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

In addition to aggregate size, the rate of aggregate change in size over time, where time corresponds to the protein product lifecycle, is a useful parameter that can provide a functional characterization of aggregates. An aggregate can initially exist as a small dimer or fragment, and progress toward larger structures, such as subvisible or visible particles, if such a transition becomes thermodynamically favorable. At any given moment, a protein may be transitioning between a thermodynamic state that favors the monomer or native configuration of the protein, and an intermediate state that favors an unfolded native-like protein configuration. Under the right set of conditions, the unfolded protein may form a complex with other native and non-native forms, gaining enough free energy to transition to an aggregated state that may become the most stable state of the new proteinaceous entity. The basis for these transitions was studied by Lumry and Eyring in the 1960s, who proposed the kinetic transitions of protein aggregates in solution, and was further modified to first-order transition reaction kinetics in a case of interferon-γ aggregation.8,9 Evaluating the rate of aggregate growth in a drug product is complex. A single drug product typically has a non-homogeneous aggregate mixture (Table 1). Stable protein preparations will have aggregates present in a solution of a heterogeneous nature, but the rate of growth is minimal compared with more unstable preparations. Aggregates that have increased rates of growth are more worrisome and more aggressive strategies of control and minimization may be needed.


Drug product aggregates may originate from multiple sources and various types of aggregates may be present in a given drug product vial. The potential for protein aggregate formation exists at all levels of protein-based pharmaceutical manufacturing. Starting with the sequence and characterization of the protein, each protein will have a physicochemical signature that can render it more or less stable. This is the case of a CHO-derived monoclonal antibody (MAb) found to aggregate in culture because of elevated levels of free thiol, which could be prevented if copper sulfate were added to the culture.10 Protein heterogeneity also can be a contributing factor for protein aggregation, as the probability of multiple protein forms interacting with their environment is increased. In the case of epratuzumab, disulfide bond scrambling favored covalent aggregate formation.11

Therapeutic MAbs are typically formulated at high concentrations that favor the increased incidence of molecular interaction, and therefore, the potential for aggregate formation. Manufacturers therefore dedicate much time and effort to developing a formulation that will keep the protein drug product stable during its lifecycle, whether in solution or lyophilized. For example, adding sucrose to interleukin-1 receptor agonist or to a lyophilized MAb inhibited or reduced aggregate formation respectively. 12,13

Bulk freezing presents a challenge to stable protein preparations because of the solute concentration effect that occurs during the freezing of solutions. An ideal strategy is to freeze the entire solution at the same time and rapidly at –80C where thermal transitions (such as eutectic melting) and glass transitions are minimized. This strategy is not practical for large-volume solutions. Changes in solute concentration and pH during bulkfreezing can promote protein aggregation.14,15 Bulk thawing also presents challenges, primarily related to surface adsorption at the ice–liquid interface. The appropriate formulation becomes critical when bulk freezing and thawing is required, and excipients may serve as protein cryoprotectants.

Fill-and-finish operations may use pumps that can mechanically denature the protein because of shear stress or introduce impurities that serve as nucleation sources of protein aggregates. Some piston-displacement pumps, for example, can interact with protein drug product in a similar way that a car motor engine piston interacts with lubricant oil. The intimate contact between protein drug product and a piston rod can disrupt an otherwise stable drug product. This was the case for an antibody drug product that was found to have increased levels of aggregate particles with more pump passes, as determined by absorbance and light obscuration methods.16 In the case of piston shedding, stainless-steel passivation should reduce the risk of introducing stainless-steel deposits to the final drug product, which has the potential to induce protein heteronucleation.17

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