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Process

Analytical

Technology

Process Analytical
Technology in
Biopharmaceutical
Production: Past Successes
and Future Challenges
Tina M. Larson and Harry Lam, Ph.D.
Biochemical Manufacturing Technology Group, Genentech, Inc.

Introduction
first difference means that the favorite PAT technology of the pharmaceutical industry, near-infrared spectroscopy (NIR), may be of limited
use in biotechnology. The second difference means that product quality measurements need to be inferred from process data, or a major
effort needs to be made to develop simpler analytical technologies.
Even with these challenges, there are PAT applications that are well
established in the pharmaceutical and food industries that could be
applied to biopharmaceutical manufacturing.
NIR as a tool for moisture detection is a relatively mature technology in the pharmaceutical and food industries. Instruments for this
application are commercially available. NIR could also be used to perform moisture detection on lyophilized protein products.
Lyophilization is a common formulation for protein pharmaceuticals,
and moisture content in final product is typically part of certificate of
analysis testing for lyophilized products. Current chemical titration
methods for moisture detection, such as Karl Fischer, require that a
representative sample of vials be removed from each production lot.
These samples are then tested using meticulous laboratory techniques
to determine moisture. The benefits of NIR would be that a larger
number of final product vials, perhaps even all vials, could be tested
using a relatively simple procedure. In addition, the testing could be
done in real-time, which would decrease lot release time. Genentech
has done preliminary investigations using four different products to
evaluate the feasibility of NIR for moisture detection in final product
vials. The results, presented in figure 1, indicate that NIR yields moisture values consistent with the Karl Fischer method of detection.
Another analyzer technology that could be easily adapted hails from
the brewing industry. They have broad experience using capacitance,
also know as radio frequency impedance, probes to measure viable cell
concentrations of suspended yeast. This technology can replace the
traditional cell count methods that use exclusion dyes to distinguis

he concept of process analytical technology (PAT) is not new,


but it has received considerable attention in recent years as the
FDA continues to unveil its PAT initiative. The FDA is optimistic that it can encourage industry to improve the state of their manufacturing technology by reducing the regulatory burden that currently hinders continuous process improvement. The FDA believes this
will benefit the public by decreasing the amount of regulatory oversight required, while continuing to ensure a high level of product quality. Of course, there are numerous challenges to realizing the FDAs
vision. The benefits and challenges of the PAT initiative are currently
being discussed at a variety of conferences and in publications [1,2,3].
This article will not focus on the PAT initiative itself; instead it will
give an overview of the technologies that show promise for improving
manufacturing in the biotechnology industry.
One good reason to leave discussion of the PAT initiative to authors
from pharmaceutical companies is that the draft guidance for the
initiative specifically excludes CDERs Office of Biotechnology
Products [4]. However, the benefits, and challenges, related to PAT
implementations are just as applicable to biotechnology companies as
they are to pharmaceutical manufacturers. Therefore, it is still worthwhile for biopharmaceutical manufacturers to evaluate their need and
ability to implement new technologies. It is also very possible that the
final version of the PAT guidance will apply to biopharmaceutical
manufacturing.

Process Analyzer Technology


Biotechnology processes present technical challenges different from
those in the pharmaceutical industry. Two important differences are 1)
the majority of bioprocessing occurs with the product in aqueous solution and 2) direct measure of protein purity typically can only be
accomplished using complex assays such as HPLC or ELISA. The
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NIR analysis of moisture content in lyophilized vials was shown to be


comparable to the Karl Fischer method for the four different proteins
studied.
Figure 1

A PLS model was developed using over 1600 fermentation process variables per batch. The model was able to predict final titer with a rootmean-square error of prediction (RMSEP) of 10%

between dead and viable cells. Traditional cell count methods require
that a sterile sample be withdrawn from the fermenter, diluted to a concentration appropriate for the current cell density, and counted under a
microscope by a trained technician. Replacing traditional methods
with capacitance probes eliminates risks from sampling, provides continuous data in real-time, and eliminates variability due to human
counting techniques. In addition, the availability of real-time viable
cell data opens up the possibility of implementing improved nutrient
feeding strategies. Capacitance probes are commercially available for
use in yeast, bacterial, animal and insect cultures.
The use of NIR for moisture detection and capacitance for viable cell
count are both examples of process analyzer technologies that are commercially available and have been proven suitable for use in GMP
environments. Despite the potential benefits of each technology, neither is commonly used in the manufacture of biopharmaceuticals. The
cost of implementation and risk of regulatory impact make the implementation of new technologies unattractive to companies. Further, it is
difficult to justify the benefit of such technologies when companies are
currently able to produce and release quality product. New analyzer
technologies clearly have the potential to improve process monitoring
capabilities; however, it is often difficult to demonstrate that the company will realize a benefit from improved process monitoring capability. On the contrary, there is often resistance to increased process monitoring because of fear that a previously unknown phenomenon will be
discovered and become open to regulatory scrutiny. If the PAT initiative realizes its full vision, this attitude will no longer be an impediment to manufacturing technology development. However, realizing
the vision of the PAT initiative will take effort from both industry and
the FDA to change the way manufacturers think about continuous
process improvement.
Assuming that biopharmaceutical companies will strive for new analytical technologies in the future, there are a number of interesting possibilities for the industry. The efforts currently underway to investigate
NIR for raw material identification in pharmaceutical production [5]
should be directly applicable to raw material characterization in bioprocess applications.
In regards to process monitoring,
the spectroscopic techniques currently being evaluated by the pharmaceutical industry, such as NIR and Raman, may also prove to be useful
in monitoring bioprocesses. A recent study of NIR for determining
amino acid concentration in chromatography pools [6]
indicates that NIR may be useful in aqueous processing streams. For
fermentation monitoring, fluorescence based sensors have shown some
early promise [7]. Preliminary research into the use of biosensors for

Figure 2
monitoring of specific nutrients or metabolic products in fermentation
has also shown potential [8]. The current focus on biosensors for
detection of biochemical warfare agents may give biosensor research a
boost that will eventually provide benefits to biopharmaceutical manufacturers.

Data Technology
In the past, process data extraction and data analysis techniques may
not have been considered part of the PAT field. However, the FDAs
broad definition of PAT in the draft guidance has brought process data
analysis to the forefront of manufacturing improvement. This is actually good news for industry because the cost of analyzing current data
points to gain additional knowledge is significantly less than the cost
of installing new process analyzers. Further, improved data analysis
techniques can often be implemented without the need to file regulatory submissions. Because the cost and risk of new data technology projects is much lower than for analyzers, this field of PAT will potentially see the most growth in the near future. The following paragraphs
will give two examples of data technology projects that have contributed to increased understanding of bioprocesses.
Fermentation continues to be the most studied aspect of biopharmaceutical manufacturing because it is both critical to product quality and
quantity, and it is still not well understood. Fermentations are a complex mixture of raw materials that are often not well characterized, and
living cells that behave in manners not fully understood. Process analyzer data (pH, temperature, pressure, dissolved oxygen, optical density, oxygen uptake rate, etc.) can be combined with supporting information (raw material analysis, timing and duration of feeds, manual
cell counts, metabolite levels, etc.) to generate large sets of fermentation data. However, using the data to draw meaningful conclusions
about the process is very difficult with traditional x-y plots and simple
statistics. This has led researchers to start turning to chemometrics in
hopes of gaining increased process understanding [9,10]. Figure 2
illustrates a partial least squares (PLS) model that was developed for a
bacterial fermentation process. The model was created with over 1600
variables per batch, and can be used to predict final titer with reasonable accuracy. By applying chemometric techniques to historical sets
of processing data, or new sets of experimental data, information can
be derived that will increase process understanding with minimal capital investment.
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Acknowledgements
The authors would like to acknowledge Tanya Moy-Lin,
Kirin Jamison and Jeff Davis of Genentech for their
contributions to the work presented in the figures of
this article.

References
1. Miller, R.W. Process Analytical Technologies (PAT)
Part 1. American Pharmaceutical Review, Vol. 5,
Issue 2, Summer 2002.
2. Miller, R.W. Process Analytical Technologies (PAT)
Part 2. American Pharmaceutical Review, Vol. 6,
Issue 1, Spring 2001.
3. Arrivo, S.M. The Role of PAT in Pharmaceutical
Research
and
Development.
American
Pharmaceutical Review, Vol. 6, Issue 2, Summer 2003.
4. PAT - A Framework for Innovative Pharmaceutical
Manufacturing and Quality Assurance Draft
Guidance.
Available from the FDA website:
www.fda.gov/cder/gmp/
5. Robbe, C. L. et. al. Exploring Pharmaceutical
Height equivalent of a theoretical plate (HETP) is calculated using continuous
Applications of Near-Infrared Technology. American
process data in order to monitor efficiency on a chromatography column. As HETP
Pharmaceutical Review, Vol. 6, Issue 3, Fall 2003.
increases (decreased efficiency), the yield drops significantly.
6. Goode, J. The On-line Analysis of Aqueous Based
Products from a Separation Column using Near
Figure 3
Infrared Spectroscopy. IFPAC 2004 Meeting Abstract,
January 2004.
7. Skibsted, E., Lindemann, C., Roca, C., Olsson, L. OnEven for processes that are relatively well understood, such as chroline bioprocess monitoring with a multi-wavelength
matography purification operations, there are benefits to be derived
fluorescence sensor using multivariate calibration. J.
from improved data technology. Successful chromatography operaof Biotech. 2001, Vol. 88, pp. 47-57.
tions are key to final protein yield and quality. Chromatography
8. Ge, X., Tolosa, L., Simpson, J., Rao, G. Genetically Engineered
column performance is traditionally measured by introducing a pulse
Binding Proteins as Biosensors for Fermentation and Cell Culture.
of high concentration solute buffer at the column inlet, and using the
Biotechnol. and Bioeng., 2003, Vol. 84, pp. 723-731.
response at the column outlet to determine the efficiency of the col9. Larson, T., Gawlitzek, M., Evans, H., Albers, U., Cacia, J.
umn. Column efficiency is given as a height equivalent of a theoretiChemometric Evaluation of On-line High Pressure Liquid
cal plate (HETP). Low HETP values correspond to highly efficient
Chromatography in Mammalian Cell Cultures: Analysis of Amino
columns. As production run rates and column flow rates increase, rouAcids and Glucose. Biotechnol. and Bioeng., 2002, Vol. 77, pp.
tine pulse-input HETP testing becomes increasingly difficult. To
553-563.
address this issue, existing in-process trend data (pH, conductivity,
optical density) can be used to determine a columns HETP value [11]. 10. Bradamante, S. et. al. Production of Lovastatin Examined by and
Integrated Approach Based on Chemometrics and DOSY-NMR.
An example of this technique is given in figure 3, where increasing
Biotechnol. and Bioeng., 2002, Vol. 80, pp. 589-593.
HETP values corresponded to decreasing step yield. Calculating
HETP in this manner requires sophisticated data extraction, transfor- 11. Larson, T. Davis, J., Lam, H., Cacia, J. Use of Process Data to
Assess Chromatographic Performance in Production-Scale Protein
mation and analysis tools. However, the information gained is of obviPurification Columns. Biotechnol. Prog. 2003, Vol. 19, pp. 485ous benefit. For the case presented in figure 3, in-process HETP val492.
ues can be used to determine the optimal time to repack the column.
Tina M. Larson is a Senior Engineer and Group Leader in the
Biochemical Manufacturing Technology group at Genentechs
South San Francisco facility. She holds a B.S. in Chemical
Engineering from Colorado State University. Her research interests include on-line nutrient monitoring in fermentations and
analysis of chromatographic production data. Correspondence
should be addressed to: tinam@gene.com
Dr. Harry Lam is the Director of Biochemical Technology at
Genentech. Before joining Genentech in 1996, he spent eleven
years in the Bioprocess R&D department at Pfizer. He has over
eighteen years of experience in bioprocess R&D and large-scale
biochemical manufacturing operations. Dr. Lam received a B.Sc.
degree in Chemical Engineering from University of Birmingham,
U.K., and Ph.D. degree in Chemical Engineering from Rensselaer
Polytechnic Institute.

Conclusions
Although the FDAs PAT initiative is not yet applicable to biotechnology products, it seems reasonable to believe that success of this initiative in drug manufacturing will have an effect on manufacturing of
biologics. There are a number of technologies that manufacturers of
biotechnology products can consider as they strive to obtain better
understanding of their processes, and to improve quality and manufacturing efficiency. The food and pharmaceutical industries have already
paved the way for implementation of many of these technologies, making it even easier for adoption into biopharmaceutical manufacturing
facilities. And even if new analyzer technologies are not embraced by
the industry, significant benefits can be derived by focusing on data
technology improvements that maximize the value of existing analyzers. Either way, biopharmaceutical manufacturing is poised to see significant improvements in process technology in the coming years.

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