Chromatography is an integral step in downstream bioprocessing, and buffered mobile-phase properties such as ionic strength
and pH are often critical process parameters for protein purification. The interactions between the mobile and stationary
phases can affect chromatographic performance, and thus the separation profile between product and impurities. Product recovery,
purity, and throughput are often marred by the variability in buffer preparation during production-scale bioprocessing. The
popular approach to biological production typically relies on postchromatographic adjustments (e.g., fractionation controlled
by ultraviolet (UV) signal and segregation of out-of-specification product through quality-control testing) to achieve the
desired product quality. Although the US Food and Drug Administration accepts this traditional practice, this post-process
approach to quality control is time-consuming, increases production cost, and does not provide the process understanding and
control from the quality-by-design (QbD) approach that FDA promotes.
Process analytical technology
In accordance with FDA's Pharmaceutical CGMPs for the 21st Century initiatives, companies should employ process monitoring and control strategies to streamline biopharmaceutical manufacturing.
Real-time process knowledge can help manufacturers avoid excessive rework and discarded product. To achieve this standard
of quality, FDA highly recommends the implementation of process analytical technology (PAT) to facilitate a QbD approach.
According to FDA, the working definition of PAT is "a system for designing, analyzing, and controlling manufacturing through
timely measurements (i.e., during process) of critical quality and performance attributes of raw and in-process materials
and processes with the goal of ensuring final product quality" (1).
Many modern technniques for process analysis such as pH measurement, near-infrared spectroscopy, Raman spectroscopy, refractive-index
changes, humidity movement, UV monitoring, conductivity detection, and dissolved-oxygen analysis are commercially available
for different applications. These analytical tools can measure physical and chemical properties of raw materials directly
in real time, in contrast with techniques such as flow-meter monitoring, which only makes indirect measurements.
Applications of PAT in downstream bioprocessing
In the past 10 years, protein-expression titers have increased 1000 times from mg/L to g/L. As upstream yields continue to
increase, downstream bioprocessing involving buffer preparations and delivery also must increase proportionally to keep pace
with demand. Therefore, conventional practices of preparing and delivering buffers have to be modified or replaced with new
manufacturing sciences (see Figure 1). This principle is especially true for industrial-scale quantities of buffers that must
be reproducibile and meet tight specifications. Scientists at Genentech (South San Francisco, CA) proposed using mass-flow
meters to dilute buffers from concentrates, which is a great improvement (2). The scientists described the dilution of concentrated
acetone, which could be monitored by an in-line optical-density meter. Additional monitoring through feedback control of conductivity
and pH can complement the design and reduce the variability of buffer feedstock, preparation, and delivery frequently encountered
Figure 1: Conventional system for buffer preparation and delivery. CGMP is current good manufacturing practices, CIP is clean-in-place,
and QC is quality control. (ALL IMAGES ARE COURTESY OF THE AUTHORS)