Dr Michael Caves, India Business Development Manager, Malvern Instruments, elaborates on optimising the efficiency and quality of biotherapeutic formulation
Most biomolecules are only slightly stable under the ambient conditions that they have evolved to work under. Since the living entities that create these natural molecules require them to be broken down quickly when they are not needed, it is very important that this is the case. However, when we are designing biopharmaceuticals and vaccines to be stored over long periods of time we must ensure their stability without having a major effect on the inherent structure (and therefore efficacy) of the molecule itself. We do this through formulation.
Though protein formulation is a well-studied field, stretching back into the early 20th century, understanding of how biotherapeutics are affected by various excipients is still relatively unpredictable. Arginine, a much-used inhibitor of protein aggregation, for instance, is known to have a deleterious effect on the structural stability of certain proteins. Whatever the nature of the biomolecule (hormone, mab, vaccine, enzyme etc.) being developed it is important that the stability of the intended formulation is fully understood prior to clinical trials and scale-up, at which point the quantity of sample available for testing may be very low and the number of formulations requiring analysis very high. Stability studies are also required for biocomparability studies, regulatory approval and batch release, studies that require accurate and repeatable measurements to be made.
Preformulation
With this in mind, Malvern offer an orthogonal approach to stability studies, with different methods targeted at different stages of the development pathway. The Viscosizer TD, for instance, is used early in the preformulation stage where the number of samples that need analysing are large and the sample volume available is often low. Innovative microcapillary technology allows viscosity and Taylor Dispersion Analysis. Viscosity analysis gives assessment of the injectability of the drug, in addition to stability(1), whilst Taylor Dispersion Analysis gives direct measurement of the presence of aggregates (through absorbance-based size analysis). Perhaps most importantly, Taylor Dispersion Analysis also measures the dynamic virial coefficient of a protein formulation, a measure of the propensity of a formulated protein to aggregate(2). The Viscosizer TD therefore presents an ideal means of high-throughput analysis of large pre-formulation matrices under storage conditions (measurements can be made at temperatures from 4 – 40 °C) using small volumes of precious sample (only 40 nl of sample is consumed by Taylor Dispersion Analysis). High measurement repeatability and accuracy also makes the technique valuable to biocomparability, with the absorbance-based nature of the measurements eliminating the interference from formulation components and large particulates often seen with light scattering methods.
Refining your formulation
Later on in the development pathway, where more sample is available and a direct measure of thermal stability is required, Differential Scanning Calorimetry (DSC) is the gold standard. The technique involves ramping the temperature of a formulation in order to measure the denaturation of the biotherapeutic at elevated temperature. DSC is easily automatable, requires no sample modification and measures thermodynamic change – a universal property common to all protein conformational changes. Malvern’s Microcal VP-Capillary DSC system performs comprehensive stability characterisation for 50-plus samples per day, with use of a tantalum cell eliminating interactions between the formulation and the cell and maximising the signal-to-noise ratio. Malvern’s Microcal DSC range gives an ideal means of stability-based formulation screening, allowing efficient development of stable formulations. The information-rich nature of Microcal DSC measurements makes the technology ideal to biocomparability testing also, particularly for complex biomolecules like mabs. Each domain of such proteins gives a signal, creating a thermal stability fingerprint that is ideal for assessing the similarity of, for instance, biosimilar formulations to the innovative medicine.
Understanding your formulation
The necessity of understanding the causes of them is underlined by the increasing implementation of Quality by Design (QbD) precepts throughout the industry. With QbD in mind, Malvern has combined Dynamic Light Scattering (DLS) and Raman Spectroscopy, giving a comprehensive assessment of both aggregation and conformational changes in a single instrument – Zetasizer Helix. Since both measurement types are performed in an interleaved manner on the same sample, the instrument is ideal for performing kinetics measurement. Detailed spectra allow analysis of tertiary structure and quantification of secondary structure changes. Zetasizer Helix provides a comprehensive understanding of the causes of biopharmaceutical instability, allowing intelligent formulation design and increasing the quality and efficiency of biotherapeutic development.
Summary
The complexity of proteins and the unpredictability of the effect of excipients on their stability makes an orthogonal approach to stability-testing essential to preformulation, formulation and biocomparability studies. Viscosizer TD detects early aggregate formulation and measures the propensity of a formulated protein to aggregate under ambient conditions (using extremely low quantities of sample), whereas Microcal DSC asses the structural stability of proteins during thermal stress. Along with the unique information provided by Zetasizer Helix, these techniques give an ideal platform for formulation characterisation at every stage of biotherapeutic development, from discovery to batch release.
References:
1. MAbs. 2015 Jan-Feb; 7(1): 77–83
2. Anal. Methods, 2016, 8: 386-392
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