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Techniques & Tools Spectroscopy, Pharma & Biopharma

Fast and Furious HOS Analysis?


The bar is rising for analytical techniques in the biopharmaceutical industry. Data quality used to be the only deciding factor, but other issues are now weighing in – notably, ease of use. Usability is becoming a defining characteristic of analytical tools for biotherapeutics labs looking to do more with less against aggressive timelines. As workflows are refined to maximize information flow, high-throughput systems with automated data acquisition and processing are becoming increasingly desirable. There is a great (and growing) appetite for innovative technologies that can serve this purpose across the biopharmaceutical lifecycle.

Higher order structure (HOS) analysis is crucial in biopharmaceutical development and commercial manufacture; such measurements characterize the secondary, tertiary, and quaternary folding and spatial arrangements that define the three-dimensional shape and interactions of biotherapeutic molecules. Those with a basic grounding in biology will know that changes in HOS impact functionality – and for biopharmaceuticals that can trigger loss of stability, increased aggregation, compromised efficacy, and increased immunogenicity. In short, quantifying and monitoring HOS across biopharmaceutical development and commercial manufacture is critical to understand, identify, and maintain conditions that will reliably deliver a safe and efficacious drug.

A raft of techniques are deployed for HOS characterization, but incumbent technology for secondary structure is currently ill-matched to industry needs (and is a primary target for improvement). Current techniques include far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. And both have limitations.

Automated CD instrumentation allows the sequential application of far-UV CD and near-UV CD (for the assessment of tertiary structure) on a single sample set. Such an approach sits comfortably in modern labs, though sample preparation is essential as UV CD is most suitable for relatively dilute and simple solutions. The removal of many common formulation excipients that interfere with the measurement is a common requirement (1).

Infrared spectroscopy has long been prized for its ability to measure secondary structures by probing the amide 1 band associated with stretching vibrations of the protein backbone. FTIR can measure more concentrated, clinically representative samples, and is particularly relevant for monoclonal antibodies because of its sensitivity to the β-sheet motif – a key feature of these clinically vital biologics. However, because of multiple limitations, it is reasonable to assert that FTIR is tolerated rather than loved by the industry… An inability to measure low-concentration samples without pretreatment, susceptibility to background drift, the need to separately collect buffer spectra (and the associated manual background subtraction), poor amenability to automation – all these issues hamper FTIR’s use. Clearly, there is a pressing need for more suitable technologies.

Enter microfluidic modulation spectroscopy (MMS). The superior performance of MMS rests on two core technical advances: a high-power quantum cascade laser and a microfluidic transmission cell. The laser enables measurement across a broad concentration range with no sample preparation. The microfluidic cell then modulates the sample with a relevant buffer to deliver automatic background subtraction in real time. MMS systems are also highly automated, with self-monitored cleaning routines and 96 well-plate compatibility. The bottom line: acquisition of sensitive and reproducible data, with substantially less effort.

Such merits explain considerable enthusiasm for MMS from the biopharma industry and point to a bright future within the biophysical characterization toolkit. Industry leaders highlight its ability to demonstrate “high accuracy, linearity, sensitivity, and reproducibility” and “to detect very small protein structural differences, enabling a level of characterization not achievable using conventional FTIR methods” (2). Whether as a replacement for FTIR, for orthogonal measurements of secondary structure, or ultimately as a primary characterization tool for protein products, MMS shows how the right analytical tools can easily find a loving home in the biopharmaceutical industry.

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  1. B Kendrick et al., “Determining spectroscopic quantitation limits for misfolded structures,” J Pharma Sci, 109 (2020). DOI: 10.1016/j.xphs.2019.09.004
  2. L Liu et al., “Automated, high-throughput infrared spectroscopy for secondary structure analysis of protein biopharmaceuticals,” J Pharma Sci [In Press] (2020). DOI: 10.1016/j.xphs.2020.07.030

About the Author

Jeff Zonderman

Chief Commercial Officer, RedShift BioAnalytics Inc., Greater Boston, MA, USA

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