Figure 1 illustrates the refold process. As time progresses the unfolded protein (peak B) is oxidized to produce the product
(peak A). The peaks designated by numbers are other impurities that are also generated during the refold process.
Figure 1. Purity profile during refolding. Chromatograms show the progressive oxidation of B to A.
We wanted to design a PAT-based control strategy using on-line monitoring that would allow refolding operations to end
at a time determined by product quality parameters (percent purity of the product or percent impurity). To generate data for
such a strategy, samples were withdrawn at fixed time intervals, quenched, and transferred to the auto sampler for analysis
using an Agilent 1100 Series high performance liquid chromatography (HPLC) system with a binary pump, micro vacuum degasser,
thermostatted autosampler, thermostatted column compartment, and a multi-wavelength detector (Agilent Technologies, Palo Alto,
Figure 2 presents profiles of the product (percent purity), reduced form of product (percent Impurity 2), and other impurities
(percent Impurities 1, 3, and 4) over the refold time. The data suggest that it is feasible to implement a PAT-based control
scheme that allows for ending refold based on product quality data. This would ensure consistency in product quality of the
refold end pool, and improve operational efficiency by keeping refold time to completion (10 h may suffice versus 16 h presently
Figure 2. Purity and impurity profiles as generated by on-line HPLC
This PAT-based control scheme does pose several problems. First, operations are simpler with time-based recipes. In the proposed
scheme, the refold time will vary from run to run so it can generate the process stream of consistent product quality. The
likely result is a need for more dynamic planning and scheduling of manufacturing steps. Second, this scheme would require
that the manufacturing operators be trained to interpret and act upon the data from the HPLC. Third, since the decision to
end the refold is now based on HPLC data, it is critical that the analytical methods have a high degree of robustness. It
may be necessary to have redundancy (e.g., duplicate analysis) built into the control scheme to ensure accuracy of the data.
Implementation of a PAT-based control scheme for a protein-refolding unit operation is feasible and will offer several benefits.
This will be particularly attractive for cases when one or more impurities are created in the refold process and excess refold
time is likely to result in higher levels of one of them.
The diafiltration step of the UF–DF process is often specified for a fixed number of diavolumes. The number is based on process
development studies and, as for refold time, the number is often set conservatively, resulting in excess buffer use and process
time. In this study, we wanted to design a PAT-based control strategy which would allow us to end the UF–DF step when the
diafiltration process is complete, as signaled by a product quality criterion. This would result in consistent product quality
at the end of the step and would also minimize the buffer usage and operation time.