Existing State of the Art
The primary analysis requirement in biotechnology downstream processing is molecular identification and quantitation; used
to identify and quantitate the product to determine its purity after separation from its production matrix by process liquid
chromatography. Therefore, the most powerful and useful analytical techniques to consider for downstream processing in the
PAT context are spectroscopy and analytical scale separation methods that can characterize analytes on a molecular basis.
A typical downstream process scale chromatographic technique not only purifies but concentrates the analyte and is operated
at high load factors and high flow rates, therefore, the associated analytical technique must have a wide dynamic range and
a fast cycle time. Typical cycle time requirements for on-line downstream assays are under 15 min, with many under 10 min.
This cycle time requirement plus the need to separate, characterize, and identify the desired product in an often complex
matrix composed of chemical species similar to the desired product, requires precise, high speed, purpose-designed instrumentation
characterized by high accuracy, reproducibility, and resolving power. This combination of requirements limits the discussion
of applicable downstream biotech PAT instrumentation to near infrared spectroscopy (NIR) and other specialty optical spectroscopy
techniques, such as mass spectroscopy, liquid chromatography, and electrophoretic techniques.
NIR has enjoyed some success in pharmaceutical manufacturing operations, primarily in blending and formulations of dry bulk
materials. Its use in on-line downstream processing applications for identification and quantitation of product is less established,
but its possibilities should not be overlooked. Practical on-line NIR analyses for biotechnology applications have been demonstrated.6 Factors that will control use of NIR for downstream process applications include probe design and placement, calibration
methodology, and heavy reliance of NIR on statistical analysis to calibrate and report concentration and identity of analytes.
In many applications, a major advantage of NIR is non-contact. In these applications, the spectrometer is not in physical
contact with the sample and, therefore enjoys long and reliable operation. In the case of a downstream process application
however, the spectrometer samples the analyte by inserting an optical probe into a product transfer line at an appropriate
sample point. This has the advantage that the sterility of the downstream system is maintained and that there is no physical
withdrawal of sample from the process line. However, with the probe in direct contact with the fluid sample matrix, proper
orientation and maintenance of this probe is critical to the technique's success. Fouling resulting from deposition of biological
material on the probe quickly renders the measurement unusable. Furthermore, the low frequency characteristics of the spectra
produced in NIR requires heavy reliance on calibration by multivariate solution of simultaneous equations. The sample and
its matrix must be fully characterized and non-variate for the calibration to remain valid. This is not a simple task in a
complex sample and matrix characteristic of biotechnology production. The speed and simplicity of instrumentation do, however,
make it appropriate for consideration for downstream process applications and early applications of this technology appear
Mass spectroscopy (MS) is a powerful and well appreciated tool, finding broad acceptance in a number of non-traditional applications
including biotechnology. For example, mass spectrometers have been installed and used in upstream applications for measurement
of headspace gas analysis in cell culture and fermentation reactors, to monitor O2 and CO2. Mass spectrometry can be used to determine absolute analyte identity and for quantitation—both desirable attributes. It
is rapid and has high resolving capability. In downstream processing, a mass spectrometer could however, acquire a sample
stream from an automated sample port or sampling system at the appropriate point in the process stream. Mass spectrometer
applications in a downstream process are effectively prohibited by typical high salt content found in many biotech process
streams. All known commercial fluid interfaces for mass spectrometers are characteristically intolerant of salt content in
the fluid stream. Development of a successful salt resistant or salt remediating interface would open downstream processing
to the power of MS. This would appear to be the primary and only limiting factor in successful, robust application of MS in
biotech process applications, notwithstanding the high acquisition and operating cost of a mass spectrometer.