Protein aggregation poses a significant challenge in the biopharmaceutical industry, affecting the stability and efficacy of therapeutic proteins. Through innovative analytical technologies, scientists can precisely analyze and characterize protein aggregates, gaining a deeper understanding of the underlying mechanisms of protein aggregation and ultimately leading to optimal protein-based drug formulations.
Recombinant protein manufacturing has been possible since the 1970s and continues to drive the need for advanced analytical methodologies to characterize these proteins as they become increasingly complex. Although numerous advancements have been made, protein aggregate characterization remains a challenge because of the unique protein construct and its impurity profile. At times, the vast amount of effort required to develop and validate these methods results in their underutilization for more complex samples generated in the manufacturing process.
Protein aggregates are high-molecular-weight protein species, such as oligomers or multimers, formed through various pathways that differ by protein and may lead to different end states.1 They can be stabilized by noncovalent interactions or covalent bonds (for example, disulfide bridges), and may be reversible or irreversible. No single method can detect all sizes of protein aggregates, which range from small, soluble dimers (~10 nm) to large, insoluble structures (>1 µm). Aggregation can originate from native or partially unfolded proteins or be triggered by impurities. It can occur throughout manufacturing and is commonly observed during formulation and stability studies, where factors such as temperature, freeze-thaw cycles, agitation, protein concentration, excipients, and storage conditions are evaluated.
Analytical Tools for Comprehensive Aggregate Characterization
Current regulatory guidelines (for example, USP2, 3, EMA4, and ICH5) require comprehensive analytical characterization of biopharmaceuticals prior to clinical trials and commercial release. Protein aggregation, categorized under purity and contaminant analysis, can be assessed using non-liquid chromatographic methods, such as analytical centrifugation and dynamic light scattering (DLS), or using liquid chromatography (LC) methods, such as size exclusion chromatography (SEC). Each method has its limitations. For instance, SEC only detects soluble aggregates that pass through column filters, while DLS has limited size resolution and is less sensitive to small particles in the presence of larger ones. Therefore, SEC is often combined with DLS and visual inspection to cover a broad aggregate size range: small (SEC), mid-sized (DLS), and large (visual). Among these, SEC will be examined in greater detail for its advanced capabilities in protein aggregate analysis.
SEC—An Extremely Accurate and Highly Quantitative Technique
Advancements in sub-3 µm particle sizes and inert surface chemistries (for example, diol) have enhanced SEC by utilizing low-dwell-volume UHPLC systems. SEC separates analytes by hydrodynamic radius, with larger species eluting first due to shorter flow paths. When paired with sensitive detectors—such as ultraviolet (UV), fluorescence detector (FLD), refractive index (RI), multi-angle light scattering (MALS), or mass spectrometry (MS)—for a fit-for-purpose method, SEC offers robust, reproducible separation. With suitable controls, standards, and an analytical quality by design (AQbD) method development in mind, SEC can accurately quantify monomers and small soluble aggregates and indirectly monitor aggregate formation via mass balance and reference standards (for example, time-zero samples in stability studies).
For most proteins (>10 kDa) and immunoglobulin G (IgG)-related monoclonal antibodies (mAbs), a 200Å pore size SEC column is suitable. As shown in Figure 1, it enables reproducible and quantitative separation of three proteins and one IgG-related mAb from their aggregates in under 12 minutes. For larger proteins, such as immunoglobulin M (IgM)-related mAbs or adeno-associated viruses (AAVs), a 700Å pore size column is more suitable (Figure 2).
Selecting the correct pore size is critical. If too small, the small aggregates won't separate from the target analyte, leading to inaccurate quantification of monomers or aggregates. Figure 3 illustrates an SEC method using 1.8x phosphate buffered saline (PBS) solution plus 0.001% Pluronic® F-68 for AAVs. It consistently quantified aggregates across five AAV8 samples, over multiple days, at various concentrations (a 10-fold range), and using three SEC columns from different batches. The method’s reproducible retention times also enabled automated data processing, minimizing human error.
Accelerated Degradation Studies
SEC-UV-FLD and SEC-RI-MALS were used to analyze the degradation of AAV8-CMV-GFP—an AAV vector expressing green fluorescent protein (GFP)—under elevated temperature and acetonitrile exposure, conditions relevant to AAV manufacturing. Both stressors caused visible degradation (see Figure 4 for temperature and Figure 5 for acetonitrile). Effective separation of degradation products enabled further characterization using UV 260/280 ratios and MALS. The blue trace in both chromatograms represents the reference (time zero).
The analysis identified that Impurity 1 is likely genetic material, Impurity 2 is a complex of genetic material and protein, and Impurity 3 (only observed in the temperature degradation study) is likely the empty AAV8 capsid. Degradation pathways differed by condition. Acetonitrile caused a dose-dependent decrease in the monomer peak and an increase in Impurities 1 and 2. Temperature led to rapid monomer loss (within 2 min), with Impurity 2 converting into Impurity 1 over time. No visible particulates were observed. All samples were centrifuged before SEC analysis.
Conclusion
SEC is effective for studying protein aggregation, as it separates target proteins from small soluble aggregates and enables precise quantitation and identification. Aggregation can be indirectly measured using mass balance with a control (for example, a fresh sample). Large aggregates are visible by inspection, and intermediate ones are visible by DLS. SEC can analyze soluble aggregates and active biomolecules after 0.2 µm filtration or high-speed centrifugation.
References
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