Mikrochim.

Acta [Wien] 1991,II, 167-176 IRikrochimica

Acta
9 by Springer-Verlag1991
Printed in Austria

Challenges in the Relation Between Analysis and

Biotechnology

Bauke te Nijenhuis*
International Bio-SyntheticsB.V.,Patentlaan 3, NL-2288 EE Rijswijk,The Netherlands

Abstract. Analytical methods and systems for biotechnological applications are

becoming increasingly important. The development of these methods and sys-
tems ask for an interdisciplinary approach. The use of analytical methods and
systems in daily practice in biotechnological research, development, application
and industrial production is essential for progress in biotechnology. Analytical
methods and systems are not solely used for monitoring of research, develop-
ment and production of biotechnological products but also goal in itself being
used in many applications like medical, environmental, etc. This lecture presents
an overview of current progress in analytical chemistry in relation to biotech-
nology. Special selected items have been treated into more detail.
Key words: analytical chemistry, biotechnology, process control, contaminant
monitoring.

When we discuss biotechnology we have to realize that this sector is not only one
of the most rapidly growing areas in industry, but also is one of the oldest technolo-
gies 'in human history.
Biotechnology by definition is the integration of the basic disciplines bio-
chemistry, genetics, microbiology and process technology in biological systems on
behalf of industrial processes and the environment. This definition not only includes
the activities of the traditional fermentation industry, but gives also entrance to new
fields of biotechnology as commercialized plant tissue culture, its use for production
of important plant related chemical compounds, the use of animal, more specifically
mammalian cells for special purposes and the rapidly growing field of monoclonal
antibodies.
More than 10000 years ago biochemical processes were already used for spon-
taneous fermentation of food, resulting in better digestibility, better taste, improved
stability and tenability, etc. Just think of the leavening of bread and the production
of a wide variety of fermented food and beverages.

Gist-brocades N.V., Researchand Development,P.O. Box 1, NL-2600 MA Delft,

* Present address:
The Netherlands
168 B. te Nijenhuis
Selection procedures through the centuries and even in this century were based
on natural selection and mutation, resulting in natural genetic modification. Suc-
cessful products from this classical biotechnology are penicillin and enzymes for
industrial, pharmaceutical and food applications. Acceleration of these in principle
slow selection processes took place by the introduction of cell fusion, recombinant
DNA techniques and protein engineering. The application of these techniques into
industrial processes resulted in the momentum of biotechnology today.
Marly new products have been introduced in several areas [i]:
- - pharmaceutical products: therapeutics (e.g., human insulin, interferons, human
growth hormones, interleukins, hepatitis B vaccine, etc.); diagnostics (over 300
new applications);
- - a g r i c u l t u r a l products: crop protection (biopesticides), animal therapeutics,
diagnostics;
- - specialty products: enzymes, specialty chemicals, BiofineTMchemicals.
In general we can state that products in the medical area, agrobusiness and industrial
business are still mainly the result of research on new applications with the aid of
micro-organisms. But the developments in plant tissue culture and mammalian cell
usage are that fast, that in due time a range of products from these sources can be
expected.

Analytical Chemistry and Biotechnology

How can analytical chemistry cope with this rapidly growing sector.
Let us first look at a break down of the main technologies in development today
(Fig. 1). One can recognize the grow-up of biotechnology from embryonal state
growing to maturity. One also may expect much work in the R&D phase in the
left-hand part of the figure, whereas control of production processes will play an
important role in mature processes.
This gives also a rapid insight of what will be expected of the analytical discipline:
guidance in the early R&D phase, assistance in the development of products and
processes and finally process and product quality control.

Classical rnicr o bi~o~4_

Fermlnt~ Enzyn

R-DNA: Micro-org ~11" Bio-detection (e)

Biocatalysis~ Immuno-analysis
R-DNA: Ani

ir'DNA-probes
Bio-sensors

Ernbryonal Growing Mature Ageing

Fig. 1. Biotechnotogylife cycle.Source:Arthur D. Little (ref. [-2])

 Challenges in the Relation Between Analysis and Biotechnology 169

Medical Food Agriculture Veterinary Environment Forensic Defence Other

Analytical
chemistry x x x x x x x x x x x x x x x x x x x x x x

Classical
microbiology xx x xx x xx xx x xx x x x? xx x
Enzymatic xx x x x x x x x? x
Immunology xx x x (x ) xx (x ) x x? x x
Biosensors (x ) (x ) (x)
DNA probes (x ) (x) x ?

Fig. 2. Current status: Technology versus application ( x x strong impact, x moderate impact,
limited impact, ( ) first product introductions). Source: Arthur D. Little (ref. [2])

What has the analytical discipline available? In the days of classical biotech-
nology and fermentation the c o m m o n physical chemical techniques were used with
an extension to microbiological methods, mainly used for contamination detection
and sometimes for activity determination. O n relatively short notice biochemical
analytical procedures have found their way into biotechnology and nowadays the
analytical range has been extended with enzymatic methods, immunology, bio-
sensors and DNA-probes, more generally called: biodetection. The current applica-
tion status is shown in Fig 2.
However, analytical chemistry is not only fast in introducing the new bio-
chemical methods in its package, but is also working very hard on applications of
existing physical chemical methods in biotechnology with surprising results. One
recognizes that modern biotechnology is not only able to produce new products for
use in society, but also capable to prepare analytical devices for testing purposes in
the biochemical discipline.
iTor more indepth information, I would like to draw your attention to a series
of symposia dedicated to "Analytical Methods in Biotechnology", the so-called
Anabiotec Symposia held in Noordwijkerhout in the Netherlands in 1984 and 1988.
This international symposium has now flown over the Atlantic and has been held in
October 1990 in San Francisco. Noordwijkerhout is the venue again in 1992 [3].
As a second option to mention is the Annual Seminars on Analytical Biotechnology
under the chairmanship of Professors Karger and Regnier, dedicated to special
subjects. This year's meeting was dedicated to monitoring of genetically engineered
product composition and purity, especially for use in the pharmaceutical drug
industry [4].

Major Analytical Techniquesin Biotechnoiogy

Although almost all classical physical chemical analytical techniques have been
applied in biotechnology, the major techniques of today are [2, 4]: HPLC, mass
spectrometry, nuclear magnetic resonance, FIA (flow injection analysis), electro-
phoresis, and combinations of techniques.
170 B. te Nijenhuis

As an extension a second group of analytical techniques, called biodetection, has

to be mentioned. Biotechnology has discovered and developed new analytical
methods and tools based on the specifity and selectivity of several types of bio-
chemical selectors such as enzymes, antibodies and DNA-probes. The biodetection
group includes also the area of biosensors. Whether these devices ever will surpass
the level of academia is still questionable. Let us have a view on how and where
some of the techniques mentioned above are used.

HPLC
HPLC is seen as the most powerful separation technique in the field of sciences. A
very broad supply of columns for all types of biomolecules is available [6]. High
speed separations, using short, small particle columns will become more common.
Combining HPLC with electrophoresis will be reality in future and sensitivities
enhancement will even override the femto- and attomole level.
For structural analysis, the combinations liquid chromatography (LC)-mass
spectrometry (MS) and capillary zone electrophoresis (CZE)-LC-MS seem to be the
most promising techniques of the future [7]. Interfacing is quite simple by the usage
of techniques like electrospray ionization, which can be used for both low and high
molecular weight compounds.
For therapeutic drug monitoring in the hospital, e.g. studies on effectiveness,
stability, biotransformation, excretion, etc., HPLC is the best choice because of its
relative good tolerance of complex biological matrices. In this respect HPLC can
compete with the also often used ELISA assays. Pharmaceuticals, monitored by
HPLC are tabulated by Kulinsky [8] (Fig. 3).
Chiral separations are extremely important because of the fact that as a rule only
one of the enantiomers is physiologically active. Several systems are available: chiral
stationary phase, chiral modifiers in the mobile phase or precolumn derivatisation
with chiral agents [7]. The FDA is proposing making the screening of all pharma-
ceuticals for optical activity a legal requirement.

Penicillin Verapamil
Levodopa p-Aminosalicylic
acid
Cephalosporin Imipramine
Codeine Isoniazid
Aminoglycosides Captopril
Cimetidine Trimethoprim
Chloramphenicol Extromethorphan
Acyclovir Wafarin
Erythromycin Carbamazepine
Cyclosporin Carbutamide
Tetracycline Chlorpromazine
6-Mercaptopurine Econazole
Clofibrate Benperidol
Theophyline Chloroquine
Dapsone Clonazepam Fig. 3. Pharmaceuticals assayed by HPLC. Source:
Kulinsky (ref. I-8])
Challenges in the Relation BetweenAnalysisand Biotechnology 171

Mass Spectrometry
MS has proven its applicability for both fermentation control and structure informa-
tion on biomolecules. Molecular weight information on protein up to about 25-
50000 Dalton can be obtained by means of plasma-desorption mass-spectrometry.
Sequence informative fragmentation of peptides have been reported as a joint effect
from industry (Gist-brocades, The Netherlands) and university (Leiden) [9]. PDMS
might thus become an alternative for Fast Atom Bombardment MS as tool for
protein sequencing.

FIA (Flow Injection Analysis)

Since its discovery by Ruzicka and Hansen the importance of FIA as a method for
both routine analysis and on-line process control is well recognized. FIA is simple,
flexible and allows combinations with many detection devices. However, on-line
control of bioprocesses causes problems, especially with sterile sampling. This is in
general a problem in on-line control of fermentation and many groups have tried
to find solutions for this sampling problem. Sophisticated filter systems, membrane
systems based on dialysis or ultrafiltration or by-pass systems are proposed [10]
and tested in practice.

Electrophoresis
Capillary electrophoresis proves to be an almost perfect separation method for
water-soluble components in small sample volumes. Three execution modes are used
in practice: capillary zone electrophoresis, capillary gel electrophoresis, and separa-
tion of unchanged molecules by complexation and inclusion methods.
Applications include separation of proteins and peptides, tryptic mapping, DNA
sequencing, serum analysis, etc. DNA sequencing is the hottest topic because of the
interest in determining the human genome [11, 12]. In combination with LC and
MS a very powerful combination can be set up.

Near Infrared Spectroscopy

A technique not mentioned normally among important analytic techniques is NIR
spectroscopy. A combination of accurate measurements, available expert files and
chemometric treatment of the data makes NIR a technique for reliable determina-
tions in known matrices. However a big minus is that disturbing, matrix-strange
components are disastrous for the measurement.
The system in itself is perfectly suited for at-line measurements and equipped
with fibre optics also suitable for on-line measurements. However, the costs of the
optical fibres is a constraint and also eventual fouling of the fibres might cause
problems. Whether the method is suitable for particular biochemical situations has
to be determined case by case [13]. Anyhow, an interesting feature for special well
considered situations.
172 B. te Nijenhuis

Biosensors
Because of the still high expectations of this special area in biodetection some critical
remarks are in order. Biosensors can be defined as devices which convert biological
events into electronic output signals. A more practical definition is "biosensors are
sensors measuring in biological systems", thus including also pH and selective
electrodes in the biosensor definitions. A lot of biosensors are known. Fig. 4 gives
an overview of which various biological elements and transducers are being used in
biosensor devices.
About 100 examples have been published now, of which some 10 are commer-
cially available. Most of the applications of biosensors are in medical and clinical
diagnosis with a spin-off to biotechnology and environment [15, 17]. Although the
enthusiasm in academia is undiminished high and the expectations in the market
place also are still high, it is doubtful whether the breakthrough will come soon
[14, 16]. Usage in biotechnology looks very limited, not only because of the fact
that biosensors cannot be sterilised, but also on other unwanted properties (Fig. 5).
Radical new approaches are needed e.g. the development and incorporation of
thermostable biological compounds.
As a conclusion one can state that because of the presented constraints the
present biosensors can only be used in biotechnology in the following modes: off-line
and discontinuous, on-line after a sampling treatment, on-line behind a membrane.

Biological elements Transducers

Organisms Potentiometric
Tissues Amperometric
Cells Conductometric
Organelles Impedimetric
Membranes Optical
Enzymes Calorimetric
Enzyme components Acoustic
Receptors Mechanical
Antibodies "Molecular" electronic
Nucleic acids
Organic molecules Fig. 4. Components that may be used to construct
a biosensor. Source: te Nijenhuis (ref. [14])

Less stability
Response time too long
Not suitable for continuous operation
Re-usability uncertain
Fouling by clogging of particulate matters
Recalibration hardly possible
Sensitivity for pH, ionic strength, temperature Fig. 5. Disadvantages of biosensors. Source: te
Nijenhuis (ref. 1-14])
Challenges in the Relation Between Analysis and Biotechnology 173

Application of Analytical Techniques in Biotechnology

Two areas are chosen here to demonstrate the use of various analytical techniques,
both physical/chemical and biochemical. Those areas are: process control in fermen-
tation, contaminant monitoring in product quality control.

Process Control in Fermentation

One may expect that because of its age process control of bioprocesses in the past
was an intuitive event and completely relied on skills of the producer. When
bioprocessing was set up in a more industrial way prevention of contamination and
growth control were the main issues. Very slowly new parameters were added to
the system.
In the seventies Professor Cooney from MIT started working on computer
control of fermentations and he developed feed back and feed forward strategies.
Nowadays control on the basis of expert system is in the experimental phase.
The goal of process analytical chemistry is to supply quantitative and qualitative
information about a process [18]. This information can be supplied in several ways:
off-line, at-line, on-line, in-line, non-invasive.
"The information can be used not only to monitor and control a process, but
also for optimisation purposes. The main bottle-neck for on-line and in-line process
analysis is the continuing lack of sensors, especially biochemical sensors, for a good
process control" [18].
In the following the state of affairs with respect to on-line and in-line bioprocess
analysis is discussed.
Production takes place in the fermenter (Fig. 6) which normally is a stirred

Agitator Air (outlet)

Nutrients
Acid/caustic I
Antifoam

Cooling water

Cooling

Air (inlet)

Product Steam Fig. 6. Fermentor

174 B. te Nijenhuis

reactor with feed systems and various instrumentation. Nutritional compounds

either in solution or as a slurry are added to the fermenter, an inoculum of the desired
microorganisms is added, the whole mash will be stirred, air for oxygen supply is
passed through the mash and the fermentation process goes on. After the fermenta-
tion the products are isolated in a recovery process. The product is the micro-
organism itself (e.g. yeast) or its metabolites like enzymes or antibiotics.
Typical properties of a fermentation process are: complex matrix, process time
1-7 days or more, monoculture, aqueous systems, variations during process (batch
process!). Due to the fact that the goal of a fermentation process is to produce
uncontaminated monoculture, high demands are necessary to prevent contamina-
tion. This not only holds for addition of the various nutrition compounds, but
also for the sampling systems and on-line analytical systems which have to be
autoclavable.
Information is gathered for process monitoring, for manual interference into the
process and/or automatic control. Parameters are determined in both gas phase and
aqueous phase.
Typical parameters are in the gas phase: oxygen, carbon dioxide, gaseous metab-
olites and products; and in the aqueous phase: pH, temperature, dissolved oxygen,
dissolved carbon dioxide, dissolved gases, nutrition compounds, biomass, product
concentration, etc.
Although still conventional methods are used, measurement in the gas phase is
now the domain of on-line mass spectrometry. Special precautions have to be taken
to prevent liquids and aerosols entering the inlet. Introduction in the industrial
production plants has already started.
Measurement in the aqueous phase is relatively difficult. A direct approach via
sensors is the best solution, and realised for pH,' temperature, but the application
of biosensors is still questionable and of limited importance. Researchers like
Mattiasson and Schiigerl work very hard to make biosensors a success in bio-
processes [2, 10, 17, 19, 20].
Because of the contamination risk, it has already been mentioned, sampling
devices have to be installed in the fermenter. Thereafter either sensors can be used
for direct measurement or analytical arrangements have to be installed for rapid
determination of the parameters wanted. The best techniques or measurements in
filtrates today are HPLC and FIA, coupled with detection devices, whereas mass
spectrometry can also play its role. A simple scheme for this type of analytical
systems in fermentation control is given in Fig. 7. Also mentioned is NIR as an
interesting approach for further consideration. Biomass measurement and determi-
nation of typical "life" components as ATP, NADH, DNA, RNA etc. are up to now
reserved for the laboratory situation.

Contaminant Monitoring
"Contaminant monitoring is a small but dynamic biotechnology segment that will
grow rapidly in importance as public awareness and concern over the contamination
of food, air, soil and water increases" [4].
Conventional methods include chromatography and mass spectrometry, but
inmuno diagnostics is a rapidly growing area. Here, special attention is devoted to
another area of contaminant monitoring: purity aspects of genetically engineered
Challengesin the Relation BetweenAnalysisand Biotechnology 175

II FERMENTER l[
I
F. . . . . . . . . . . . . . . . . . . . . . . . 1

F. . . . . . . . ~-----l F. . . . . . .......... l
I GAS P ~ S E I I AQ~OUS P~SE I
L. . . . . . . . T__3 L ..... T. . . . . . . . . U

I
F- V~IOUS ~- SENSORS
I I
I I
I ~- (NIR)
I I
L_ MS I

] SAMPLING SYSTEM I
L ..... T . . . . . . . . . . . ]

I
~- SENSORS
I
I
~-~LC+DETECTORS
i
~- FIA+DETECTORS Fig. 7. Analytical systems in fer-
I
L_ MS mentation control

product compositions and the tests on absence of all kind of compounds related to
the production system: say, contaminants of the cell cultures used and the produc-
tion process. Residual DNA, protein and polysaccharides, esp. toxins subdivided in
endotoxins and mycotoxins are considered in this respect [22]. This area is not only
of analytical importance, but also highly politically influenced. The EC is very active
in this field and the European Organisation for Quality Control in Pharmaceutical
and Cosmetic Industries is trying to influence the setting of the rules in such a way
that the limits are in line with reasonable criteria and analytical feasibility.
Here once again the limits of analytical chemistry today are approached and
immense efforts have to be made in order to be capable to detect the foresaid
compounds at the desired level. It can be foreseen that the conventional methods
are not good enough for the targets set. Most probably HPLC-MS and CZE-MS
can reach the low levels asked for, next to biodetection methods.
For residual DNA commercially available systems are being tested very seri-
ously. Levels of two picogram seem to be attainable which is close to the present
demands of the FDA. For residual protein pyrochemoluminiscence is claimed to be
a good technique. Most simple, of course, is a determination in what is supposed
to be a nitrogen-free preparation, but problems arise when that is not the case. For
endotoxins the microlumilus test is a good alternative whereas for mycotoxins
immuno-assays are recommended next to GC-MS determinations.
Not explicitly touched in this contribution is the importance of quality and
consequently of validation of methods, but it is assumed that the extreme impor-
tance for research and production is generally recognized.

Conclusion
The final goal of industry and the prior research is to produce safe and effective
products which are properly controlled during the production process. To achieve
176 Challenges in the Relation Between Analysis and Biotechnology

this goal, the usefulness of a well developed analytical chemistry in biotechnology

was stressed and implicitly the great challenges still to be treated for its further
development were mentioned.

Note
BiofineTM chemicals is a registered trademark from International Bio-Synthetics B.V. The products
involved are speciality intermediates with at least one biocatalytic transformation step during synthesis.

Acknowledgements. The author gratefully acknowledges Drs. J. G. A. M. Raaijmakers (Gist-brocades

N.V.) for his excellent assistance in the preparation of this lecture.

References
[1] R. E. Shamel, J. J. Chow, Chem. Eng. Prog. 1989, December, 33.
[2] A. D. Little, Bioteehnology and the Dutch Industry, Strategic study on behalf of Dutch Ministry
of Economic Affairs, December 1989 (in Dutch).
[3] (a) Proceedings Anabiotec 1984: Anal. Chim. Aeta 1984, 163, 1-326; (b) Proceedings Anabiotec
1988: Anal. Chim. Acta 1988, 213, 1-282.
[4] Agenda 4th Annual Seminar on Analytical Biotechnology, June 11-14, 1990, Crystal City (Arling-
ton), Virginia, USA
[5] J. L. Glajch, Anal. Chem. 1986, 58, 385A.
[6] R. E. Majors, LG/GC International 1990, 3 (4), 12; LG/GC International 1990, 3 (5), 10.
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[i1] V. Berry, LG/GC International 1990, 3 (6), 46.
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[13I J. G. A. M. Raaijmakers, Personal communication.
[14] B. te Nijenhuis, Proceedings Eleetro Finn Analysis Conference, Turku, 1988.
[15] Bisensrs Fundamentals and Applicatins (A. P. F. TurnerI. Karube G. S. Wisn eds.) xfrd
University Press, Oxford, 1987.
[16] A. P. F. Turner, Contribution to Eurosensors II, 4th Symposium on Sensors and Actuators,
November, 1988.
[17] Proceedings Biosensor Symposium, Enschede, December, 1988.
[18] J. B. Callis, D. L. Illman, B. R. Kowalski, Anal. Chem. 1987, 59, 624A.
[19] G. Schmidt, Biotec 1990, April, 6.
[20] K. Schfigerl, T. Lorenz, A. Liibbert, J. Niehoff, T. Scheper, W. Schmidt, Trends in Biotech. 1986,
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[21] J.G.A.M. Raaijmakers, Personal communication; Process Analysis in the Fermentation Industry
(in Dutch), (lecture presented to the Dutch Section for Analytical Chemistry), 1988.
[22] Quality Assurance in the Manufacture f Prducts Derivedfrm Bitehnly EQC Cnference
Frankfurt, December, 1988.

Received August 31, 1990. Revision February 4, 1991.