Monoclonal antibody (mAb) products have emerged as an extremely important and valuable class of therapeutic products for the
treatment of cancer and other diseases such as rheumatoid arthritis, auto-inflammatory disease, allergic asthma, and multiple
sclerosis (1, 2). The proliferation of this product class has led to significant advancements in the production processes
that are used to manufacture them. However, product aggregation remains a crucial issue and results not only in product loss
but also affects product safety (3).
Figure 1: Illustration of the different pathways that result in mAb aggregation.
Aggregates are typically large, tangled clusters of denatured antibody molecules that are irreversibly formed either during
product expression in the cell culture, product purification in downstream processing, or storage as drug substance or drug
product (see Figure 1). The process of aggregation is influenced by the biochemical and biophysical properties of the mAb itself, as well as the
physicochemical environment to which the mAb is exposed during processing and storage (4).
Aggregation can cause formation of subvisible particles and may expose normally unexposed epitopes, leading to increased immunogenicity.
The subvisible particles have become of increasing concern to the regulatory agencies because of the difficulty in detecting
them and their potential for causing unintended immunogenicity. Subvisible particles, defined as particles larger than 0.1
µm but too small to be visible to the unaided eye (i.e., < 100 µm), may form either during product manufacture or storage.
Of particular concern are particles less than 10 µm in size, which are not well detected by current analytical methods used
to monitor degradation of mAb products. Purification processes for mAb products should be capable of achieving low aggregate
levels (i.e., 0.5–2%). The relatively large, irreversible aggregates are different from the often reversible dimers and trimers
that are often less of a concern. Individual antibodies vary widely in their propensity to form aggregates (5). Extremes of
pH, ionic strength, temperature, concentration, shear forces, and other processing conditions can lead to increased aggregation.
As a result, aggregation has to be monitored throughout process development and manufacturing of a mAb product.
During manufacturing of protein therapeutics, the protein is exposed to various stresses. The cell-culture phase of a typical
mAb production process improves secretion of the protein from the cell into the medium, which contains the cells, buffer constituents,
nutrients for the cells, host-cell proteins (including proteases), dissolved oxygen, and other species. This cellular suspension
is held at near neutral pH at temperatures above 30 °C for several days. Once a sufficient amount of protein has been expressed,
the cell-culture fluid is harvested and purified with Protein A chromatography. The resulting pool is often held at acidic
pH for viral inactivation. Polishing steps that typically follow include cation-exchange chromatography (CEX), which is performed
under conditions of high conductivity, and anion-exchange chromatography (AEX), which is performed at high pH. Finally, the
protein is formulated using ultrafiltration/diafiltration and filled in its final form. Throughout the production process,
the protein solution is pumped, stirred, and filtered and is exposed to a variety of materials including stainless steel,
glass, and plastic. All of these factors can potentially result in aggregate formation (6).
Elements of Biopharmaceutical Production
This article, 29th in the "Elements of Biopharmaceutical Production" series, presents a discussion of the factors that result in formation
as well as control of mAb aggregates during processing.
Anurag S. Rathore, PhD, is a consultant, Biotech CMC Issues, and a member of the faculty in the department of chemical engineering at the Indian Institute of Technology. Rathore is also a member of BioPharm International's Editorial Advisory Board.
Articles by Anurag S. Rathore, PhD