FACTORS THAT CAUSE AGGREGATION
During product expression, accumulation of high amounts of protein may lead to intracellular aggregation either because of
interactions between unfolded protein molecules or because of inefficient recognition of the nascent peptide chain by molecular
chaperones responsible for proper folding (7). The temperature during cell culture is generally high (> 25 °C) and can cause
partial misfolding of the protein product, further leading to increased hydrophobic interactions and aggregation. The choice
of components in the cell-culture medium may affect aggregation by influencing the ability of the protein to fold to its native
structure. During protein production, disulfide bond formation occurs in the endoplasmic reticulum of cells. Proper disulfide
bond formation, which is typically an enzyme-catalyzed process, is crucial for folding of native protein structures. Formation
of the disulfide bond typically requires an oxidative environment and in the absence of this environment, the free thiols
on the cysteines may remain unpaired, leading to improper folding, which results in aggregation. Aggregation because of
cell culture can also be affected by amino acid sequence, variation in clone characteristics of the cell line, and operating
conditions of the bioreactor such as pH and agitation (6, 7).
Centrifugation is commonly used for efficient removal of mammalian cells and can handle large volumes. Particular concerns
during centrifugation include controlling exposure of the protein product to zones of intense shear forces at locations close
to where the feedstream enters the centrifuge rotor. Shear can cause cell lysis, denaturation, and aggregation. Use of microfiltration
as an alternative has also been examined, but if not appropriately designed, it can result in the protein molecules undergoing
stress-related deformations, leading to aggregation (4).
During purification, the protein product may be subjected to extensive changes in environment such as pH, protein concentration,
ionic strength, and contact materials. These changes by themselves and in combination may result in production of aggregates.
Protein A chromatography and low-pH viral inactivation are the two purification steps where the product is exposed to low
pH. Low pH has been known to cause protein aggregation by altering the physiochemical properties of the product and causing
instability of the primary structure (8–11).
During crossflow filtration, the protein may be exposed to shear rates from 1000–10,000 s-1 for a few seconds as it passes through the membrane (12). Adsorption to the membrane during filtration can also sometimes
cause aggregation, as demonstrated by the deactivation of aminoacylase by adsorption to an ultrafiltration membrane (13).
Meireles at al. have studied the effect of different pump heads and observed an increase in turbidity of an albumin preparation
over time when pumping at room temperature using a screw pump (14). Cavitation during pumping leads to generation and destruction
of air bubbles and hence an increase in the air-water interface, resulting in the formation of protein aggregates (15).
Characteristics of the buffers used, such as composition, pH, ionic strength, concentration, and presence or absence of salts
can also affect protein stability. Use of strong acids and bases in buffer to adjust the pH induces degradation as compared
with buffers containing weak acids and bases. Both high and low pH can initiate aggregation (16). While insoluble aggregates
are formed at higher pH because of lower net charge, aggregates remain soluble at lower pH due to the higher net charge (17).
Another important characteristic of buffer solutions used in biopharmaceutical manufacturing is their ionic strength. Aggregation
is faster at high ionic strength than at lower ionic strength (18). In formulation, the effect of buffer salt on protein stability
is strongly ion-specific and pH-dependent (19).
Equipment contact material
During different processing steps, proteins may be adsorbed at the solid surface of the equipment, and in the adsorbed state,
protein residues can undertake non-native interactions with the exposed functional groups at the solid-surface interface.
The adsorption process is mostly driven by either hydrophobic interactions or electrostatic interactions between the surfaces
and the protein (20). Because of partial unfolding of the protein on the surface, structurally perturbed molecules can potentially
form aggregates on the surface or in the bulk solution after desorption. An increase in α-helical structures accompanied by
a reduction in β-sheet and β-turn conformations has been reported upon adsorption of a mAb product on Teflon particles (21).
The product may adsorb on the glass or stopper surface, especially after losing its three-dimensional structure. It is often
difficult to detect this phenomenon. Although it may result in loss of only a small amount of protein initially, the adsorbed
protein may catalyse protein unfolding and aggregation rapidly over time (4).
Freeze-thaw and storage
Freezing is often required for shipping and storage of proteins and their formulations followed by thawing at the time of
use (5). However, when protein products undergo a freeze–thaw cycle, they experience several stress factors, which in turn
can lead to aggregation. Freezing or low-temperature denaturation of proteins increases solubility of hydrophobic residues
and causes increased hydrophobic patches and ultimately some unfolding of the protein. Furthermore, aggregation caused by
a decrease in pH of the formulation buffer during freezing has also been reported in some studies (22, 23). Exposure of mAb
products to an acidic environment can result in conformational changes and aggregation. During freezing, the protein molecules
can also adsorb at the container surface or ice-liquid interface, resulting in unfolding of proteins and subsequent aggregation
(24). Furthermore, perturbation in the protein conformation can be caused by cryo-concentration of protein, buffer salts,
and excipients or by phase separation. In addition, process conditions such as freezing and thawing rates play an important
role with respect to the stability of a protein upon freeze–thawing (25).
Therapeutic proteins are exposed to shear at the end of production during the vial filling operation. Dispensing a protein
solution into vials through a 20-gauge-by 10-cm needle at a rate of 0.5 mL/s exposes the protein to a shear rate of 20,000
s-1 for approximately 50 ms and can lead to aggregation (26).