Aggregates in MAbs and Recombinant Therapeutic Proteins: A Regulatory Perspective - FDA perspectives on specifications and effective control strategies - BioPharm International


Aggregates in MAbs and Recombinant Therapeutic Proteins: A Regulatory Perspective
FDA perspectives on specifications and effective control strategies

BioPharm International
Volume 21, Issue 11


Table 2
The biopharmaceutical industry has experienced an increase in the number of analytical methods available to detect, characterize, quantify, and monitor aggregates in biopharmaceutical protein products. A list of the most common methods used to detect, monitor, and study aggregates appears in Table 2. Although not meant to be comprehensive, Table 2 offers a general concept of the analytical methods and their main advantages and limitations. Test methods available can be clustered into two groups: those that detect small aggregates such as dimers, LMW, HMW, soluble aggregates, and protein fragments (first section of Table 2), and those that detect large aggregates, such as insoluble subvisible and visible particles (second section of Table 2). Within the group of test methods used to detect small aggregates, size exclusion chromatography (SEC) is commonly used for routine detection and monitoring of aggregates during lot release. Methods that do not lend themselves to lot release can be used for additional characterization or as confirmatory tests.

Size exclusion high-pressure liquid chromatography is the most widely used analytical method for protein aggregates such as dimers and LMW, and HMW species. The method is amenable to validation for high throughput analytical testing with good sensitivity, precision, resolution, and accuracy. However, as a chromatographic method, it can also induce aggregation, cause existing aggregates to be removed during sample preparation, or underestimate the presence of aggregates when HMW species cannot be discriminated or recovered. Perhaps the main limitation of SEC is the need to inject samples at low concentrations (e.g., 1 mg/mL). For therapeutic proteins, this often means a 100-fold dilution causing reversible soluble aggregates to disaggregate. This concern leads to more general questions, such as, How relevant is the aggregate profile obtained? Does it represent the aggregates present in the final drug product? As noted elsewhere, there is not a single analytical method capable of evaluating all aggregates present in a given protein solution.21 A combination of methods is usually needed to cover the microscopic and visual range of aggregates that may be present. In addition, because of the limitations of each method, orthogonal confirmatory methods may be needed. For example, SEC results may require confirmation with other orthogonal methods, such as analytical ultracentrifugation (AUC). AUC can be used as a confirmatory method because it provides a good separation of aggregates, and it does not require sample dilution or sample preparation.

Among the methods used for aggregate characterization, field flow fractionation (FFF), and dynamic light scattering (DLS) are capable of evaluating aggregates directly in solution (without dilution). FFF has a detection range broader than that of SEC, but data interpretation of concentrated samples may be difficult. DLS is a good quantitative method, but the read-out is proportional to surface area so large aggregates can mask detection of small ones if differences in size are not enough. Calorimetry is very useful to evaluate the stability of a protein solution because it can detect protein unfolding. Some methods may be combined to have a more powerful tool, such as mass spectrometry coupled to chromatography, which provides information on chemical and physical degradation. In addition, DLS in combination with SEC can overcome the limitations that may be encountered using either method.

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