I recently took part in a question and answer session with BioPharm International about techniques for measuring and characterizing protein aggregates. The full discussion can be viewed here, however I thought I would also use this blog post to summarize the main points.
Essentially, there is a pressing need within the biopharmaceutical industry to understand aggregation pathways, and the risk factors that can induce aggregation, right from the start of the drug development process to align manufacturing processes with Quality by Design (QbD) principles. Given the link between protein aggregation and immunogenicity, companies are under ever increasing pressure from regulatory agencies to minimize immunogenic risk and better characterize any aggregated species in their products.
The FDA Guidance for Industry document “Immunogenicity Assessment for Therapeutic Protein Products” issued in August 2014 states that “Methods that individually or in combination enhance detection of protein aggregates should be employed to characterize distinct species of aggregates in a product”. The guidance also indicates that as more analytical methods become available, there should be a move to characterize particles in smaller (0.1–2 microns) size ranges. However, there are currently very few instruments that can provide quantitative sizing in this range, and there is no single instrument that can cover the full measurement range required for sub-visible particles. Consequently, different analytical approaches must be applied, each with their own distinct sizing range.
Methods such as Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS) are well-established within the biopharmaceutical industry. While both SEC and DLS provide crucial information about the state of a biotherapeutic, further characterization is required to satisfy the guidance document provided by the FDA. This additional characterization can be provided, at least in part, by the new additions to Malvern Instruments biopharmaceutical product portfolio. Nanoparticle Tracking Analysis (NTA) uses a high resolution digital camera and specially designed software to track the movement of particles under a microscope to determining hydrodynamic size, generating high-resolution particle size distribution and concentration. Resonant Mass Measurement (RMM) in the form of the Archimedes system, can detect and accurately count particles in the critically important size range 50 nm – 5 µm, and to reliably measure their buoyant mass, dry mass and size. Furthermore it can also distinguish between proteinaceous material and contaminants such as silicone oil by means of comparing their buoyant masses. Indeed, the identification of particulates and contaminants is of huge importance, as non-protein material can be introduced into products from many different bioprocessing steps and product contact surfaces. More detailed product characterization is provided by the Morphologi G3-ID system, which combined imaging with Raman spectroscopy to automatically measure particle size, shape and chemical identity from 1 µm to 1000 µm.
One of the newest analytical systems for protein characterization (Zetasizer Helix) combines industry-leading DLS technology, for high sensitivity aggregate sizing, with Raman spectroscopy, which allows monitoring of changes in secondary and tertiary protein structure. The combination of DLS and Raman spectroscopy enables measurement of protein size and structure from a single small volume sample, providing unique insights into protein folding, unfolding, aggregation, agglomeration and oligomerization. Ultimately, this can lead to identification of the degradation pathways that result in the formation of aggregates, and identify high risk processes and parameters. Such detailed information supports both the effective application of QbD and the efficient development of biosimilars.
Due to the complex nature of protein therapeutics, characterization tools that improve detection, understanding and/or progression of aggregation can greatly facilitate product development, process optimization and trouble-shooting. Increasing product understanding is central to minimizing immunogenic risk and improving product stability, which ultimately reduces the cost to manufacture and supply biotherapeutics.
Read the BioPharm article: Analyzing Protein Aggregation in Biopharmaceuticals
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