Innovative Spectroscopy Is Enhancing Protein Structure Determination
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Proteins are central to many disciplines within the life sciences. Understanding how they are formed, how they function, their role in health and disease and importantly the ways in which they respond to therapies and pharmaceutical intervention are all key aspects.
Whilst proteins are essentially created from our genetic code, the picture is far more complicated, and they are not simply a 2D representation of a genetic template. Transcription and translation are part of the picture but beyond this there are modifications, secondary and tertiary structures, all of which are influenced by the environment surrounding the forming protein. The same is true of protein-based pharmaceuticals, changes in which could have significant impact on efficacy and safety. Therefore, being able to understand what forms of a protein are present in their natural conditions, even if present only at low levels, is important for analysts.
We spoke to Julien Bradley, CEO of RedShiftBio, about their technology, the problems in protein structure analysis it overcomes and the importance of being able to make accurate and representative measurements.
Karen Steward (KS): Can you tell us a bit about your technology and how its development came about?
Julien Bradley (JB): Microfluidic modulation spectroscopy (MMS) as found in the AQS3™pro from RedShiftBio was developed in response to an unmet need in the biopharmaceutical analytical testing space. It is well known that secondary structure can be deduced from classical spectroscopic techniques, such as circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy techniques, along with several others such as Raman and nuclear magnetic resonance (NMR) spectroscopy. These techniques, particularly the more incumbent CD and FTIR suffer from several limitations that make them of little use to quantify a fully formulated biopharmaceutical. These limitations include concentration and incompatibility with formulation components.
RedShiftBio developed MMS as an advancement on the basic principles of infrared spectroscopy. The company beginnings were in laser and optical systems, where they focused on applications of the quantum cascade laser. Utilizing this technology to focus on the Amide I band of proteins, the technology brought about almost 100-fold sensitivity increase, facilitating the measurement of samples that otherwise could not be measured.
KS: What are the problems with existing solutions and how does your platform overcome them?
JB: Traditional spectrophotometers used to assess protein secondary structure have limitations in both concentration range and chemical compatibility. In addition, there are limited automated solutions available, with FTIR being particularly labor intensive. Biopharmaceutical drug products are often high in concentration, and consist of complex formulations with buffers, surfactants, antioxidants, and more being part of the final product. These complex formulations are not compatible with CD. FTIR presents its own limitations, background referencing can be challenging due to drift. With manual sample preparation, measuring a single sample can be labor intensive.
The AQS3™pro utilizes MMS, a technique that rapidly alternates sample and buffer through the detection zone. Combined with a quantum cascade laser, the system measures absorbance of a protein across the Amide I band with over 100 x greater sensitivity relative to traditional FTIR. In addition, automated background referencing removes background drift, providing high quality spectra. With the addition of the AQS3™delta software package, processing of raw data to the final results has been streamlined eliminating the complexities of traditional spectral processing.
KS: Why is the accurate measurement of protein secondary structure important?
JB: Change in protein structure is directly linked to change in efficacy. One of the major risks associated with biopharmaceutical products is the presence of protein aggregates, commonly caused by instability in the protein higher order structure (HOS). These aggregates are associated with multiple risk factors such as injection site swelling, anaphylaxis, and the production of anti-drug antibodies, reducing the efficacy of the drug. As a fundamental building block of protein HOS, changes in secondary structure can be an early warning sign of longer-term stability issues. Secondary structure can be affected by many stressors including oxidation, deamidation, shear stress, cavitation, pH changes and more. For this reason, monitoring of secondary structure through the entire development and manufacturing process is critical to ensuring quality drug products.
KS: Who do you see as being your end user? Could anyone operate the system?
JB: Knowing how critical secondary structure can be to the quality of a biopharmaceutical, the AQS3™pro can provide critical quality data throughout the entire process of discovery through development into manufacturing. With the addition of automated sample analysis, the platform can be a workhorse for even the most demanding labs. Key applications of the system include formulation development, process design and development, quality assurance and control, biosimilar development and more. The AQS3™pro provides critical assessment of structural similarity and comparability, providing a direct measure of protein stability.
KS: In what areas is it being taken up? Can you tell us about some interesting applications to which your technology is being applied?
JB: Although interest in the AQS3™pro has been shown all across the drug development pipeline, the most interesting applications have been focused around observing effects of excipients such as polysorbate80 on secondary structure, as well as monitoring the effect of purification stages including viral inactivation and protein A purification stages where significant pH shifts are required, in some cases for extended periods of time.
In conjunction with Janssen, the AQS3™pro was used to monitor a viral inactivation step for a drug substance, where a shift to pH 3.5 resulted in observable changes in β-sheet content. Interestingly, the data showed that over an extended period of about 15-18 hours, the secondary structure composition returned to that of the starting material, providing evidence of stability under these extreme conditions.
KS: What areas still pose challenges? Are there aspects in which you would like to expand in the future?
JB: While today we are focused on protein secondary structure, we could potentially leverage MMS for other molecule types in future (mRNA for example).
Julien Bradley was speaking to Dr Karen Steward, Science Writer for Technology Networks.