Malvern Instruments in the article details the solutions provided for each step along the path of biopharmaceutical development, from discovery to quality control
A number of unique properties make the development of effective protein therapeutics particularly challenging. An evolutionary trade-off between function and stability has led to most proteins being only slightly stable under ambient conditions, making storage problematic. Biotherapeutics tend to be administered directly into the blood stream, making it essential that impurities are removed or at least extensively characterised in order to minimise the potential of immunogenic reaction. The very process of injection itself has also been implicated as a cause of adverse patient reactions.
‘The pharmacologic activity of protein products can be evaluated by…binding assays, and enzyme kinetics’
FDA Guidance for industry 2012 – Scientific considerations in demonstrating biosimilarity to a reference product
Upon discovery of a potentially therapeutic molecule it is important to understand where the therapeutic properties come from in order to efficiently develop a high quality biopharmaceutical. For instance, the optimisation of the structure of the molecule in order to control the manner in which it binds to the target (a process known as lead optimisation) requires analysis of not only the affinity but also the mechanism of binding. Through measurement of the Binding affinity (Ka), enthalpy change (DH), stoichiometry (n), and Gibbs free energy (DG) and entropy change (DS) of an interaction, Malvern’s Microcal range of isothermal titration calorimeters allow intelligent lead optimisation through elucidation of the binding mechanism (Figures 1 and 2). Interactions are studied in a biologically relevant environment by measuring a universal property common to all binding processes, making the Microcal iTC200 not only a highly effective drug discovery tool, but also an ideal means of demonstrating comparability or batch quality.
Formulation and stability
‘Excipients should be evaluated for their potential to prevent denaturation and degradation of therapeutic protein products during storage’
‘Some antibodies generated by aggregates containing native protein can inhibit or neutralize product activity. In contrast, some antibodies to denatured/ degraded protein…cause anaphylaxis, but do not inhibit or neutralise activity of the native protein’
FDA guidance for industry 2014 – Immunogenicity assessment for therapeutic protein products
After discovery and lead optimisation begins the formulation process, with preformulation screening allowing the detection of any potential barriers that may crop up during the development of a lead candidate, minimising the risk of developing a drug that cannot be successfully marketed.
As a screening step, preformulation requires automatable analysis. The Malvern Viscosizer 200 allows automated viscosity analysis of formulations with protein concentrations up to and above 300 mg/ml. Viscosity is calculated as a function of the speed of a sample moving through a microcapillary (similar in diameter to a hypodermic needle), measured using two spectrophotometric detection windows. Viscosity data from the Viscosizer 200 is used to establish injectability and processability; if the viscosity of a formulation is too high for it to be injected into a patient, it must either be developed further or dropped – however, stable the formulation is. Likewise, however, an easily injectable biopharmaceutical with low stability is also of limited marketability, and the same dual-detection set-up that allows viscosity analysis also allows monitoring of size by taylor dispersion analysis (TDA). As an absorbance-based method, TDA can be used to assess protein in complex formulations. The Viscosizer 200 therefore gives a truly orthogonal means of formulation analysis.
The gold standard for formulation stability screening is differential scanning calorimetry (DSC). 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 (Figure 3). 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 measurements (Figure 4). Detailed spectra allow analysis of tertiary structure and quantification of secondary structure changes. The light scattering function, in addition to aggregation analysis also allows stability prediction through measurement of virial coefficients and zeta potential. 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.
‘Subvisible particulates in the size range of 0.1–10 microns have a strong potential to be immunogenic’
‘All therapeutic protein products should be evaluated for their content of…proteins and nonprotein components’
FDA Guidance for Industry 2014 – Immunogenicity Assessment for Therapeutic Protein Products
Subvisible particles (generally defined as being from 0.1 – 10 µm in size) are currently the subject of much regulatory scrutiny. Since immunogenicity is dependent not only on particle size, but also on morphology, composition and concentration, regulatory authorities are increasingly advocating an orthogonal analytical approach to characterising subvisible particles.
NanoSight Nanoparticle Tracking Analysis (NTA) uses an advanced video camera to track and size subvisible particles individually but simultaneously using their light scattering signal (Figure 5). Hydrodynamic size is calculated from the Brownian diffusion speed of the particles, with resolution of individual particles allowing concentration calculation. Measurement of the scattering intensity of each particle allows differentiation of particles of different compositions in complex mixtures. The particles can also be tracked using their fluorescence signal, with fluorescence presenting another means of compositional differentiation. The ability to monitor individual particles allows the generation of high resolution size profiles without fractionation, whilst visual validation gives confidence in any process optimisation that such data may inspire.
Archimedes Resonant Mass Measurement (RMM) also presents a means of accurately measuring subvisible particle concentration and size, as well as differentiating between different materials. Sample flows through a cantilever resonating with a specific frequency, with particles causing a change in the resonant frequency based on their buoyant mass. Size can be calculated from the magnitude of the change in frequency, with concentration being directly measured from the number of such frequency changes. Particles are identified based on their density, with the differentiation of subvisible protein aggregates and silicon droplets in pre-filled syringes being an excellent example of the power of Archimedes (Figure 6).
The importance of morphology to particle immunogenicity is becoming increasingly understood as the options for analysing this parameter improve. Microscopy can be used to assess the morphology of sub-visible and visible particles, though automation is essential for such characterisation to be performed efficiently and without operator bias. The Morphologi G3-ID system (Figure 7) allows a range of morphological parameters to be assessed in a hands-off manner using static image analysis, with both the collection and analysis of data being completely automated. The Morphologi G3-ID also comes fitted with a Raman spectroscope, with the capability of acquiring Raman spectra of individual particles. Comparison with a robust spectral library (incorporated within the Morphologi G3-ID software) containing Raman spectra of common biopharmaceutical contaminants such as silicon, cellulose and rubber, allows unequivocal chemical identification of each particle measured.
Quality control and comparability
‘Methods that individually or in combination enhance detection of protein aggregates should be employed to characterise distinct species of aggregates in a product’
FDA Guidance for industry 2014 – Immunogenicity assessment for therapeutic protein products
The problem of proving quality or comparability requires a simple, robust and highly sensitive solution. Since protein aggregates and other large particles are possibly immunogenic, and that in many cases protein’s aggregate upon loss of function, sensitive analysis of aggregates and impurities gives an excellent means of assessing quality. Since larger molecules scatter more light than smaller molecules, light scattering methods are an ideal means of performing such analysis.
Size exclusion chromatography (SEC), such as that performed by Malvern’s Viscotek systems, allows high-resolution aggregate analysis by separating monomer and small oligomers (dimer, trimer etc.), with the use of light scattering detectors to analyse the eluent giving the best chance of detecting such aggregates. Malvern’s SEC-MALS 20 multi angle light scattering detector uses 20 light scattering detectors arranged around a vertical flow cell to calculate absolute molecular weight without the need for column calibration (Figure 8). In addition to working in conjunction with Malvern’s Viscotek SEC systems, the MALS 20 detector is also compatible with third party SEC systems. The simplicity and high resolution of SEC explains why its use is advocated for aggregate analysis in ICH Topic Q6B.
Dynamic light scattering (DLS) measurements performed using Malvern Zetasizer systems give a highly sensitive means of assessing aggregates and impurities without fractionation. Although, relative to SEC, DLS is a low resolution technique, the simple cuvette-based nature of a Zetasizer measurement explains the wide usage of the instrument alongside SEC-MALS as an orthogonal biopharmaceutical characterisation strategy.
The Malvern Zetasizer range allows sizing over a very wide range (from 0.0003 – 10 µm), and includes the APS system (Figure 9), an automated plate sampler capable of performing efficient QC and comparability analysis on large numbers of samples.
Orthogonal characterisation from discovery to batch release
Though biopharmaceuticals have obvious advantages over chemically synthesised drugs, including high potency and fewer side effects, protein therapeutic development involves a number of novel problems. Malvern Instruments provides a solution for every stage of the biopharma development process, from lead optimisation and formulation development to proof of quality and comparability.