ABSTRACT
Case studies were run to test Process Analytical Technology applications for protein refolding, diafiltration, and cation
exchange chromatography. It is shown that it is feasible to design control schemes that rely on measurement of product quality
attributes and thereby enable real-time decisions. Implementation of these schemes should result in consistent product quality
and high operational efficiency. However, these advantages are balanced by requirements of a higher level of process understanding
for designing these schemes, and increased operational complexity during implementation. Increased consistency in product
quality is likely to increase variability in step recovery, a parameter that is commonly considered an indicator of process
consistency.
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The biopharmaceutical community is interested in using Process Analytical Technology (PAT) for continuous real-time quality
assurance. PAT has the potential to improve operational control and compliance. The operational definition of PAT is as "a
system for designing, analyzing, and controlling manufacturing through timely measurements (i.e., during processing) of critical
quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product
quality."1
Several case studies demonstrating the utility of this approach in formulation unit operations such as granulation and compression
have been recently published.2 Fewer studies have addressed bioprocessing unit operations mainly because they are difficult to control, and subject to
the increased variability and relatively limited understanding of raw and feed materials.3,4
A desired goal of the PAT framework is to design and develop well-understood processes that will consistently ensure a predefined
quality at the end of the manufacturing process.
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A process is generally considered well understood when (1) all critical sources of variability are identified and explained;
(2) variability is managed by the process; and, (3) product quality attributes can be accurately and reliably predicted over
the ranges of acceptance criteria established for materials used, process parameters, manufacturing, environmental, and other
conditions.
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This article focuses on the feasibility of creating and implementing control schemes that enable real-time and product quality
decisions for commonly used biotech unit operations, which include protein refolding, process chromatography, and ultrafiltration–diafiltration.
This is the sixth article in the "Elements of Biopharmaceutical Production" series.5 We present below three case histories that use the PAT framework to improve process quality.
PROTEIN REFOLDING
Protein refolding typically runs with a time-based recipe. The time is set based on the rate of refolding (as determined by
process development studies) and operational constraints (manufacturing shifts). There are two consequences of performing
refolds by fixed time. The refold step produces varying product quality as measured by percent purity or percent impurities
(e.g., reduced and oxidized forms of product, misfolds, etc). The refold rate will vary from lot to lot due to variability
in feed materials, and so refold time is often set conservatively. This makes production operating costs higher than optimum.
We ran a study to evaluate PAT methods for controlling a protein refolding step. In our test, frozen cell paste was resuspended
using a Silverson L4R high-shear mixer (Silverson Machine, Chesham, Bucks, UK). A Model 100Y Microfluidizer (Microfluidics,
Newton, MA) was used to perform cell lysis. The lysate and the subsequent wash suspension pellets were spun down using a Beckman
J6-B bucket centrifuge with a JS 4.2 rotor (both from Global Medical Instrumentation, Ramsey, MN). The refold was performed
in a Biostat MD 12 fermentor (Sartorius BBI Systems, Bethlehem, PA). Post-refold filtration was carried out using a CUNO 60
SP depth filter (CUNO, Meriden, CT).
