1543–1547.
This case study, from Eli Lilly, describes the utility of several HOS methods as investigational tools during the purification process development. An atypically high level of residual detergent in a development drug substance batch of a therapeutic monoclonal antibody triggered a root cause investigation. Several orthogonal biophysical techniques were used to uncover and characterize a specific interaction between the detergent and the antibody. Isothermal titration calorimetry (ITC, MicroCal VP-ITC) was used to quantify the molar ratio and affinity of the binding event, and circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC, MicroCal VP-DSC) were used to evaluate corresponding impacts on secondary/tertiary structure and thermal stability. As detergents are used routinely in biopharmaceutical processing, this case study highlights the value and power of HOS data in informing technical investigations and underlines the importance of HOS characterization as a component of overall biopharmaceutical analytical control strategy.
Case study 4:
Arthur, K. K., Dinh, N. and Gabrielson, J. P. (2015), Technical Decision Making with Higher Order Structure Data: Utilization of Differential Scanning Calorimetry to Elucidate Critical Protein Structural Changes Resulting from Oxidation. J. Pharm. Sci., 104: 1548–1554.
DSC is a useful tool for monitoring thermal stability of the molecular conformation of proteins at all stages of discovery and development. In this case study, scientists from Amgen presented an example using MicroCal VP-Capillary DSC to evaluate changes in stability arising from oxidation, a common chemical degradation pathway that can lead to protein aggregation. Six protein products from three structural classes were evaluated at multiple levels of oxidation. For each protein, the melting temperature (Tm) decreased linearly as a function of oxidation. Differences in the rate of change in Tm, as well as differences in domain Tm stability were observed across and within structural classes. For one protein, analysis of the impact of oxidation on protein function was also performed.
DSC was shown to be a leading indicator of decreased antigen binding (using potency assays) suggesting a subtle conformation change may be underway that can be detected using DSC prior to any observable impact on product potency. Detectable changes in oxidized methionine by mass spectrometry (MS) occurred at oxidation levels below those with a detectable conformational or functional impact. Using MS, DSC, and relative potency methods together, the intricate relationship between a primary structural modification, changes in conformational stability, and functional impact can be elucidated.
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