Beyond the regulatory and compendial requirements for determination of subvisible particles in the > 10 micron and > 25 micron range, strong concerns have been raised recently about the micron-size particles being correlated to the potential risk of immunogenicity. FDA has become increasingly concerned with the safety of therapeutic proteins and has recommended that subvisible particles between 0.1-10 μm be monitored in protein formulations. As a result, a new USP monograph, Chapter <787> “Subvisible Particulate Matter in Therapeutic Protein Injections,” has become effective in 2014. In this new monograph, several improvements such as smaller particle size detection reporting and reduction of volume required for testing have been proposed. The small- volume light obscuration (LO) does not only require less product to perform the method, but also makes it possible to determine vial-to-vial or syringe-to-syringe variability of particle counts. In addition, the information Chapter <1787> “Measurement of Subvisible Particulate Matter in Therapeutic Protein Injections” recommends the measurement of 2-10 μm (≥ 2 μm and ≥ 5 μm) subvisible particle concentrations and the use of orthogonal methods to characterize these subvisible particles. It underlines the need to distinguish, and when possible, identify extrinsic (e.g. fibers) and intrinsic (e.g. silicone oil) particles from other inherent protein-containing particles.  In the same way, the Guideline on Development, Production, Characterisation and Specifications for Monoclonal Antibodies and Related products published by the EMEA has stated that “[the] formation of aggregates, subvisible and visible particulates in the drug product […] should be investigated and closely monitored on batch release and during stability studies. In addition to the pharmacopoeia test for particulate matter, other orthogonal analytical methods may be necessary to determine levels and nature of particles.” LO is the most widely used subvisible particle assay in the biotech industry and is the preferred compendial method (USP, EP, JP). It detects particles based on scattering of light by individual particles and provides particle size and counts, assuming spherical shape of particles. In addition to this compendial method, many techniques have been developed to characterize and identify these particles, which have become essential for data analysis and interpretation. Some examples of orthogonal analytical methods to LO for characterization of subvisible and submicron particles that are commonly used depending on particle size range and nature of information sought (qualitative versus quantitative), are: DLS/SLS, Nanoparticle Tracking Analysis (NTA), Flow-Imaging (FI) technologies, Electrical Sensing Zone (ESZ), Resonant Mass Measurement (RMM), Light and Electron Microscopy, while Fourier Transform Infrared (FTIR) Microscopy and Raman Microscopy can provide information on chemical identity of complex particles, secondary structure, particle distributions, images and morphology. 

With increasing concerns about immunogenicity, the FDA has approved a Guidance for Industry on the Immunogenicity Assessment for Therapeutic Protein Products that states “[assessment] should be made of the range and levels of subvisible particles (2-10 microns) present in therapeutic protein products initially and over the course of shelf-life”. As more and more methods become available, the FDA also recommends that “sponsors should strive to characterize particles in smaller (0.1 – 2 microns) size ranges. 

How did you combine biophysical methods in formulation development and what were the outcomes?

A number of chromatography, spectrophotometry and microscopy techniques providing different types of information on the higher order structure modifications and interactions between protein molecules are implemented. These are the so-called “orthogonal” techniques, which means techniques providing different, non-directly related, complementary information.  For instance, efficient combination of techniques and tools have been developed, such as using Raman spectroscopy and DLS techniques. These techniques provide information from a different point of view; for instance, Raman spectroscopy gives information on what happens within the protein in terms of modification of secondary or tertiary structures, while DLS gives information on the interactions between protein molecules in solution and the kinetics of aggregation. The challenges here are for instance how we select and implement relevant techniques, and get a representative result at an early development stage. Indeed, at an early development stage, there is a quite restricted amount of drug substance available, and then specific dilution or concentration conditions that are not necessarily significant and representative of what we will get for the final commercial formulation are used. The real challenge is to make sure that the methods used at the early development stage are relevant and the results stay valid as well, once you get to the full formulation development at the targeted concentration for commercial formulation. This is the reason why my recommendation is to combine techniques where you don’t have to significantly dilute the protein solution to perform the analysis and techniques that do not use too much material; for instance for the characterization of the interactions between proteins. In my opinion, these are the biggest challenges associated with anticipating protein aggregation.

In your opinion, why is there a need for and implementation of a set of orthogonal methods for successful QbD formulation development? 

In the field of drug product development, and more specifically for proteins, the industry has now clearly shifted their mindset from Quality by Control to Quality by Design. As for proteins, it means that once physical instability and aggregation have occurred in the formulation, it is already too late. Conversely, the industry wants to be able to anticipate and predict what the stability will be in key operations, like downstream processing, formulation, fill and finish operations, storage etc. There is a need to identify the design space of formulation components (nature and concentration) and process parameters for formulation stability with regards to the main parameters, such as pH, protein concentration, shear stress, shaking and so on, but also excipients that can be used to enhance physical and chemical stability. A Risk Assessment should be carried out based on anticipated degradation pathways and instability-triggering stresses (e.g. temperature, shear, shaking, oxygen, etc.) and definition of potential Critical Quality Attributes (pCQAs) and Critical Process Parameters (pCPPs). These attributes and parameters will then be confirmed or not as CQAs and CPPs during formulation development. Anticipating aggregation propensity means to select and implement appropriate analytical and characterization tools that make it possible to evaluate pCQAs and pCPPs in late-stage development, anticipate the aggregation of proteins and get a first draft design space that will, of course, need confirmation and consolidation later, in late-stage development. As you know, protein stability is a complex attribute, involving many different mechanisms and impacting so many different formulation parameters. Then, it gives us the real opportunity to implement the appropriate set of methods that will provide us with relevant and different types of information; the so-called orthogonal methods. I think the understanding of protein aggregation - what happens during protein aggregation, what mechanisms make protein aggregation happen - is getting better and better every day. This is related to the implementation of the combination of these orthogonal methods, and to the increasing use of a systematic QbD approach. 

What would you like to achieve by attending the 9th Annual Biologics Formulation Development and Drug Delivery Forum conference?

My goal in attending this conference is to have the opportunity to meet with strong, highly recognized experts in the field of protein formulation and stability, to have exciting discussions on the challenges for formulation of complex biologics, i.e. ADC, fusion proteins, bispecific Abs, and co-formulation of biologic therapeutics. I would also be interested in discussing the most recent regulatory recommendations and guidance on biologics, as well as the innovative approaches to biologic delivery, such as 3D printing microneedle arrays for transdermal drug delivery or alternative routes of delivery for biologic therapeutics (oral, nasal, etc.) and innovative medical devices.  The conference shows a really great scientific programme and expert speaker panel, so I am really excited by attending this major event in the field.