Biotherapeutic Protein Stability Screening with the AQS3pro
Sensitively Measure Protein Conformational Change, Under Formulation Conditions To Understand And Control Protein Aggregation
A detailed understanding of the mechanisms of protein aggregation is essential to control stability and ensure a safe, effective drug product. Developing this understanding is a primary motivation in formulation, which is driven by high throughput analysis and intense information gathering.
Formulation scientists use a core set of analytical techniques to quantify the colloidal, chemical and conformational stability parameters that define the stability of a biotherapeutic. However, this is a toolset with wisely recognized gaps, notably an inability to measure protein conformational difference with high reproducibility in clinically representative formulations.
The AQS3pro is a Microfluidic Modulation Spectroscopy analytical technique commercialized for measuring the secondary structure of proteins. It offers:
- The ability to measure across four decades of concentration and characterize clinically representative formulations without dilution or preparation.
- High sensitivity. Repeatability and reproducibility in excess of 99% for most samples allow the detection of even minor changes in conformational structure.
- Fully automated measurement and data handling for seamless integration with existing formulation workflows.
The AQS3pro capabilities have the potential to accelerate and enhance formulation by supporting the development of a clear understanding of the impact of formulation conditions on protein structure.
In combination, colloidal, chemical and conformational stability parameters define the stability of a biotherapeutic formulation. The analytical techniques that elucidate colloidal and chemical stability are relatively well-established:
- Traditional light scattering techniques – dynamic light scattering and static light scattering – are the ‘go to’ choice for measuring many colloidal properties.
- Mass spectrometry and capillary electrophoresis are well-matched to investigations of chemical stability.
In contrast, incumbent solutions for the detection and elucidation of conformational change are less than optimal.
For example, techniques such as differential scanning calorimetry and differential scanning fluorescence provide evidence of unfolding and global structural information, giving insight into the result of the aggregation process, but not the associated pathway. The inability of these techniques to provide information relating to secondary and tertiary structure means that they cannot directly elucidate conformational change.
The AQS3pro fills the gap for an efficient tool for measuring conformational stability.
Identify a better biotherapeutic formulation, faster with the AQS3pro
For formulation scientists the defining features of this technique/system include:
Observe Higher Order Structure in Excipient Screening
Obtaining protein secondary structure using near-UV CD spectroscopy becomes difficult once you introduce excipients or additives to a protein formulation. Excipients such as surfactants commonly have strong signals in a CD spectrum, making structural measurements by CD extremely difficult. Understanding the impact of excipients on protein secondary structure has to date been a challenging measurement to obtain for this reason.
To understand the effect of Polysorbate 80 on secondary structure, monoclonal antibody (5mg/mL) provided by Amgen was formulated with varying concentrations of polysorbate 80 (PS80) from 0.01 % to 0.1 % w/v. Analysis of the second derivative spectra and higher-order structure composition can be seen in the figure above.
Using the spectrum of the 0.01% sample as a reference, the similarity of the triplicate spectra obtained at each concentration was 99.6% for both 0.05% and 0.1% PS80 samples.
Statistical analysis of the higher-order structural data shown above (right) indicated less than 0.2% variation in β‑sheet content, and less than 0.1% variation in turn, unordered and α-helix content across the entire sample set. This data provides strong evidence that the addition of PS80 does not cause a change in the secondary structure of the antibody.
Reproducibility, Linearity, and HOS
Dilution of IgG in Buffer
(left) overlay of second derivative data of mAb formulated with 0.01, 0.05 and 0.1% PS80. (right) summary of HOS data obtained from mAb formulated with PS80
AQS3pro For Analyzing Protein Secondary Structure And Stability In Formulation Studies
The AQS3pro is a Microfluidic Modulation Spectroscopy analytical technique that fills a recognized gap in the existing biopharmaceutical analytical toolkit. AQS3pro enables the precise measurement of protein secondary structure throughout the drug development pipeline.
Take another look at what the AQS3pro has to offer
For formulation scientists, the AQS3pro offers the opportunity to:
- Add rapid, routine, repeatable measurement of the secondary structure of proteins to the analytical armory for formulation
- Directly and clearly measure conformational change and stability, rather than infer it
- Detect change with a sensitivity and clarity beyond that of alternative techniques, in clinically representative formulations