Changing the Way We Measure ‘Undetectable’ Differences in Protein Structure

How do you resolve an issue in a product when your analytical methods fail to indicate any difference between batches? Consider the ramifications should your pharmaceutical product cause harm despite your best efforts of your procedures, not to mention the need to analyse in non-native conditions. Most basic assessments cannot delineate conformational effects from colloidal effects . 

Making complex processes simple

Biophysical characterisation of proteins can be a daunting task, given the number of methods required to elucidate the results for Aggregation, Quantitation, Stability, Similarity and Structure. RedShift Bio’s AQS³ pro, powered by Microfluidic Modulation Spectroscopy (MMS), can perform these 5 measurements in a single analysis which is impressive. Sensitivity is improved up to 30 times that of conventional FT/IR instrumentation with a desirable concentration range of 0.1mg/ml to over 200mg/ml and with protein analytics that is equally enviable to the operator. Savings in sample testing time can be more than 80% thanks to the fully automated multi-sample capability.

Often the buffers used in a formulation are not compatible for the analytical method, as seen with spectropolarimetry. The AQS³ pro experiences no interference from excipients in the buffer. This is a game changer, to measure at concentration, and in the final drug conditions, removing the guesswork from formulation and de-risking many steps.

What is the magic behind the AQS³ Pro system?

There are 3 key components in the AQS³ pro that allow the system to achieve all this and separates it from all other systems,

1. a mid IR tuneable quantum cascade laser
2. a thermal, electrically cooled detector
3. a Y shaped microfluidic transmission cell. 

The Tuneable laser provides an optical signal almost 100X brighter than the conventional light source used in FT/IR allowing the use of a simple thermal electrically cooled detector without the need for liquid nitrogen cooling. Given the intensity of the laser, the system is amenable to low concentration samples as low 0.1mg/ml. The measurement differs from the conventional as well. The sample and the reference stream are injected alternately through the Y shaped microfluidic cell passing through the observation zone. Alternating at a rate of 1 – 5hz, the absorbance of the reference and sample are measured almost simultaneously allowing the reference absorbance to be subtracted from the sample absorbance in real time resulting in the collection of reference corrected absorbance spectra. Such a real time buffer subtraction and auto-referencing greatly enhances the sensitivity method and produces an almost drift free signal. 

These innovations create the 30X sensitivity boost. The speed of the AQS³ pro impresses. Where it may take 30 mins per sample for the FT/IR, the AQS³Pro system with MMS technology can measure samples in less than 1.5min. The AQS³ delta analytics packages are refreshingly simple and intuitive, applying advanced analytical tools enabling its use in a vast range of applications across the industry.

What does the AQS³ pro actually measure?

This is an infrared system that measures the Amide 1 band of a protein which is in the wavenumber range of about 1580 – 1720. It takes about 32 data points across the range alternating between buffer and sample, plotting a differential absorbance which is interpolated into a spectrum, which in turn is converted to a second derivative affording fine detail changes from spectrum to spectrum to be elucidated. Flipping the spectra shows the baselines and area of overlap plot enables you to look at similarity and trends between spectra. This can then be deconvoluted using a normal gaussian deconvolution, giving rise to secondary structure motifs, the percentage of the components such as α-Helix, β-Sheet, and Anti-parallel β-Sheet Higher Order Structures (HOS).

Case study: complexity of a vaccine.

The diverse composition of a vaccine makes biophysical characterisation challenging. Not only does a vaccine have antigens and antibodies, they can also have an array of excipients such as preservatives, stabilisers, and buffers plus they can contain antibiotics. The concentrations of the components vary by orders of magnitude and the particle size distribution of a final formulation can range from nano to micron. 

The additional ingredients can introduce protein and non-protein, organic and inorganic materials. If you add up the numbers of tests and instrumentation required to perform these tests it can be incredibly diverse. MMS is a single technology that provides unique insights into many of the parameters required to fully understand these biophysical properties of the sample, from looking at interaction effects of antigen and adjuvant or the effect of looking at varying stabiliser concentrations or altering the buffer pH. All these steps can be performed to understand their effect on the protein secondary structure.

The total characterisation of a protein-based vaccine should include:

Biophysical Characterisation to understand Protein antigen properties such as pH, ionic strength, HOS, and aggregation propensity.

Stabiliser Screening These convey stability by addition of amino acids, surfactants, proteins, sugars and antioxidants to improve shelf life.

Adjuvant Screening Adjuvant surface chemistry (eg alum) as well as adjuvant-antigen interactions should be characterised.

Process Design & Control Process design and vaccine-surface interactions of process equipment should be understood – ie protein loss on filtration substrates as well as denaturation of protein samples.

Stability Studies Real time and accelerated stability studies ensure antigen is in chemically and physically stable state

With this in mind, let’s look at the tools and parameters needed for such biophysical characterisation of protein antigens; from molecular weight in a chromatography system to surface charge on an electrophoretic light scattering system. A few are listed below.

LC – HPLC and SEC Liquid Chromatography in the form of reverse phase, ion exchange, size exclusion can be used to assess chemical and physical stability.

DSC- Differential scanning Calorimetry Thermal stability properties of antigen for insights into formulation conditions.

MALDI – Maldi TOF  High resolution molecular weight providing information about primary structure and post translational modifications.

ELS – Electrophoretic Light Scattering – Surface charge of pure adjuvant versus adjuvant in formulation will identify protein adjuvant interactions.

DLS – Dynamic Light Scattering Particle size distribution of antigen, adjuvant and complex mixtures, Colloidal stability parameters KD and B22

It is important to note – all these parameters need to be measured at some point during development of a protein vaccine.

Where does MMS fit into the development of vaccines? 

Given MMS measures protein secondary structure, we can leverage this capability to measure in final formulation conditions to assess HOS as stabilisers are varied to determine if they impart a stabilising effect and prevent aggregation processes. The AQS³ pro could be used for adjuvant screening, investigating the interaction and to determine if it is damaging to the antigen. Process design can be studied to establish, for example, if a filtration membrane causes a negative effect on the structure of the antigen, or to understand the stability of the formulation using long-term stability studies. It is now clear that MMS can be applied across the entire development pipeline with a wide range of measurements to help understand the full properties of protein-based vaccines.

De-Risk Drug Development

MMS’s sensitive, accurate measurements coupled with a robust data analysis package provides simple, accessible, and reliable results to de-risk and accelerate drug development workflows. The AQS³ pro can identify at-risk candidates far earlier than traditional methods, clearly saving time and resources.

ATA Scientific are proud to have the RedShiftBio AQS³ pro within our suite of instrumentation. Should you require further information on the AQS³ pro or indeed many of the techniques cited above, please do not hesitate to contact us.