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 by a variety of biophysical methods including:

With complementary and orthogonal techniques, HOS data are used to make decisions on which drugs to move forward in development, how best to formulate the molecules, for 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 also included in regulatory submissions.

In recent years, Quality by Design (QbD) has been suggested by the US FDA and the International Conference on Harmonization (ICH). QbD is defined as “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management” (ICH website). Benefits of QbD include:

  • Better design of product
  • Fewer problems in manufacturing
  • Understanding and mitigation of risk
  • A reduction in overall cost of manufacturing
  • Faster regulatory approval
  • Enabling continuous improvement
  • Providing a better understanding of processes

QbD involves a complete understanding of the biopharmaceutical product and the processes in producing the drug, including cell culture, purification, filtration, formation, and packaging.  Proteins have attributes which can be susceptible to structural modifications, which can be affected by the above processes.  It is important that processes are designed to control the key protein attributes which are most critical for protein structure and activity.  Using HOS methods in QbD involves linking any structural modification to the protein to changes in biological function.

The intent of QbD is to encourage pharmaceutical companies to develop sufficient understanding of their products and manufacturing processes, ensure that their processes are robust, and demonstrate this enhanced understanding to the pharmaceutical regulatory agencies.

Oxidation of amino acid side chains is a major degradation pathway for protein therapeutics. As a result of oxidation, product efficacy and stability can be compromised; so careful method selection should be applied to aid in evaluating this quality attribute.

Dr. John Gabrielson of Elion Labs, a contract research organisation based in Louisville, Colorado, recently presented a webinar “Using DSC to investigate the impact of oxidation on protein structure” describing the use of DSC to look at the structural changes associated with methionine oxidation, demonstrating the sensitivity of DSC to oxidation-induced conformational stability changes across multiple protein classes. For all proteins evaluated, the Tm determined by DSC decreased linearly with increasing MetOx levels. In comparison, orthogonal spectroscopic structural characterization methods (near-UV CD and fluorescence spectroscopy) were far less sensitive to oxidation-induced conformational changes, suggesting the methods have different capabilities and monitor different properties of the molecule. DSC is a more appropriate structural characterization method for monitoring protein oxidation compared with these spectroscopic methods.

Monoclonal antibody IgG2 has multiple structural domains, and several methionines on the antibody’s heavy chain are potential targets for oxidation and methionine sulfoxide formation.  M253 (on the CH2 in the Fc region) was the methionine residue which was the most susceptible to oxidation for the IgG2B molecule studied in this research. DSC was able to detect this specific methionine oxidation by changes in Tm by DSC. These changes in Tm were found to precede a loss in relative potency, demonstrating that DSC is a leading indicator of decreased antigen binding.

These results suggest that the Tm measured by DSC can be a useful and predictive tool for elucidating the impact of any production and manufacturing process changes on protein HOS, and relating conformational changes to potential functional impact. This can have a positive impact on QbD for biopharmaceutical quality.

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