Insight

The formation of protein particles in drug substance (DS) and drug product (DP) manufacturing of antibodies and recombinant proteins has been widely reported. Typical manifestations include elevated turbidity of protein solutions caused by ultrafiltration and diafiltration, subvisible particles (SvPs) generated during filling with rotary piston pumps, as well as protein aggregation and SvPs resulting from silicone oil shedding on the inner surface of pre-filled syringes.

Given the immunogenic risks posed by protein particles, relevant regulations have set stringent limits on particulate matter in injectable preparations. The specified limits for SvPs measuring ≥10 μm and ≥25 μm are 6,000 particles per container and 600 particles per container respectively. Although current regulations have not imposed control requirements for SvPs smaller than 10 μm, non-filterable nanoparticles may act as nucleation sites to trigger further protein aggregation, potentially compromising the safety and efficacy of finished products throughout their shelf life. Accordingly, efforts shall be made to minimize particle generation and strictly control particle counts during production.

Essentially, protein particles consist of aggregated protein molecules, each particle containing thousands to millions of protein monomers. Protein aggregation generally proceeds in two sequential stages: nucleation and aggregate growth. Initially, conformational changes of protein molecules lead to the formation of dimers or oligomeric nuclei. These nuclei then grow progressively, eventually developing into subvisible or visible protein particles.

During manufacturing, protein molecules are subjected to multiple stresses, mainly including interfacial stress, mechanical shear, cavitation, foreign nanoparticle contamination and thermal stress, which are the primary drivers of protein particle formation. Combined with current research findings, this article elaborates on the mechanisms of particle formation induced by each type of stress.

Interfacial stress is categorized into gas-liquid interfacial stress and liquid-solid interfacial stress, both of which are prevalent in DS and DP process operations. It is generally acknowledged that proteins tend to adsorb and accumulate at phase boundaries, triggering conformational alterations and further forming aggregates, protein clusters or protein films. Under mechanical actions such as stirring, pumping and fluid stretching, these aggregates, clusters and film fragments detach into the bulk solution and continue to grow into protein particles.

Nucleation of protein aggregates is primarily initiated by interfacial stress. Nevertheless, interfacial stress alone is insufficient to induce intact protein particle formation. Synergistic mechanical stress is required to release the formed nuclei into the bulk solution and facilitate continuous generation of new nuclei at interfaces; the suspended nuclei in the solution subsequently aggregate to form mature protein particles.

Solid materials significantly affect particle formation associated with liquid-solid interfacial stress. For instance, pipelines modified with hydrophilic copolymers used in tangential flow filtration (TFF) can reduce protein particle formation while improving filtration flux. Distinct differences in SvP levels have also been observed between systems using stainless steel and alumina surfaces. These findings provide practical references for the selection of consumables and process equipment in DS and DP production.

The addition of surfactants such as Polysorbate 80 (PS80) and Polysorbate 20 (PS20) to protein solutions can effectively inhibit protein aggregation and particle formation. Surfactants exert protective effects by competing with protein molecules for adsorption at gas-liquid interfaces or preferentially adhering to liquid-solid interfaces.

Mechanical shear generated during stirring is a common cause of protein aggregation. The combined effects of shear stress and interfacial stress have been extensively investigated for years. The susceptibility of proteins to shear-induced aggregation varies greatly among different molecules. For proteins with high structural stability, high shear stress alone rarely induces aggregation in the absence of interfacial stress.

In a relevant study, test proteins were exposed to an average shear rate of 1.6×10⁶ s⁻¹ — 3 to 5 orders of magnitude higher than typical shear rates encountered in biomanufacturing — yet no noticeable increase in aggregation was detected. Pure high shear force is unlikely to alter protein conformations to form aggregation nuclei. Instead, it amplifies gas-liquid and liquid-solid interfacial effects, accelerating nucleation, transporting nuclei within the solution and promoting aggregate growth.

Accordingly, protein particle formation results from the combined action of mechanical stress and interfacial stress. These two stresses nearly always coexist in practical production processes, making it difficult to quantify their individual contributions. Both are recognized as essential factors for particle generation. Adjusting process parameters to reduce mechanical shear is the most commonly adopted and technically feasible strategy to mitigate protein aggregation in commercial manufacturing.

Hydrodynamic cavitation is a distinctive fluid phenomenon characterized by the formation of vapor cavities (bubbles) in liquids due to pressure fluctuations, which frequently occurs under turbulent flow conditions. A sudden pressure drop gives rise to cavitation bubbles, which then collapse violently when exposed to elevated pressure. The continuous formation and implosion of cavities generate intense shockwaves, accompanied by transient localized high temperature and high pressure.

Such extreme conditions may cause protein unfolding and aggregation, or induce free radical generation that triggers covalent cross-linking of proteins. Relevant reports have confirmed that mechanical impact and subsequent cavitation — even from dropping vials containing monoclonal antibody formulations from a height of 10 inches — can induce protein particle formation. Hydroxyl radicals produced during cavitation accelerate protein cross-linking and increase SvP levels. Additionally, cavitation increases the contact between protein molecules and gas-liquid interfaces. Overall, cavitation-induced protein aggregation and particle formation arise from the combined effects of mechanical stress, chemical stress and gas-liquid interfacial stress.

Abrasion and friction between contacting solid components may shed particulate contaminants into protein solutions. A typical example is rotary piston pumps: friction between pistons and pump housings releases stainless steel nanoparticles. These foreign particles act as exogenous nucleation sites. In accordance with heterogeneous nucleation theory, proteins rapidly accumulate on these particles to form mature particles, bypassing the early endogenous nucleation stage of protein molecules.

Another underlying mechanism involves liquid-solid interfacial stress: proteins adsorb onto solid surfaces and form aggregates under interfacial effects. These aggregates detach from surfaces and re-enter the bulk solution via turbulent mixing, serving as new nuclei to drive further particle growth. In this scenario, the surface area of solid components and flow turbulence are the dominant influencing factors. Foreign particulates promote protein particle formation through dual mechanisms: acting as direct nucleation cores and exacerbating interfacial stress.

Friction and abrasion between solid components generate frictional heat and induce localized thermal stress. Heat is produced by atomic interactions at contact surfaces, and the resultant temperature rise is sufficient to trigger conformational changes of adsorbed proteins. However, such transient thermal effects are extremely difficult to monitor.

Conventional temperature detection methods including infrared thermography only measure macroscopic temperatures, failing to capture microscopic temperature variations at friction zones. Moreover, bulk liquid rapidly dissipates transient heat generated during mixing, further hindering accurate measurement. The recorded bulk temperature therefore cannot reflect the actual thermal conditions experienced by proteins. Proteins near friction points may be exposed to extreme high temperatures for an extremely short duration (less than 1 millisecond), leading to protein degradation. This phenomenon becomes more pronounced with enlarged contact areas, elevated operating speeds or prolonged mixing time.

This theory has been validated in both small-scale stirrer tests and large-scale mixing operations. Studies on bottom-mounted agitators indicate that friction between rotor and stator bearings is a key trigger for particle formation, which is primarily attributed to localized thermal effects.

Protein aggregation and particle formation constitute a complex process, governed by the intrinsic structural stability of proteins as well as manufacturing parameters and operational practices. Consumable selection and routine operations such as stirring inevitably expose protein molecules to multiple stresses, which often coexist in a single unit operation. For example, peristaltic pumping involves interfacial stress, mechanical shear, foreign particle contamination and thermal stress simultaneously. To date, protein particle formation in biomanufacturing is generally regarded as the outcome of synergistic actions of multiple stresses. With in-depth research advancing the understanding of relevant mechanisms, more optimized strategies will be developed to refine manufacturing processes.