Comparative analysis of the three processes
In Process 1, a relatively stable in-process intermediate is produced after the diafiltration step with a log reduction value
(LRV) of ~3 for DNA relative to the clarified cell culture supernatant. For some antibodies, the capture step reduces HCP
levels to below 100 ng/mg, allowing the development of a two-step non-affinity purification scheme. For others, HCP levels
remain above 500 ng/mg after CEX and the process may require more than one polishing step. Therefore, to convert the majority
of processes to two-step non-affinity schemes, either direct contaminant precipitation or selective antibody precipitation
is necessary. Because the additives used for precipitation are small molecules that are generally accepted in protein purification
processes, additional clearance steps are not necessary as the additives are removed in the first column's flow-through and
In process 2, depth filtration can separate the bulk feed containing the target protein from the precipitated contaminants,
and this bulk can be loaded directly onto the first capture column. There is no reduction in the harvest volume from the precipitation
step compared to TFF, so the CEX cycle time is extended during loading. Fast-flow high-binding ion exchange resins can compensate
for this increase in process time, however, especially for low-volume, high-titer processes (>25 g/L) where further dilution
may not have a significant impact.12 Precipitation does not require any specialized equipment and is easily transferable to existing large-scale manufacturing
facilities. Further purification of the antibody by CEX chromatography achieves consistent product recovery and quality (Figure
2). With reference to HCP removal after the first column, process 2 was more efficient than processes 1 and 3 for many HuMAbs.
Process 2 also takes the best cost advantage of disposable chromatography.
In process 3, the direct precipitation of the antibody by additive 2 (based on its hydrophobicity) also results in significant
contaminant removal. However, this technique is prone to greater variation during scale-up and HCP levels are higher in CEX-purified
material. Although the contaminant level was high, a single Q membrane polishing step was sufficient to reduce HCP to undetectable
levels. The qualitative nature of the contaminants differs substantially between process 3 and process 1. The amount loaded
onto the Q membrane is much lower than in process 2 (Table 1) to accommodate the scalability and robustness of the two-step
non-affinity process. Overall, the process exploited hydrophobicity as well as CEX and AEX orthogonal separations to produce
therapeutic quality material. The advantages of process 3 include volume reduction, faster processing time, and easy adaptation
to facilities equipped with centrifugation or gravity filtration.
Table 1. Productivity comparison of the three non-affinity purification schemes
Protein precipitation is an efficient replacement step for primary recovery TFF and can be integrated easily into the two-step
non-affinity purification process for many antibodies. The precipitation-based process can help minimize the cost of antibody
therapy by increasing productivity and reducing operating costs. Similar viral clearance strategies can be implemented for
both ion exchange processes with either diafiltration or precipitation of contaminants (Table 2). Process 2 has the potential
to clear adventitious agents by a LRV of >4 during the precipitation step.13 Significant virus removal was accomplished by membrane chromatography in addition to other orthogonal viral inactivation
and removal methods to produce therapeutic-grade antibodies.
Table 2. Viral clearance comparison for three non-affinity purification schemes