During the development, commercial production and clinical application of protein drugs, protein aggregation stands as one of the major challenges. Protein aggregation may occur throughout the entire lifecycle of protein drug development and manufacturing, including cell culture, protein purification, sterile filling and storage; it can also emerge during clinical administration by patients. Protein aggregates exert significantly negative impacts on product quality and biological activity. While certain reversible protein aggregates can dissociate to restore biological activity in vivo, most aggregates exhibit reduced or complete loss of bioactivity and trigger immunogenicity, ultimately leading to therapeutic failure of the drug.
1 Formation Pathways of Protein Aggregates
Protein aggregates refer to polymers composed of two or more protein molecules cross-linked via covalent bonds, hydrogen bonds and other intermolecular forces, with an extremely complex formation mechanism. Aggregates can be classified from multiple perspectives:
By chemical bonding type: Non-covalent aggregates stabilized by weak intermolecular forces such as hydrogen bonds; covalent aggregates mainly linked by disulfide bonds.
By reversibility: Reversible aggregates and irreversible aggregates.
By particle size: Soluble small oligomers (dimers, trimers, tetramers, particle size: tens to hundreds of nanometers); subvisible insoluble particles (hundreds of nanometers to 50 μm); visible foreign particles (particle size above 50 μm).
1.1 Oligomer Formation
Proteins are charged amphipathic substances, and spontaneous formation of reversible oligomers has been widely documented for numerous proteins. The diversity of protein intermolecular interactions complicates the aggregation mechanism. The dominant driving forces for natural protein oligomerization include electrostatic interaction, hydrophobic interaction, hydrogen bonding and van der Waals force. The propensity for protein aggregation is governed by intrinsic protein structure, solution composition and ambient conditions.
1.2 Aggregate Growth
Initial small oligomers can further develop into larger aggregates through multiple mechanisms, following a non-linear growth pattern over time. The growth rate of protein aggregates is primarily determined by colloidal stability, conformational stability and intermolecular interaction strength of proteins. With the increase in particle size, aggregates eventually evolve into insoluble particles with diverse morphological characteristics.
2 Factors Affecting Protein Aggregation
2.1 Intrinsic Protein Structure
The primary amino acid sequence, quantity and arrangement of hydrophobic amino acids greatly influence aggregate levels. Hydrophobic amino acid residues tend to form aggregation-prone regions. Protein aggregation propensity is regulated not only by aggregation-prone peptide sequences but also by the amphipathic ratio of oligopeptide segments. Introduction of one or more distinct hydrophobic amino acids at specific sites can markedly accelerate protein aggregation.
Protein glycosylation is critical to the structural stability of many biomolecules. Glycosylation in the CH2 domain of monoclonal antibodies stabilizes the CH2 domain as well as the overall antibody architecture. Deglycosylation may disrupt the tertiary structure of the CH2 domain, destabilize Fab and Fc regions, and thereby accelerate thermally induced aggregation. Even glycan profiling can alter aggregation tendency by modulating protein conformational stability.
Protein secondary structure also plays a regulatory role in aggregation propensity. For proteins with free disulfide bonds, secondary structure modulates the rate of disulfide bond exchange, further affecting aggregate formation kinetics.
2.2 Chemical Degradation
Proteins undergo multiple chemical degradation pathways throughout drug development, manufacturing and storage. Many degradation events (e.g., oxidation, deamidation) do not induce detectable tertiary structural changes, yet they may alter protein conformation and colloidal stability, thereby modulating aggregation propensity.
Enhanced aggregation after protein oxidation is mainly attributed to three aspects: (1) increased surface hydrophobicity; (2) promoted inter-aggregate interaction; (3) reduced conformational stability.
Aggregation elevation upon deamidation results from protein destabilization and/or intensified protein-protein interaction. Additionally, negative charges introduced by deamidation may alter overall electrostatic interaction between protein molecules and further aggravate aggregation.
Antibody-drug conjugates (ADCs) have gained increasing clinical applications in recent years. ADCs generally exhibit poorer stability than native monoclonal antibodies. The conjugated hydrophobic small-molecule payload increases antibody surface hydrophobicity, and elevated drug-to-antibody ratio (DAR) progressively enhances aggregation tendency.
2.3 Temperature
Temperature modulates protein aggregation by affecting multiple solution properties, including molecular diffusion rate, protein-protein interaction, conformational stability and chemical degradation kinetics. Elevated temperature accelerates chemical reactions such as oxidation and deamidation, increases molecular collision frequency, and facilitates aggregate formation. Excessively high temperature unfolds protein higher-order structures, exposes internal hydrophobic groups, and triggers massive aggregation.
2.4 Solution Environment
Protein conformational and colloidal stability are strongly correlated with solution conditions, among which pH value is the most critical factor. pH simultaneously regulates conformational stability, colloidal stability, and the rate and pathway of chemical degradation (typically deamidation). Adjustment of solution pH alters native protein conformation and intermolecular interaction forces.
Ionic strength is another key solution parameter affecting aggregation. It modulates aggregation propensity by regulating conformational stability and the nature/intensity of protein-protein interaction, and its regulatory effect is highly pH-dependent. Cations and anions interact with proteins via specific binding or non-specific charge shielding to regulate aggregation, and their effects do not strictly follow the Hofmeister series.
Excipients are commonly formulated to mitigate aggregation, including arginine, polyols (sucrose, trehalose, sorbitol) and surfactants. Notably, some conventional protein stabilizers may promote aggregation under specific conditions. Studies have shown that sugars such as sucrose and trehalose can induce protein aggregation under stirring conditions. High-concentration trehalose (> 500 mM) may cluster on protein surfaces, compete for bound water molecules, and promote protein-protein association. Surfactants such as polysorbates effectively inhibit aggregation and particle formation induced by shaking and freeze-thaw cycles. However, impurities in polysorbates (insoluble fatty acids, peroxides, POE fatty acid esters, fatty lipids, aldehydes) may conversely trigger aggregate generation.
2.5 Protein Concentration
Protein molecules perform random Brownian motion in solution. Increased protein concentration enhances intermolecular interaction and collision probability, leading to elevated aggregate levels.
3 Analytical Methods for Protein Aggregates
A growing panel of analytical techniques is available in biopharmaceutical industry for the detection, characterization, quantification and real-time monitoring of protein aggregates. Each method has inherent limitations, and rational selection or combined application is required based on aggregate characteristics.
Size-Exclusion High-Performance Liquid Chromatography (SEC-HPLC) is the most widely adopted method for quantifying dimers, multimers and fragments. It features high sensitivity, precision, resolution, accuracy and high-throughput capability. Nevertheless, limitations remain: aggregation may be induced during sample dilution for analysis; separation resolution for fragments is limited with inherent quantification errors; the stability of target protein in mobile phase and column matrix must be verified to ensure the results reflect the authentic aggregation status in drug products. Cross-validation with orthogonal methods is usually required.
SEC-HPLC results are commonly confirmed by Analytical Ultracentrifugation (AUC), including two core modes: Sedimentation Velocity (SV) and Sedimentation Equilibrium (SE).
SV is a hydrodynamic technique: samples are subjected to high-speed centrifugation, and molecular migration rate is recorded to calculate sedimentation coefficient, which correlates with molecular weight, size and conformation. Heterogeneous components can be differentiated by distinct sedimentation coefficients.
SE is a thermodynamic technique operated at low rotational speed. Aggregation-dissolution equilibrium is established between centrifugal sedimentation and molecular diffusion, and the steady-state concentration distribution is only dependent on molecular mass, independent of molecular shape. Post-run data processing via SEDFIT/SEDPHAT software enables precise characterization of macromolecular aggregation state and accurate quantification of aggregate content. The drawbacks of AUC include high equipment cost, requirement for professional operation and maintenance, long testing time and inapplicability to large-scale batch testing.
Asymmetric Flow Field-Flow Fractionation (AF4) is another powerful separation technology for aggregate characterization. A perpendicular force field is applied perpendicular to the sample flow to separate molecules by size and molecular weight. The permeable membrane at the channel boundary forms a parabolic laminar flow velocity profile. Analytes are driven toward the boundary by the vertical force field, while Brownian motion counteracts this migration, allowing smaller particles to maintain a farther equilibrium position from the boundary. AF4 offers a wide separation range and compatibility with diverse eluents.
Dynamic Light Scattering (DLS) detects particle size based on Brownian motion of colloidal particles. Smaller particles exhibit faster Brownian motion, causing Doppler shift of scattered light. Particle size distribution is calculated by monitoring intensity fluctuations of scattered light.
For subvisible insoluble particles, mainstream detection methods include Light Obscuration, Light Microscopy and Micro Flow Imaging. Light Obscuration and Light Microscopy are compendial methods specified in the Chinese Pharmacopoeia and USP <788>. Light Obscuration enables automatic counting of insoluble particles of different sizes via photoelectric conversion, yet it cannot identify particle properties or elucidate aggregation mechanisms for process control. Light Microscopy suffers from low throughput and counting errors caused by visual fatigue.
Micro Flow Imaging is an emerging technology integrating digital microscopy, microfluidics and image processing. It captures high-resolution images of particles passing through the flow cell and establishes a characteristic database covering particle count, size, transparency and morphology. Particles can be classified into protein aggregates, glass debris, silicone oil, air bubbles, rubber stopper fragments and fibers, providing critical clues for tracing particle origins and formation mechanisms.
4 Regulation Strategies of Protein Aggregation
4.1 Protein Molecular Engineering
Intrinsic primary sequence and higher-order structure determine aggregation propensity. Rational molecular design and developability assessment at the early stage are the most efficient strategies to mitigate aggregation. Removal or modification of aggregation hotspots can alleviate protein self-association, which can be achieved via sequence analysis and identification of hydrophobic aggregation-prone regions. Modulation of surface charge also improves protein solubility and reduces aggregation tendency. Single amino acid mutation is often sufficient to suppress aggregation significantly. However, modification of key functional residues may compromise biological activity. Process optimization is required if ideal molecular properties cannot be obtained via protein engineering.
4.2 Cell Culture Process Optimization
Aggregate levels can be controlled by adjusting physicochemical culture conditions such as temperature, pH and osmotic pressure. Studies have shown that supplementation of 30 mM cysteine in culture medium reduces multimer content by 40%; reduction of copper ion concentration from 50 mM to 0 mM decreases aggregation by 43%. Lowering culture temperature from 35 ℃ to 32 ℃ upregulates the expression of binding immunoglobulin protein (Bip) and protein disulfide isomerase (PDI) by 89%, enhances intracellular post-translational modification capacity, and reduces secreted antibody multimer content by 38%. Aggregation levels decrease with elevated culture pH and osmotic pressure; increasing sodium chloride concentration to raise osmotic pressure is an effective regulation approach. Shortening culture duration also lowers aggregate content, so harvesting at qualified product titer without over-cultivation is recommended.
4.3 Protein Purification Process Optimization
For high aggregate levels from cell culture, purification process optimization is adopted for aggregate removal. Buffer composition is a critical influencing factor. Acidic elution buffer used in Protein A affinity chromatography readily induces aggregation. Replacing conventional citrate, glycine or histidine buffer with arginine-supplemented buffer effectively reduces aggregate content in eluates. Cation exchange chromatography, hydrophobic interaction chromatography and mixed-mode chromatography are also applied for aggregate clearance. Size-exclusion chromatography can remove high aggregates in early research stages but suffers from low yield loss and limited scalability, thus rarely used in commercial antibody manufacturing.
4.4 Formulation Development
After purification to high purity, solution formulation directly governs aggregate formation during subsequent storage and application. Core strategies include pH and ionic strength optimization, and application of stabilizers. Screening of optimal pH and buffer type is the priority of formulation development.
Protein stabilizers mitigate aggregation by enhancing conformational stability, colloidal stability and solubility. Sugars are widely used stabilizers against aggregation under various stress conditions. Arginine serves as a highly effective excipient: it is preferentially excluded from protein-protein encounter complexes via electrostatic and hydrophobic interactions, inhibiting molecular association and aggregation.
Non-ionic surfactants are commonly formulated to suppress aggregation induced by shaking and freeze-thaw stress. They reduce protein self-association, bind to hydrophobic surface regions, and compete with proteins for air-water hydrophobic interfaces to prevent structural unfolding and interfacial adsorption. Polysorbate 20 and Polysorbate 80 are the most widely used surfactants in biotherapeutic formulations.
Trace metal ions inevitably enter protein solutions during manufacturing and pre-filled syringe storage, catalyzing the degradation of amino acid residues including Met, Cys, His and Trp. Proteins may also be exposed to disinfectants such as hydrogen peroxide during production, or radical-generating conditions including light irradiation and mechanical shock during storage. For proteins sensitive to metal ions and oxidants, chelating agents (e.g., EDTA) and antioxidants (e.g., methionine) are supplemented in formulations for protection.
4.5 Lyophilization Process Optimization
If formulation screening fails to control aggregation and degradation to acceptable levels, lyophilization is adopted as an alternative strategy. Nevertheless, the lyophilization process itself may induce aggregation and involves high time and energy consumption. Rational lyophilization cycle development (optimization of freezing rate, primary drying temperature and pressure) is essential to minimize lyophilization-induced aggregation and reduce production cost. Annealing treatment can lower surface fraction and global molecular mobility, reducing aggregation rate during long-term storage.
Formulation compatibility with lyophilization must also be evaluated: some stabilizers are unsuitable for lyophilized products. For instance, phosphate buffer undergoes pH shift during freezing; acetate buffer may volatilize during lyophilization; salt stabilizers may depress collapse temperature. Synergistic optimization of formulation composition and lyophilization cycle is required to maximally inhibit protein aggregation.
