In this commentary, we, as representatives of EIP-PCS, the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.
Despite the high quality of current therapeutic biotech products and the resemblance of recombinant human proteins and antibodies to endogenous human proteins, protein immunogenicity remains an important concern. Among the several factors playing a role in immunogenicity, the presence of aggregates is considered an important product-related factor that may increase the risk of an immune response 12.
Although little is known about which aggregate species trigger the immune system, it is believed that aggregates are more easily recognized by the immune system than the native parent protein. They can be formed during Validating sec-ls aggregation methods, storage, shipment or delivery to the patient.
Aggregation can occur because of exposure to air-liquid or liquid-solid interfaces, e. Also, solution contact with ice during adventitious or deliberate freezing can cause aggregation 56. Moreover, protein aggregates may in some cases be induced by foreign particles, e. The challenge in analyzing protein aggregates lies in "Validating sec-ls aggregation methods" unknown nature of the formed aggregates as well as the Validating sec-ls aggregation methods size range of up to six orders of magnitude, from a few nm to a few mm in diameter.
Since no single one of the currently available techniques is able to cover this size range, a combination of several
Validating sec-ls aggregation methods is necessary. However, each technique has its own strengths and weaknesses.
Moreover, the available methods differ in the physical measuring principle and, consequently, in the results and type of information obtained. The aim of this commentary is to discuss the currently available analytical methods to characterize protein aggregates in relation to product quality and also the interpretation of data resulting from these methods.
Moreover, we propose approaches to use these methods for the characterization of protein therapeutics from early product development through to commercialization. Many techniques are available for the analysis of protein aggregates or proteinaceous particles and particles in general.
These techniques rely on different separation as well as different detection principles and range from relatively basic techniques such as visual or microscopic inspection, through to high-tech methods such as analytical ultracentrifugation AUC or mass spectrometry 4. Furthermore, this table summarizes the basic principles, describes types of analyses, e.
The consequence of these Validating sec-ls aggregation methods analytical features e. Based on discussions among the members of the EIP-PCS, Validating sec-ls aggregation methods identified techniques which are commonly used in the industry as analytical quality control QC and extended characterization assays. These methods and their advantages and disadvantages in protein aggregate analysis are discussed in more detail in the following paragraphs.
Depending on the detection principle, the particle is determined in length or mass units; b Possible chemical degradation when working in the far UV range; Abbreviations used: Furthermore, the manufacturers are recommended to have procedures in place that describe the Acceptable Quality Levels AQL of the inspection process and
Validating sec-ls aggregation methods for the maximum number of rejects per batch.
Originally, the compendial guidance was primarily focused on particles originating from external sources such as manufacturing materials and primary packaging components. However, for protein products, visible particles may also be product related. That is, protein products may contain insoluble visible particles derived Validating sec-ls aggregation methods the protein product itself. In these cases, sterile filtration is often not effective because insoluble protein particles may reform over time.
Nowadays, regulators expect that manufacturers try to limit the presence of Validating sec-ls aggregation methods particles by applying good formulation strategies and practices However, in specific cases, the presence of particles may be accepted as an inherent product attribute, provided that these particles do not pose a quality or clinical safety concern.
One example of this is insulin. In the EP it is stated that Validating sec-ls aggregation methods solutions may form insoluble particles over time. Moreover, intermediate-acting and long-acting insulin suspensions are particulate by definition.
Nevertheless, insulin has proven to be a very safe product for many years in a large number of patients. Also, for patients with a compromised immune system, a product with non-optimal appearance may be acceptable, provided that clinical data do not reveal major safety issues. Assay of visible particles is typically done by trained operators under controlled conditions.
Automated or semi-automated instrumental methods may be applied, but even then the human factor may be relevant, e. The EP describes a specific visual inspection method the black and white box. The threshold of visibility is believed to be ca. However, detection of visible particles is a problematic process 14and in daily practice the detection limit very much depends on the individual operator, the inspection device, the inspection time as well as the morphology, number and refractive index of the particles.
Companies may use various additional, non-EP methods for manual visual inspection to improve the sensitivity or ergonomics of the process, such as the use of aids such as magnifying glasses or differences in lighting conditions or observation times and swirling procedures. These operations may have a pronounced effect on reject percentages. Although visual inspection itself is pretty straightforward, interpretation of final inspection results can be very difficult.
For instance, abnormal reject rates may be observed after GMP batch manufacture in a large-scale commercial facility, whereas no problems were noticed for clinical batches produced in a small-scale Validating sec-ls aggregation methods. In such a situation a root cause investigation can be very laborious and time-consuming because the abnormal reject may be caused by either a manufacturing failure, a change in manufacturing conditions, a difference in visual inspection conditions between the large- and small-scale facilities, or a combination of these factors.
In order to minimize particle problems as much as possible, it is recommended to use harmonized inspection procedures and acceptance criteria throughout development especially for release testing and to evaluate the performance of the visual inspection method before manufacture of GMP batches.
The pharmacopoeial guidance with regard to assessing subvisible particles is applicable Validating sec-ls aggregation methods all parenteral solutions or lyophilizates, including those containing proteins.
Interestingly, these criteria are different between the two methods, reflecting the differences in detection principle. The measuring range of most light obscuration instruments is ca. Potential pitfalls of the method lie in the sample preparation, which has to ensure that no sample contaminations or other artifacts occur. Air bubbles and silicone oil droplets from the primary packaging may mistakenly be counted, non-spherical "Validating sec-ls aggregation methods" cannot adequately "Validating sec-ls aggregation methods" measured, and the technique is unable to discriminate proteinaceous particles from unrelated, non-proteinaceous solid particles.
The microscopy method uses a suitable binocular microscope. The sample is filtered, and the filter-dried particles are then counted manually or automatically on the filter. With this method, potential contaminations can occur. Furthermore, the method is extremely labor intensive, which limits its use for routine, large throughput applications.
The current compendial acceptance criteria for subvisible particles are usually met for modern injectable solutions. However, it should be realized that these criteria were originally not set to control proteinaceous particles. An animated discussion is currently ongoing between regulators, industry and academia to determine whether subvisible particles could pose a significant immunogenicity risk 15 Unfortunately, the current compendial methods for subvisible particle testing have several limitations regarding robustness, sensitivity and type of particles that can be quantified accurately.
Therefore, currently the industry is evaluating technologies such as micro-flow imaging However, whether these new technologies really perform better and can be applied in a QC environment remains to be established. The basic principle of high performance size exclusion chromatography SEC is simple.
Solute molecules are passed through a column containing porous beads. Small molecules penetrate the pores of the beads, while larger molecules or aggregates do not; therefore, small molecules take a longer path through the column and elute later. Nominally, the ability to enter the pores is dictated by molecular size, so if it is assumed that the analyte molecule is spherical, the elution position relative to molecular weight Validating sec-ls aggregation methods may be "Validating sec-ls aggregation methods" to estimate molecular weight.
However, any such estimate is an approximation, as most molecules tend to not be spherical, and actually the hydrodynamic radius, rather than the molecular weight, is measured Aqueous, non-denaturing buffers are compatible with SEC. The basic method may be modified to gain insight into the quality aggregates, for instance by addition of denaturant to the eluent This can disperse non-covalently associated aggregates.
Alternatively, samples may be treated prior to SEC, Validating sec-ls aggregation methods instance by reduction and reaction Validating sec-ls aggregation methods cysteinyl thiols with an alkylating agentto break disulfide bonds. The addition to the eluent of fluorescent dyes that bind to denatured protein and so inducing an enhancement of the fluorescence allows detection of very low amount of denatured protein Quantification is not possible, however, since the fluorescence depends on both the type and extent of denaturation.
Finally, additional detectors may be added to give complementary information, such as molecular weight and stoichiometry of complexes from use of on-line light-scattering, UV absorbance, and refractive index detectors 21 Various factors may affect the results of SEC, as discussed recently by Carpenter et al. For instance, low Validating sec-ls aggregation methods ionic strength e.
Addition of arginine to the eluent may inhibit Validating sec-ls aggregation methods between solute and column matrix Detergents in the sample as opposed to the eluentthough of small molecular weight, can behave as large molecules if they form micelles above their critical micelle concentrationappearing in the chromatogram as UV-absorbing peaks 25 and potentially also giving rise to light scattering and fluorescence signals.
There is an upper limit to the size of aggregate detectable by SEC, because larger aggregates can be filtered out by frits in the system or by the column itself. As a consequence, large material large protein aggregates may disappear and be overlooked in the analysis.
They also build up on the top of the column and gradually degrade its performance, seen as broadened peaks, poorer resolution and decreased yields smaller peaks. Another form of aggregate that may be missed is that formed by very low affinity intermolecular association, as these may dissociate into monomers following a change in conditions from those of the sample to those experienced during chromatography e.
For detection of such low affinity aggregates other methods could be used, such as AUC, or method conditions of SEC could possibly be adjusted. SDS-PAGE is a very common, fairly robust method that is easy to perform and can supply information on approximate molecular weight and quantity, when using a suitable method of quantitative staining and gel scanning. The presence of SDS means that non-covalent aggregates are disrupted, so the method only detects covalent aggregates.
If reducing conditions are used, SDS-PAGE can discriminate between aggregates Validating sec-ls aggregation methods together by disulfide bonds and those held together by
Validating sec-ls aggregation methods non-reducible covalent bonds. With CE-SDS, Validating sec-ls aggregation methods can achieve similar results in a technically slightly different way, with automation of of samples and quantification by UV absorption rather than dye-binding.
DLS is a non-destructive technique for the characterization of colloidal systems like protein solutions, allowing a re-use of the sample for further characterization, possibly useful if only a small amount of product is available, such as in early stage development.
The concentration ranges between 0. Measurements of highly concentrated solutions are becoming feasible owing to the application of photon cross-correlation approaches 28for example. Although the sensitivity of Validating sec-ls aggregation methods technique for detection of large particles in particular is unsurpassed, quantification is not possible.
DLS yields qualitative results, not quantitative results. AUC, mostly used in sedimentation velocity mode SV-AUCis a powerful technique to characterize the sedimentation behavior of Validating sec-ls aggregation methods and the presence of aggregates in solution.
Recently, Philo reviewed AUC as a method for characterizing non-particulate, soluble protein aggregates, including its strengths and weaknesses One of the major advantages of AUC is that protein therapeutics can often be characterized without sample manipulation in relevant solutions such as their formulation buffer, with some Validating sec-ls aggregation methods exceptions e.
Quantification of aggregate species is possible, while formation or disruption of aggregates due to sample preparation, dilution or matrix effects is limited. Among the several practical and experimental parameters that influence the accuracy and precision of AUC experiments, cell mis alignment and the quality of the centerpieces especially seem to be major reasons for variations in quantification of low amounts of aggregates 30 — Therefore, regular calibration and intensive maintenance of the system and its accessories are required
Validating sec-ls aggregation methods assure their proper functioning.
Furthermore, it should be recognized that the mathematical calculations behind AUC analyses i. Nevertheless, because of some strong advantages over SEC or AF4, like the absence of interactions of the molecules of interest with or membranes, AUC can be a valuable tool for a qualitative cross-check or analysis of trend of data obtained by SEC or AF4. Therefore, we suggest using AUC only as a qualitative or relative method comparison of relative aggregation levels but not as an absolute quantitative orthogonal method, at least not until the technical issues of equipment and component quality resulting in limited precision and reproducibility are solved.
Here in part II of our series on assessing protein aggregation, we provide an overview of best practices for achieving this goal, including the. The combination of SEC with light-scattering (SEC/LS) Validating sec-ls aggregation methods was The Arrhenius approach over-predicted the aggregation rate for 5°C.
method exists to cover the entire size range or type of aggregates which may appear. trained analysts and special effort for of the data analysis. the combination of SEC–LS The use of multi- angle laser.