Keeping Aggregation Under Control

Characterization of protein aggregates is a vital aspect of biopharmaceutical development. Early on in the development cycle, protein instability, caused by aggregation or degradation, can have a detrimental effect on the immunogenicity and efficacy of the biopharmaceutical product. During the later stages, developers are required to control protein stability in a continuously evolving and complex environment to minimize potential issues.

To learn more about protein aggregation, including risk factors, importance of addressing the formation of aggregates, available techniques and their limitations, specific containment considerations, and regulatory guidance on the issue, BioPharm International spoke with Matthew Brown, bioscience applications manager, at Malvern Panalytical.

Causes and risk factors

BioPharm: Could you run through some of the main causes and risk factors of aggregation in biologics?

Brown (Malvern Panalytical): There is a tendency to view protein aggregation as occurring via one of two mechanisms; colloidal interactions and perturbation of higher order structure. Colloidal interactions describe how individual protein monomers behave as they come into contact and are driven by the effective charge at the surface of the protein in the buffer. If protein monomers tend to remain attached as they come into contact, they form reversible, native aggregates, with higher order structure (HOS) maintained. However, if they remain attached over an extended period of time, the proteins start to spread, disturbing the higher order structure and eventually becoming irreversible aggregates. Importantly, colloidal interactions are driven by the charge of the protein itself, but also the formulation components, allowing a degree of formulation optimization to identify favorable conditions. Colloidal interactions are typically analyzed using the light scattering derived parameters B22 and kD. 

Perturbation of HOS can expose hydrophobic patches from the protein interior, causing irreversible association and the formation of irreversible aggregates. Although such irreversible aggregates are often induced in response to some type of stress, such as temperature, pH, or interaction with interfaces, the formulation composition can influence the integrity of the HOS and, therefore, how quickly the stress can disrupt the structure. This is where formulation optimization can help identify the best excipients to take forward. 

Importantly, these two pathways to the formation of aggregates are not mutually exclusive. As described above, the stabilization of native aggregates can cause disruption of the HOS and, therefore, structural instability and the formation of irreversible aggregates. Similarly, once a protein starts to unfold, how quickly it aggregates will be determined by the colloidal behavior of the denatured intermediate. 

Once we define the causes of aggregation as either colloidal or structural, we can start to identify risk factors. The key risk factors for the formation of native, reversible aggregates are low effective charge, typically caused by high salt concentration and/or pH close to the isoelectric point, high protein concentration, and storage time. For structural instability, the risk factors would include physical conditions such as temperature and pH, and handling effects such as agitation, shaking, and freeze thaw.

Importance of addressing aggregation

BioPharm: Why is it important to identify and address aggregation in biologics?

Brown (Malvern Panalytical): The main driver for addressing the formation of protein aggregates is immunogenic risk; the likelihood of the host immune system raising anti-drug antibodies (ADAs) against the administered therapeutics. This response can cause the therapeutic product to become less effective and less efficacious, and in the cases where the protein is being used to supplement endogenous protein, can cause an autoimmune response with additional clinical complications. While the mechanism driving ADA formation is still being studied, there is a growing body to evidence to suggest that large protein particles with repeating structures on the surface are the most immunogenic, due to structural similarities to viruses. 

Of course, from the perspective of a biopharmaceutical company, the driver is commercial; ensuring product can satisfy the release specifications and can be released to market. Situations where atypical or unusual levels of protein aggregation are present in products can lead to product recalls or product monitoring programs, which are hugely expensive.

Available techniques, limitations, and potential improvements

BioPharm: What techniques are currently available to address or control aggregation?

Brown (Malvern Panalytical): Aggregation issues are typically controlled through predicting the propensity to aggregate and quantifying aggregate levels during, or after some type of stress (e.g., temperature, storage, agitation, etc.). As mentioned earlier, colloidal stability is typically predicted using light scattering techniques, with kD derived from dynamic light scattering and B22 derived from static light scattering. Structural stability prediction is often performed using thermal ramp methods, such as differential scanning calorimetry, differential scanning fluorimetry, or spectroscopy-based technologies such as circular dichroism. Used together, these techniques allow optimization of candidates and formulation excipients to minimize aggregation issues later in the development process. 

Quantification of aggregates is performed using size-exclusion chromatography (SEC) for soluble aggregates (below 200 nm), while particle analysis is performed to quantify much larger particles, typically above one micron.

If aggregation problems start to materialize later in the development pipeline, it is important to identify the source of the instability and modify the formulation or the processing conditions to minimize the issues. For example, the use of microscopy combined with spectroscopy, such as morphologically directed Raman spectroscopy, allows determination of particle identity, and so can be very beneficial for root cause analysis studies.

BioPharm: Are there specific limitations to the aforementioned techniques, and if so, are there ways of overcoming these limitations?

Brown (Malvern Panalytical): It is important to appreciate all techniques have their strong points as well as their limitations. This is one of the reasons there is a growing need to utilize orthogonal and complementary approaches towards any protein characterization method. For example, although SEC is a standard tool to quantify levels of soluble aggregates, the method conditions, such as sample dilution, mobile phase (if different from formulation), and association with the stationary phase, mean sometimes SEC data needs to be verified with analytical ultracentrifugation. SEC will also only quantify aggregates that are below typically 200 nm, as larger particles are filtered out by the guard column. Consequently, relying solely on such data can result in misleading interpretations. To provide a more complete picture of the aggregate state of protein products, SEC should be combined with particle analysis tools, to provide particle concentration from submicron size ranges all the way up to visible particles.  

It is also important to place protein stability into context. The mechanisms leading to aggregation will depend on the stress inducer, and therefore different technologies can inform stability profiling under different circumstances. As described above, colloidal parameters can predict shelf life and higher concentration stability whereas structural parameters can inform resistance to stress, and so the combination of both screening types can improve the successful selection of candidates and formulations.

BioPharm:Are there any new capabilities in development to help address aggregation in biologics?

Brown (Malvern Panalytical):The bio/pharma industry can sometimes be cautious to adopt new technologies until they have been proven in the field or are forced upon them by the regulatory agencies. However, given the impact protein instability can have on biotherapeutic development and commercialization, new capabilities in this space will always generate interest. One recent development in light scattering has been multi-angle dynamic light scattering (MADLS). This advancement on traditional DLS combines data from three angles to produce a higher resolution size distribution, allowing a more robust assessment of aggregation state for protein-based biopharmaceutical products. Perhaps most importantly, this technique allows calculation of particle concentration, something that would be impossible with conventional DLS. This ability is particularly important for more complex therapeutics, such as Liposomes, viral vectors (e.g., adeno-associated viruses) and other drug delivery vehicles.  

Considering containment

BioPharm: Could you discuss the specific considerations of containment systems for biologics and any associated aggregation risks?

Brown (Malvern Panalytical): The container closure system used for any particular product tends to be driven by the commercial requirements rather than considerations around protein stability. If the product is intended to be a self-administered therapeutic, it will require autoinjector pens or pre-filled syringes, while intravenous infusion systems utilize vials of product, often requiring reconstitution prior to administration. 

Irrespective of the container system used, products are typically in contact with glass, rubber stoppers and silicone oil. This last component could be of interest, not so much in terms of silicone oil being unsafe, but in terms of distinguishing between silicone oil droplets and protein aggregates. From an immunogenic perspective, silicone oil is of minimal concern, while large protein particles can be particularly immunogenic. Therefore, being able to identify particles as either proteinaceous or silicone oil allows a more complete assessment of safety and immunogenic risk. Currently, resonant mass measurement and microfluidic flow imaging are two techniques that allow silicone oil droplets to be distinguished from protein particles. 

Apart from possibly being mistaken for protein particles, silicone oil is one example of another aspect of container closure that can play a role in protein stability; interfaces. Interfaces between aqueous and non-aqueous phases can act as sites for the stabilization of denatured intermediates, ultimately leading to the formation of irreversible, denatured aggregates. While silicone oil droplets can serve as such an interface, a far more problematic interface is the air-water interface. The presence of air bubbles or headspaces in the container closure can have a dramatic impact on protein stability, causing a significant increase in the aggregation rate. This can be particularly problematic when combined with agitation and shaking. The effect of interfaces can be reduced through the addition of surfactants to the formulation.

Regulatory requirements

BioPharm: Could you highlight some important regulatory requirements in terms of aggregation and ways in which companies can adhere to these requirements?

Brown (Malvern Panalytical): FDA is clear on the requirements for aggregation quantification within specific sizing ranges. Below around 200 nm, where SEC is the industry standard for the quantification of soluble aggregates, companies must specify and quantify the amount on monomeric protein and the amount of high molecular weight species. All biopharmaceutical products being released to market must have a validated SEC released assay as part of quality control (QC) testing. 

For particles above 10 µm, companies must adhere to United States Pharmacopeia (USP) <788>, which specifies the number of particles permitted above 10 µm and above 25 µm. For this testing, light obscuration is the standard technology used, and is specifically named in the USP documentation. Above this size range, all products are subject to visual inspection to detect visible particles, typically above 100 µm. Products should be essentially free of visible particles. 

In the sizing gap between 200 nm and 10 µm, there are no specific regulatory specifications that should be met, although companies have been provided with recommendations from FDA. In the sizing range from 200 nm to 2 µm, companies are recommended to characterize and control the number of particles present in their products. In this sizing range, the available technologies for analysis are nanoparticle tracking analysis, resonant mass measurement, and microfluidic imaging, although each covers a different sizing range. Between 2 µm and 10 µm, companies are encouraged to quantitate and control particle concentration, with microfluidic imaging being the main technology used in this sizing range. 

While the quantification of particles within parenteral products is an important part of controlling and assessing aggregation and product safety, another important aspect is chemically identifying the particles, and therefore their source. Recently, there have been reports of companies being issued warning letters from regulatory agencies for not identifying the source of particles present in their products, and so there is a growing interest in using tools to identifying the chemical composition of particles. Currently, technologies that can satisfy this requirement utilize microscopy coupled with Raman spectroscopy.