Overcoming a development challenge: Characterising aggregated material in formulated product

Dr Michael Caves, India Business Development Manager, Malvern Instruments and Dr Namrata Jain, Product Technical Specialist — Nanometrics Bioscience, Malvern Instruments in this article give an insight on the use of two novel instruments for subvisible particle characterisation and viscosity screening of bioformulations

Therapeutic antibodies represent one of the fastest growing sectors for novel biopharmaceuticals, necessitating development of new analytical platforms for faster and better antibody selection and characterisation. Early quality control and risk assessment of biophysical parameters such as aggregation, viscosity and stability can help prevent failure in the later stages of development, and thus can reduce costs and save time. Malvern Instruments provides a growing range of solutions focused specifically on critical path of testing and manufacturing requirements, which take into account the pressures of time and cost, to speed you through characterisation studies, formulation development and process development, through to commercial manufacture. We report on the use of two novel instruments from Malvern biotherapeutic development initiative for subvisible particle characterisation and viscosity screening of bioformulations.

The solution

Characterisation of subvisible particles in biotherapeutics has been the subject of intense interest due to concerns regarding immunogenic responses that compromise drug safety and efficacy. It is therefore essential to monitor aggregation in the development, manufacture and storage of therapeutic proteins. Subvisible aggregates are coming under increasing regulatory scrutiny, but are often inaccessible to optical methods due to limits on resolution. Protein formulations are also often complicated by the presence of leachables such as silicone oil droplets and other foreign particulate matter introduced during product manufacture, handling and storage. An ideal analysis method for biopharma formulations would therefore clearly distinguish contaminating matter from the aggregate content.

Archimedes is an innovative, high-performance system which harnesses the technique of resonant mass measurement in order to count particles in the size range 50nm – 5µm and to measure particle mass and size. The system is also capable of providing information on sample concentration, density and volume, and distinguishing between negatively buoyant proteinaceous particles and positively buoyant contaminant such as silicone oil (Figure 1), fulfilling the regulatory demand for differentiation of protein and non-protein particles. The system is based on a MEMS (Micro Electro-Mechanical Systems) technology sensor, allowing sensitive, reliable and reproducible measurement of aggregates one by one.

Even with the current regulatory concern over sub-visible particles we must maintain our focus on smaller aggregates also. Many current aggregate sizing techniques (such as those that rely on light scattering) are not selective enough to fully characterise complex formulations. Commonly used biopharma excipients such as arginine, sucrose and PEG can all interfere with light scattering measurements. The early stages of biopharma formulation development also require work with small amounts of expensive sample.

Malvern’s Viscosizer TD uses an absorbance-based, selective sizing method (Taylor Dispersion Analysis) to characterise aggregates without interference from formulation components or bias towards larger particulates. Taylor Dispersion Analysis with UV absorbance area imaging of microcapillary flow enables ultra-low volume analysis, each measurement typically consuming only 40 nl of sample. Viscosizer TD uses fully automated methodology to analyse the stability of peptides and proteins in formulation, and is selective enough even for analysis of small molecule drug size.

Screening for stability allows selection of the most stable formulations for further studies (such as clinical trials), and the Viscosizer also affords another means of formulation screening. By measuring the speed at which sample flows through the microcapillary (using exactly the same hardware as for the sizing measurement) relative viscosity can be calculated according to Poiseuille’s law. Viscosity issues often accompany parenteral administration. High viscosities can be unsuitable for injection and its discovery at later stage of bioformulation development have significant cost implications. Viscosizer TD can accurately and reproducibly discern differences between the relative viscosities of different protein formulations at low concentrations during the early stage of their production and therefore identify abnormal viscosity concentration profiles (Figure 2).

Proof of concept

This article further describes monoclonal antibodies formulation study using the two technologies. Figure 3 shows quantification of sub-visible particles using RMM pre and post syringe-induced shear stress. Prior to syringe stress, the number of particles detected by the Archimedes system is very low, demonstrating the sample is reasonably pure, with very few large protein aggregates. This is to be expected, as the sample had been filtered. However, this does demonstrate the low noise baseline of the technique, enabling accurate analysis of pure samples. Following shear-stress the number of particles detected increases significantly, with particle sizes ranging from 500 nm up to 1700 nm. The particle size distribution provides important data regarding the response to the type of stress induced. Such data can be used to compare different stress conditions to provide an overall picture of the degradation profile for the biopharma of interest.

Figure 4 shows the variation in self-association and oligomeric state of Insulin in a selection of different formulation buffers as measured by TDA. These data follow the same trends that are well characterised within the literature. Monomeric and Dimeric Insulin are accurately characterised, as is mixture of insulin oligomeric states, without bias towards larger aggregates or interference from formulation components. Such mass-based sizing can be used to fully characterise the different oligomeric states of important hormone and antibody drugs.

The malvern orthogonal approach

The complexities involved in biotherapeutic development (aggregation for instance) make an orthogonal approach to characterisation essential. This article outlines two of the many solutions Malvern offers for the challenge of biopharma development, focussing on both large and small aggregates. Together with our customers, we have created a suite of instruments which provide access to a raft of biophysical information on your product, to speed you through characterisation studies, formulation development, quality control and beyond.