Liquid chromatography has been successfully employed in pharmaceutical PAT applications for decades. Of particular note are
the pioneering examples of on-line high performance liquid chromatography (HPLC) applications by Eli Lilly in biotechnology
applications.7 On-line downstream applications of HPLC for biotechnology are now widely accepted and deployed. Commercial systems for biotech
process monitoring are available from Dionex and other suppliers. This technique is mature, well characterized, and well accepted
in all fields of chemistry separation including biotechnology. The system also requires capture of a physical sample from
the process stream while maintaining sterility of the stream. Recent advances in on-line sampling for biotechnology, including
introduction of commercial products that maintain process sterility, will further increase utilization of the HPLC technique
in downstream applications.8 The chief drawback of this technique for process characterization appears to be the complexity of fully automated HPLC systems
and their attendant control processes. These include column switching, fast column equilibration, and high flow systems, all
of which are strategies employed to reduce cycle times to remain consistent with downstream needs.
Electrophoretic techniques, most notably slab gel electrophoresis for protein and genetics analysis, have been a mainstay
of biotechnology development analytical laboratories since its commercial inception in the 1970s. Slab gel electrophoresis
is simple in concept, inexpensive to acquire, does have high operating costs, and has cycle times inconsistent with the needs
of downstream processing. Capillary electrophoresis (HPCE), was introduced in the late 1980s and automates the electrophoresis
process in a format similar to HPLC. Many commercial instruments are available today. Data are quantitative in contrast to
slab gel techniques. It is rapid. It has recently found widespread application for protein analysis in off line laboratories
operated in support of downstream applications, specifically for protein product quality, characterization, and quantitation.
The technique is inherently fast, versatile in the biotechnology environment, and amenable to the demands of protein assay
in the downstream process environment. The technique is employed daily in this capacity, but still has failed to gain wider
appreciation and acceptance for downstream processing assay requirements because commercial instruments have been designed
exclusively for standard benchtop laboratory use. Manual sample acquisition, preparation, sample introduction, and data reduction
are the rule as is the fact that most HPCE systems must be operated by HPCE specialists. These instruments have remained exclusively
in the province of remote laboratories operating in support of downstream operations and these instruments do not promote
the advantages of PAT technology in downstream operations.
New Downstream Technology
We are introducing an on-line and at-line process analysis technology specific for protein identification and quantitation
in a downstream process application. This instrument9 fully automates the entire process and is purposely built for on-line assays for protein identity and titer as envisioned
by the FDA PAT initiative. The instrument incorporates and can be used on-line with an automatic, sanitary sample port interface,
or can be used at-line with samples introduced by the instrument's autosampler. In either case, the entire sample introduction,
preparation, separation, detection, and data reduction are performed automatically by the instrument without operator input.
Once the sample is introduced to the instrument it is transferred directly to the sample preparation module, which delivers
an appropriately prepared sample to a HPCE module that automatically performs the separation. Detection is performed by an
ultraviolet absorbance detector. Data reduction and reporting are performed by a data system based on standard HPLC data and
user interfaces. The concentration dynamic range of the instrument is from ?g/mL to g/mL due to the internal automatic dilution-concentration
system component of the sample preparation module. Molecular weight range for the instrument is 10 to 200 kD. Calibration
of the instrument is through a timed, automatic introduction of calibration standards in a manner typical of calibration for
similar HPLC systems. Cycle times are consistent with the needs for most downstream processing.
Biotechnology manufacturing, particularly downstream processes, has many operations and opportunities suited to the premise
of the PAT initiative—namely, improving production efficiency, yield, and product quality. Several technologies have been
implemented in downstream processing for the purpose of on-line assays that have proven the concept. New, versatile instrumentation
technologies for downstream process applications will continue to emerge as the PAT initiative becomes more fully implemented
in biotechnology and biological manufacturing.
George Barringer, PhD, is president and CSO of Groton Biosystems, 85 Swanson Road, Boxborough, MA 01719, 987.266.9222, fax 978.266.9223