SELECTIVITY AND PURITY
Table 2. Challenges of downstream processing to produce large amounts of MAbs, addressed by second-generation, high-flow agarose
Protein A resins
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Selectivity plays a capital role in platform technologies because it reduces the number of purification steps, thereby shortening
overall production times and increasing recovery. Specific binding of the target molecule to a ligand-substituted resin enhances
productivity by rapidly removing the bulk of the impurities from the feedstream and concentrating the product in one step.
This point is supported by the successful use of immobilized Protein A as the capture step in the production of most approved
therapeutic monoclonal antibodies as well as hundreds in development. Protein A binds human IgGs at the Fc region, which facilitates
the isolation of monoclonal antibodies from cell culture harvest. Purity levels are 98% or greater after this one unit operation,
greatly simplifying the challenges further downstream and allowing robust, economical purification. Successful therapeutic
applications of MAbs have put great demands on production quantities, where expression levels of more than 1 g/L are commonly
reported. A new generation of high-flow agarose-based Protein A resins has been designed to handle large volumes of feed with
high titers (Table 2).
CLEANING AND SANITIZATION
Figure 6. Stability of novel Protein A agarose resin to sodium hydroxide concentrations up to 0.5 M and contact times from
15 to 60 minutes
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The potential for carryover of product and impurities from one run to another must be addressed for packed columns, and the
cleaning protocol must be shown to remove residual materials. Many cleaning and sanitization protocols use NaOH. Another factor
is the stability of resins, columns, and associated equipment in sufficiently harsh conditions for cleaning and sanitization.
In addition, column packing at large scale is time-consuming and costly; maintaining a packed column is essential for economical
production of consistent product. Repacking columns requires large quantities of water and buffers, column qualification exercises,
and time. If reserve columns are not available, the failure of a column due to bacterial contamination, for example, could
result in a production downtime that alters in-process holding times, complicates scheduling in the plant, and creates an
overall decrease in productivity. Furthermore, effective cleaning and sanitization, with preserved performance, are essential
to reduce the raw material costs of resins. Protein A-based resins used for the capture of MAbs are more expensive than ion
exchangers, but their use becomes economically favorable if they can be used for many production cycles. An example of how
increased resin stability can extend the number of cleaning cycles that can be performed in situ is shown in Figure 6. Although
the dynamic binding capacity of a conventional Protein A resin was below 90% of the original capacity for a polyclonal antibody
after approximately 20 cycles, the novel Protein A resin, designed to tolerate NaOH, was observed to maintain a capacity of
90% up to almost 200 cycles when 0.1 M NaOH was used, and up to about 60 cycles when cleaned with 0.5 M NaOH for 15 minutes.
The resin uses a genetically engineered Protein A ligand designed for improved stability under alkali conditions. Other properties
were also optimized for MAb capture.
In a case study, a clarified Chinese hamster ovary (CHO) cell culture feedstream containing IgG1 was purified on a column packed with the alkali-stabilized Protein A media for 150 cycles, with cleaning-in-place every cycle
for 15 minutes with 0.1 M NaOH. The yield was consistently 95–100%, and purity levels were consistent, as measured by removal
of host cell proteins. There was no detectable carryover and no signs of discolorations or deposits on the resin. The dynamic
binding capacity remained at greater than 85% of the initial value.
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