Advanced Analytical Methods for Pharmaceutical and Diagnostic

ANALYTICAL SCIENCE IN SWITZERLAND 705

CHIMIA 5/ (1997) Nr. 10 (Ok.obcr)

Chimia 51 (1997) 705-713 cific sites on mRNA and, therefore, © Neue Schweizerische Chemische Gesellschaft block the expression of cell proteins, lSSN 0009-4293 required novel methods in order to allow a fast detection of the parent compounds and the enzymatic degra- Advanced Analytical Methods dation products. Capillary Electro- phoresis and Optical Biosensors have for Pharmaceutical the potential to address these issues. - The discovery of new molecular mark- and Diagnostic Applications ers to diagnose specific diseases such as Fatty Acid Binding Protein (FABP) for acute myocardial infarction, or the Markus Ehrat*, Gert L. Duveneck, Gerhard M. Kresbach, Peter Oroszlan, and identification of RNA and DNA se- Aran Paulus quences to identify viral infections or genetic diseases require complicated assay procedures in order to achieve Abstract. Driving forces for the development of novel analytical technologies in the limits of quantitation in the range of 10 life-science industry are described. Technologies which either were developed in Bio- to 1000 molecules. With signal or tar- Analytical Research or brought to a reliability required for routine applications will be get amplification methods such as elucidated and, on the basis of practical examples, the impact of modern analytical Polymerase Chain Reaction and use of technologies on the industrial research and development will be discussed: Optical labeled branched DNA such limits of biosensors based on evanescent excitation of luminescence allow for real-time moni- quantitation can be achieved today. toring of the binding of active compounds to specific biomolecular recognition sites. However, many of these amplification Molecular imaging technologies have the potential to gain rapid access to physical methods are not suitable for fast diag- maps of genomic materials. Capillary electrophoresis or affinity gel electrophoresis are nostic point of care applications. Opti- well suited for the fast determination of oligonucleotide mixtures in nl amounts of cal biosensors based on evanescent samples. Integrated capillary electrophoresis on chips will allow to multiplex capillary excitation of fluoresence are consid- systems at low costs and results in high separation efficiencies. MALDI- TOF MS is an ered by us as a potential technology for easy to operate non-scanning mass spectrometric instrumentation for the analysis of ultrasensitive and fast detection of ol- high molecular weight biopolymers such as immunoglobulins. igonucleotides and DNNRNA strands. - The investigation of bioaffinity interactions is not only an impoltant task Introduction nologies allows not only the efficient iden- for the chemical optimization of the tification of molecular drug targets but potency of drug interaction with a re- Analytical technologies playa key role also opens novel ways for diagnosing in- ceptor but also for the detection of in the research and development process fectious and genetic diseases. In the last binding properties of drugs to blood of pharmaceutical products: in the research years activities in our department were proteins. This type of analytical infor- phase they are indispensable for the iden- focused on evaluating, adapting and fur- mation can be obtained in an easy and tification of the molecular targets, the se- ther developing advanced analytical tech- accurate way by the application of lection of a substance with a suitable bind- nologies and methodologies in those are- evanescent wave biosensors. ing affinity to the target or the char- as. Examples below shall identify the major - Biotechnology and molecular biology acterization of the molecular structure. In driving forces for such developments as in general, the human genome project the development phase analytical tasks well as their technical implementations. in particular had a tremendous impact are the identification of metabolites, the - Novel, more potent drug candidates on the development of advanced ana- parameters of drug-protein interactions, which are administered at lower doses lytical techniques. The high molecular and the determination of pharmacokinetic and, therefore, result in much lower weight of many biopolymers, the com- parameters. There typically large num- blood concentration levels set new plex mixtures in biotechnological pro- bers of animal and human samples have to standards in terms of the detection lim- cesses as well as the very small vol- be analyzed. In close context with pharma- it of analytical methods used for mon- umes in which minute concentrations ceutical applications is the development itoring their metabolic and pharma- of analytes have to be determined, re- of analytical technologies for human diag- cokinetic behavior. For example, the quired a reevaluation of existing ana- nostics. The enormous progress made in daily dose of the new aromatase inhib- lytical methods. For the determination molecular biology within the past years itor Letrozole (Femara®) is only 2 mg of biopolymers in complex samples it together with advanced analytical tech- compared to 250 mg for aminogluteth- was Matrix Assisted Laser Desorption imide (Orimeten®) which results in Ionization Time of Flight Mass Spec- blood concentrations in the nanomolar trometry (MALDI- TOFMS) which did range for Femara compared to micro- anow a fas't analysis without a tedious *Carrespandence: Dr. M. Ehrat molar for Orimeten. . sample preparation method. We will Navartis Pharma AG - Novel chemical classes of drug com- demonstrate how MALDI- TOF MS in BioAnalytical Research pounds often require more powerful combination with a simple sample prep- K-127.156 CH-4002 Basel separation and detection methods. The aration method was applied to monitor Tel.: +41 6\ 6966069 introduction of antisense oligonucle- the antibody production during a cell Fax: +41616964504 otides, typically oligonucleotides with cultivation. Approaches to establish E-Mail: [email protected] 20 to 25 bases which hybridize to spe- functional physical maps, e.g., to in- ANALYTICAL SCIENCE IN SWITZERLAND 706

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vestigate the localization of genes and mate senSItIVIty, a threefold selectivity Planar waveguides provide different to monitor the expression of mRNA approach was incorporated: possibilities of detection configurations. are currently under development. 1) The evanescent wave sensing princi- Both the fluorescence that is excited in the - Novel approaches in drug research such ple for spatial selectivity, evanescent field, but isotropically emitted as combinatorial chemistry, random 2) the immobilized biological recogni- ('volume detection') and the portion of screening or the assessment of genetic tion elements for (bio)chemical selec- fluorescence light, that is coupled back information pose increasing demands tivity, and into the waveguide, can be detected. With on analytical technologies in terms of 3) the luminescent labeling for amplifi- a second grating the guided emission light small sample volumes and high sam- cation and specific signal generation. can be coupled out of the waveguide (' grat- ple throughput. Miniaturization, mul- The evanescent field penetrates only ing detection') and directed towards the tiplexing and automation allow to reach some 100 nm from the sensor surface into detection system (Fig. 1). these goals. We will introduce the con- the solution and thus allows a real-time cept of Integrated Capillary Electro- monitoring of binding kinetics. As in the Immuno and Oligonucleotide Assays phoresis, demonstrate how such chips well accepted and widely used solid-phase Target analyte classes, in general, can can be obtained at low costs and - bioassays, the immobilized biorecogni- be any molecules specifically binding to using lambda phage digests- how DNA tion elements on the transducer surface their immobilized affinity partners by fragments can be separated at picomo- can be used to selectively capture the (bio)specific recognition or leading to a lar concentrations on 20 mm short chan- analytes.The fluorescent label of the trac- specific change of a physical-chemical nels. er, added at known concentrations, allows property of the recognition element. Until for an easier differentiation between spe- now, the list of affinity systems adapted to cific and nonspecific binding events, pro- the FOBIA system reflects a large part of Optical Biosensors vides superior sensitivity and makes the the spectrum of known biochemical affin- sensors signal independant of the molecu- ity interactions (antigen-antibody, recep- Outline of Technology lar weight of the analytes. tor-ligand, complementary DNAJRNA, ol- The demand in life sciences and hu- igonucleotide-strands, protein-drug inter- man diagnostics for analytical methods Transducer Layout and Optical action) and could be extended to synthetic adequate to measure biospecific interac- Configuration affinity systems as well. Due to the inher- tions at extremely low concentrations, in The transducer consists of a thin ent extremely high sensitivity and real- small sample volumes and with a mini- waveguiding layer « 200 nm) with a high time measuring capability of FOBIA, its mum of sample preparation is the driving refractive index (> 2.2) on a glass or plas- application in new target areas, like trac- force for the development of a novel gen- tic substrate. Excitation light is coupled ing RNA/DNA targets in biological sam- eration of biochemical sensors [1]. FO- into the waveguide by means of a grating ples (e.g. genetic diagnostics, genotyp- BIA (Fast Optical Biospecific Interaction structure in the waveguiding layer at one ing), or for kinetic studies of biospecific Analysis) is a planar waveguide based end of the sensing area. The light propa- interactions (e.g. drug-protein) could be optical sensor system dedicated to selec- gates a few mm in the waveguide generat- beneficial. tively measure minute amounts of ana- ing an evanescent field. All fluorophores Multiplexed sensor systems allow to Iytes in complex media, such as, e.g., bound to the surface and thus located analyze many samples without compro- blood serum, plasma or tissue extracts, on within the evanescent field are selectively mising sensitivity, in parallel thus opening a real time basis [2]. To achieve the ulti- excited. the perspective for new applications re- questing a high throughput. The feasibility of using optical sensor technology for studying sequence specific Planar Waveguide Sensor Design DNA hybridization, including quantitative hybridization assays and real-time kinetic investigation of DNA hybridization have already been demonstrated [3]. The example below shows results of the FOBIA antisense oligonucleotide hybrid- a) Detection of backcoupled ization assay designed and developed for and guided luminescence: quantitative analysis of human serum sam- "grating detection" ples in preclinical studies. To achieve maximum sensitivity, pref- erence was given to the so-called adapter Laser Diode Detectors assay which is schematically depicted in Fig. 2, using 3'-amino-substituted oligonucleotides covalently attached to epoxy- b) Detection of isotropically silanized planar waveguide chips. The emitted luminescence: "volume detection" adapter assay format involves the compe- tition between the Antisense Target Mol- ecule (ATS) and the tracer (Cy5-labeled Fig. 1. Fundamental detection configurations available for planar waveguide biosensor systems. ATS) for hybridization to a longer oligo- Both the isotropically emitted fluorescence that was excited within the penetration depth of the planar waveguide ('volume detection') and that part of the fluorescence, that was coupled back and nucleotide (adapter). The adapter medi- propagated as a guided mode in the wave-guiding film can be detected, using a second grating for ates the binding of the complex to the outcoupling the emission ('grating detection'). capture oligonucleotide immobilized on ANALYTICAL SCIENCE IN SWITZERLAND 707 CHI MIA 51 (1997) Nr. 10 (Oklober) the surface of the sensor chip. Modified oligodeoxynucleotides with higher stabil- ity in serum were used throughout. Capturesequence (16"'; 2'-MeO-RNA) FOBIA provides a unique opportunity 3'- _ - 5' for fast assay optimization. The kinetic feature of the evanescent field sensor tech- Adapter (36"'; 2'-MeO-RNA) nology (i.e. determination of association 5' - !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!liiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii - 3) and dissociation rate constants of molecu- larinteractions) allows modeling of abind- 3' _ Tracer (Cy5 - 20'" P=S) - 5' ing assay by means of using kinetic and thermodynamic constants of the given af- Target sequence (20"';P=Sj finity partner and varying assay parameters, such as tracer and binder concentration and assay time for optimizing assay Fig. 2. FOBIA Adapter assay format for quantitation of antisense oligonucleotides. The length and performance [4][5]. Investigating binding the modification for each oligonucleotide is given in parentheses. kinetics of various Cy5-labeled ATS's (tracer) to the immobilized RNA (binder) with the FOBIA system, the equilibrium

dissociation constant determined for a le+OO6 P = S-ATS-2'-MeO-RNA tracer - cap- 1--Ie 50 = 4.4e-9 M I 10 ture combination was 5.10- M (kass = 40 5 5 1.10- 1/Ms,kdiss = 5.10- 1/s). According 8e-tOO5 to the theory of competitive binding assays, sensitivity of the hybridization as- -. '"Q., 1 says should be in the range of -2-5.10- ] ~ 6e+OO5 \. M, using 0.5-1/K concentrations of the .5- ,. If. tracer as well as the binder (note, that OJ)= \ t lowering the concentration of the tracer 'iii WQ = 7e-IO M t················ 20 4e+OO5 :, and/or binder would not result in further : , sensitivity improvement of the assay). :. ', o ",."n +/. 2 std •• (n=4)~ \. Measurements ofthe adsorption of ATS 2c+OO5 to plasma proteins in 10% human plasma .•. - precision profile t- ... -.. ~-.•. pool (HPP) in dependence of the addition - of proteinase K, an enzyme with high o o proteolytic and no nuclease activity, re- sulted in dose-response curves almost Ie-OIl le-OIO le·OO9 le·008 le·007 le·OO6 equivalent to the dose-response curves analyte (M) observed in buffer. Very good signal-to- signal reproducibility was obtained using Fig. 3. Dose-response curve and precision profile of the FOBIA antisense oligonucleotide assayfrom a sample matrix containing 10% human human plasma (the figure shows true analyte concentrations; samples were 10 times diluted prior plasma with sensor lifetimes longer than measurements) eight weeks. The performance of the adapter assay on the planar waveguide biosensor system was tested by measuring five consecutive dose-response curves in buffer at a concentration of adapter and tracer of 0.1 and 0.5 nM, respectively. The resulting data limit of d tection (LOD): . are shown in Fig. 3. Sensitivity achieved with this assay matches perfectly with the value theoretically obtained from the kinetic measurement (LOD = 2.10-10 M, 10 LOQ = 7.10- M). The precision profile 104 shows coefficients of variation ofless than 6.2% in the ATS concentration range of 1 nM to 200 nM. Whereas the above assay, performed 103 in a competitive format, was an example for an application of high pharmacologi- 0.001 0.01 0.1 1 10 cal relevance, the principle system sensi- tracer concentration [pM] tivity is demonstrated for a noncompeti- tive assay. An example of a direct binding Fig. 4. Sensitivity of planar waveguide sensor system in model assay: Dose-response curve for the assay using the binding of Cy5-labeled binding ofCy-5labeled immunoglobulin to immobilizedproteinA. The detection limit was determined immunoglobulin (IgG) to immobilized from the threefold signal noise, when the baseline was recorded. ANAL YTICAL SCIENCE IN SWITZERLAND 708

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protein A isgiven in Fig. 4. For this model mation of the target to establish physical interrogation tasks. Fig. 5, a and b give a bioaffinity system, the detection limit was maps and to allow for a differential display short schematic overview and comparison reached, after a binding period of only 4 of, e.g., wild type and mutant DNA, in of both approaches. min, at a 4-femtomolar concentration of Fiber FISH a higher level of information is The experience gained during the ini- labeled IgG. A total amount of 4 attomoles required to select or create the appropriate tial phase of OMT development was ap- (2.4 x 106 molecules in 1 ml of sample labels to be used for the more specific plied to define and establish a building solution) had been applied to the sensor.

a) Molecular Imaging Technologies ~ stretched and fixed DNA

To gain simple and fast access to physical maps of genomic material, processes like Optical Mapping Technology (OMT) coiled DNA [6][7] and Fiber Fluorescence in situ Hy- --.---.------bridization (Fiber FISH) [8][9] approach- cleavage using restriction enzymes es are evaluated and further developed. Both approaches are based on the singu- larization, linearization, orientation, and controlled adsorption of DNA molecules on specially treated surfaces. In OMT tension partly released markers are introduced by (enzymatic) . endonuclease digestion resulting in the insertion of physical gaps into the mole- ordered DNA map ~ cule. The resulting fragments stay local- fluorescence labeling ized on the surface and are stained by intercalating fluorescence dyes. When visualized by epifluorescence microscopy b) physical genomic maps can be directly 'read' . linearized and fixed DNA In the Fiber FISH approach the elon- gated and adsorbed DNA molecules are thermally denatured and subsequently hybridized with single stranded - labeled coiled DNA - complementary DNA fragments. The hybridization with oligonucleotide probes hybridization event can be detected by epifluorescence microscopy after a signal contlg assembly ., ,. .. amplification, e.g., by either applying a determination of gaps --- cascade of f1uorescently labeled antibod- localization of functional sites labeling with first antibody ies or by alternately adding biotinylated anti-avidin and fluorescently labeled bi- otin to the sample. As eDNA or fragments of genomic DNA are used for the hybridization process 'functional' positions can be marked and visualized on the DNA further signal amplification steps labeling with second antibody strand. Whereas OMT can be considered an Fig. 5. a) Schematic visualization of the OMT process; b) schematic visualization of the Fiber FISH 'ab initio' method requiring little infor- process

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Fig. 6. Lambda DNA (48 500 base pairs, equivalent to ca. 16/l11l in length) stretched on silanized surfaces. Left: plain stretching and staining; center: additionally Bam HI digestion; left: Kpm I digestion performed. ANAL YTICAL SCIENCE IN SWITZERLAND 709 CHIMIA 5/ (1997) Nr. 10 (Oktober) block approach to guide the development of surfaces better suited for the OMT, Fiber FISH, and mRNA expression ar- rays. This model is based on the assump- CZE tion that different factors contribute to the C3pilllll) lone Fleclrophorc i DNA - surface interaction, that - in an ideal case - these factors can be influ- enced individually, and that the correct CGE balance of functionality, density of func- C/lpllillr) Gel flcclropllor i\ tional groups, and background polarity is responsible for the successful use in the different processes. CITP Based on this model several alterna- ~----~-I tive surface modification approaches are being developed. Starting from simple to produce - but hard to tune and stabilize - ClEF aminopropyltriethoxysilane coated glass Capillar) "CleleClric Foell,inl( better suitable surface modification meth- pttvodiml' ",••pH --)Illt:~ pH ods were introduced. More complex si- lane and polymer based coatings allowed for controlled manipulations of large ECC strands of DNA on surfaces. Using the Micellar Icclroklmlic apillal') hrom lojtraph.l same model a self-assembled monolayer approach currently is being pursued. To facilitate, control, optimize and potential- ly automate the processes involved in CE OMT/Fiber FISH appropriate microfluid- Cllpillar) r1cclr chromalU!:r ph) ics currently is being developed. Know-how developed for the imaging Fig. 7. CE Separation modes of genomic DNA - ranging from surface chemistry, microfluidics, to epifluorescence detection - currently is being ap- mAU plied for the development of fluorescence 80 antisense oligonucleotide based technologies in an array format suit- 70 p(dT) 6 -25 [P=S] able, e.g., for quick development of oligonucleotide hybridization assays and espe- 80 cially for monitoring of mRNA expres- 50 sion. 40 with organic additive

Capillary Electrophoresis 20

Outline of Technology 10 without organic additive Capillary Electrophoresis (CE) [10] is , an automated analysis technique, combin- 10 15 20 min ing features of high performance liquid chromatography (HPLC) and electro- Fig. 8. CGE of phospho rothiate oligonucleotides pd(T)6-25 with a replaceble polymer sieving matrix, phoresis. Contrary to HPLC, an electric with (upper trace) and without (lower trace) 20% acetonitrile in 200 mM Tris, 100 boric acid, pH 8.5 field of several hundred V/cm is used to buffer; capillary: /00 J.1m lD; effective length: 25 cm, total length: 35 cm mobilize the solutes and, in free solution applications, an electroosmotic solvent flow (EOF). The typically 50-100 j..Illl molecular weight compounds including depend on both its charge and size. Similar diameter of the separation capillary, the pharmaceutical active substances and their to HPLC, a multitude of techniques were small injected sample volumes of just a chiral resolution. Proteins, which tend to developed to adapt to a specific separation few nl and the plug shaped flow profile of easily adsorb to surfaces can be separated problem as shown in Fig. 7. These differ- the EOF allow fast and high efficient CE in capillaries with a hydrophilic coating ent modes allow to emphasize the charge separations. Separation performance is according to their charge to mass ratio. or size aspect in the more electrophoretic often at least one order of magnitude better Since most nucleic acids have pro- modes or the sample hydrophobicity in the than in HPLC typically, theoretical plates nounced size differences but similar chromatographic modes. In Capillary Zone in the order of several 100000 to a few charge-to-mass ratios, sieving media such Electrophoresis, e.g., small charged com- million per capillary can be obtained. as agarose and polyacrylamide gels have pounds are separated in a free buffer solu- In electrophoretic separations the ana- been very popular for their analysis. If tion in untreated fused silica capillaries. lytes are typically separated according to these sieving media are filled into a capil- Typical applications range from peptide their electrophoretic mobilities, which lary, their porous nature will decrease the analysis to the analysis of charged low ANAL YTICAL SCIENCE IN SWITZERLAND 710

CHIMIA 5/ (1997) Nr. 10 (OkLobcr) electrophoretic mobility as a function of ponents are stacked according to their sodium dodecyl sulfate (SDS) allows their solute size. Capillary Gel Electrophoresis electrophoretic mobility. ITP is often used partition between micellar and buffer phase (CGE) [11] is used predominately for the as a preconcentration technique. If one is similar to chromatography. CEC is a true analysis of double-stranded DNA frag- only interested in the charge differences, chromatographic technique with electric ments and single-stranded oligonucle- the addition of ampholines can create a field solvent pumping. MECC and CEC otides with smaller fragments moving fast- pH-gradient for capillary isoelectric fo- have a similar broad application range as er through the polymer network. cusing (ClEF) [13]. For the separation of HPLC with the additional benefit of high- The separation mechanism of Capil- neutral solutes Micellar Electrokinetic er separation performance. lary Isotachophoresis (CITP or ITP) [12] Capillary Chromatography (MECC) [14] We have focused our attention to the is again based on the charge-to-mass ratio. or Capillary Electrochromatography separation of nucleic acids ofCE. With the In contrast to CZE, the sample is sand- (CEC) [15] are the methods of choice. The antisense approach and gene therapy as wiched between a leading and a terminat- interaction of the non-charged analyte in potential drug development programs in ing buffer. Upon focusing, sample com- MECC with a charged surfactant such as the pharmaceutical industry the need for automated quantitative analysis of nucleic acids increased. In addition, problems with on-column detection are not envisioned, a) because nucleic acids exhibit very high extinction coefficients for UV detection at 260 nm. For a more sensitive laser induced fluorescence (LIF) detection, labeling with fluorescent nucleotide primers or interca- lation for double-stranded DNA is readily available.

Oligonucleotide Separations by CE b) For the separation of oligonucleotides, at first, sieving media similar to slab gel formulations were developed using cross- linked polyacrylamide formulations. How- ever, the difficulties associated with cross- linked polymer capillaries, in particular restricted capillary life time through 'bub- ble' formation, let to the exploration of hydrophilic replaceable polymer matrices. If the polymer concentration exceeds the entanglement threshold, a porous net- c) work similar to cross-linked gels is formed. Since the viscosity of the polymer solu- tions is low, the separation matrix can be pumped into a capillary prior to the separation and flushed out after the analysis. This approach is less error prone with better migration time and quantitation reproducibility and simplerto transfer into a routine environment. Also, a possible car- d) ry-over from previous injections is avoided since the capillary is replenished before each analysis. Fig. 9./CE Chipfabrica- Sieving matrices for the separation of tion procedure: a) silicon small oligonucleotides with 10-200 bases master wafer with posi- have been developed. Fig. 8 shows the tive surface relief, b) pre- separation of a phosphorothioate test mix- mixed solution of Poly- ture with a polyethylene based polymer dimethylsiloxane (POMS) matrix. Compared to phosphordiesters is poured over the master, (data not shown), phosphorothioates show c) cured PDMS slab is peeledfrom the master, d) a different migration rate. Therefore, it liquid reservoirs are can be suggested that the phosphorothio- punched into the cured ate solutes interact with the polymer in a PDMS chip, e) ready to chromatographic way. However, as dem- lise device placed on a onstrated in Fig. 8, this interaction can be slab of PDMS (reprinted utilized to manipulate the separation by with permission from [17], Copyright 1997 the addition of an organic modifier, e.g., American Chemical So- acetonitrile. ciety) ANALYTICAL SCIENCE IN SWITZERLAND 711

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Integrated Capillary Electrophoresis

1.0 1353 The formation of capillary channels on planar substrates allows for an arrange- ment of multiple parallel capillaries on one single substrate to achieve a high throughput. In addition, sample and fluid 1078 handling procedures can easily be integrated due to the ease of interfacing the 872 281 channels without introducing extra band- 271 broadening because of excessive mixing 603 volumes. This approach, initiated in our '.i department in 1989 [16] will have at least 0.5 ~i 310 the separation power of capillary electro- 234 phoresis combined with the multiplexing capabilities of slab gel electrophoresis. 194 For low cost fabrication of microchips for integrated capillary electrophoresis sys- 118 tems polydimethylsiloxane silicone elas- 72 tomer was molded against a microfabri- cated master. Constrains of glass plate bonding at elevated temperature could thus be avoided. Fig. 9 demonstrates the dis- crete steps of such a simple molding pro- o cess. The performance of the devices is dem- o 50 100 150 200 250 onstrated by separating X -17 4/Hae III DNA restriction fragments labeled with Time (sec) the intercalating dye YOYO-l and shown in Fig. 10. Fig. ]O.Separation oj'A-DNA restriction fragments infree buffer solution. Channel cross section: 50 /lffi X 20 /lffi. An electrical potential of 86 Vfcm was applied and the fragments separated on a channel Capillary Affinity Gel Electrophoresis length of only 20 mm. 150 pI of a 41 pM DNA solution was injected resulting in an injected amount of 6. J zmol (ca. 3700 molecules) (reprinted with permission from [17] Copyright 1997 American Chemical Society). The complexity of antisense samples in biological matrices demands for improvements in selectivity, resolution and sensitivity of the established separation methods. Based on our experiences with mAD sieving media, we developed a novel ap- 3.0 proach to effectively combine the size separation with a biorecognition step, dubbed Capillary Affinity Gel Electro- 1.0 phoresis (CAGE). A single stranded oligonucleotide as recognition element was -1.0 chemically bound to a polymer. Upon introduction of this polymer into the capillary and injection of a heterogeneous sample, all solutes with a complementary sequence would bind to the recognition 5.00 10.00 15.00 20.00 min element at low temperature. All non related oligonucleotides as well as non DNA related sample components would, at low Fig. 11. Electropherograms showing the separation of dAI5 from oligonucleotides with 1,2, or 3 temperature, migrate through the polymer mismatches, using the CAGE approach. CAGE Conditions: 3 %T of a dTl5-affinity polymer, network. Only upon raising the tempera- dissolved in ]00 mM TB. Concentration of each oligo was 5· ]0-4 OD/ml. Co-injection of dAIS for 6 s at -6 kV and a mixture of d(TA TA6T), d(A TA6T) and d(A TA ) for 9 s at -8 kV. ture above the melting temperature of the 6 7 7 7 double-stranded complex, the bound solutes would dehybridize and start to migrate. In this way, a true two step-system pdT chemically bound to a polymer. With eluted. Using a step temperature program is created, where selectivity can be this matrix, the complementary pdA]5 single oligonucleotide mismatches with controlled over the temperature as well oligonucleotide was hybridized at low 1, 2, or 3 adenylic acids exchange for as by the composition of the sieving temperature. Only upon raising the thymidylic acids, differing in their melt- matrix. Fig. 11 shows an example with temperature above the melting point of ing temperature as little as 10 could be a model system, consisting of a IS-mer the AT duplex, the pdA wmer could be separated. ANAL YTICAL SCIENCE IN SWITZERLAND 712 CHIMIA 51 (1997) Nr. IO(Oktober)

MALDI- TOF Mass Spectrometry out discarding ions produced in the ion Currently two main modes of ion pro- source during the ionization process. This duction are established in combination Outline of Technology significantly higher transmission rate of with TOF: matrix assisted laser desorp- In the last years the performance of the produced ions results in a significant tion and ionization (MALDI) and electro- non-scanning mass spectrometric instru- increase in sensitivity when full spectrum spray ionization (ESI). In MALDI-TOF- mentation - with time-of-flight (TOF) mass data are compared. MS introduced in 1989 by M. Karras and spectrometry as the predominant repre- In TOF mass spectrometry ions pro- F. Hillenkamp [18] (Fig. 12) samples are sentative - has dramaticall y improved and duced in an ion source are accelerated with embedded and cocrystallized in an ap- thus this type of mass spectrometers has the identical kinetic energy (E = l/zmvZ). propriate matrix like sinapinic acid. Subse- successfully penetrated the analytical mar- In a field free region ion packages then quently, this matrix is exposed to pulsed ket. In contrast to classical magnetic and 'fly' for a certain period of time according laser light (typically 337 nm, 3 ns, 5 mJ). electrical field based mass spectrometry to their mass (m prop. l/vZ) and are detect- During this process the matrix is volatil- where - with the exception of some spe- ed when hitting a microchannel plate de- ized and the isolated sample molecules are cial modes - a mass range of ions is tector. In contrast to magnetic instruments gently ionized. Even large and labile bio- scanned over time, in non-scanning MS the obtainable mass range theoretically is molecules are desorbed and ionized with- full spectral information is gathered with- not limited. out any fragmentation. This special be-

attenuator beam mirror splitter nitrogen laser 337 nm. 3 ns. 2·6 ~J

lens

high vacuum « 10.6 torr) pream plifier M+/- > ) >

sample time-of-flight tube (1.7 m) MCP detector introduction ion optics (-5 kV) (+1· 30kV)

digital oscilloscope (1 GHz) dal, ood 000'''' IIuII system g 1111• I 0000 Fig. ]2. Schematic drawing of a MALDI- TOFMS

o • N-acetyl-glucosamine Positive ion spectrum • mannose II • galactose .0• Negative ion spectrum .: fucose with DAHC o sialic acid +22 Da shifted .....1ft H

~Hi,-"-1 . a / •••••••• u ..u. D.I. •••y c •••• •••·0 • • ,.... ~ '-·--T• -.• / ,. ell :

1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 Fig. 13. Analysis of an oligosaccharide m/z library of human secretory component ANALYTICAL SCIENCE IN SWITZERLAND 713

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havior of the ionization process renders MALDI -TOF MS an ideal tool for a quick off-line analysis of mixtures of complex 4.0 composition especially of biological ori- Q -+- In:ulin gin. CUlTent state of the art is the charac- ~ L l terization of molecules from 300 to 250000 ~'" -+- Antibody ....,.~ .....•- Cell Protein Oa in amounts as low as I fmol. Due to c ~.O recent improvements in source design and -t:: E} -x- Light Chain ion ejection MALDI- TOF MS offers now c •• •• -t:: 2.5 a resolution (M/OM) of up to 20000 (de- ~ termined for Cytochrome C). .Ii) •.. 20 Its technical simplicity, ruggedness and ~ nevertheless high information content of Q", IccOIn~ "lIh meow the data obtained made MALDI -TOF MS 'B' 1.5 an instrument well accepted by research- .-•..C \:l ers. It is used preferably in research and Q::; 1.0 S,.ln \\llh,' ,'ulii\ali\1n ~ In Ih~ hHI~3~1\1 quality control laboratories as no complex t: fragmentation patterns have to be resolved. .!:p 0.5 In the last several years MALDI -TOF MS '" I has - especially due to the increase of 0.0 spectral resolution as a consequence of 100 150 200 250 300 3 0 400 improved ion extraction - found more and Process in h more acceptance in different areas of life science. Characterization of synthetic prod- Fig. l4. Monitoring of key proteins during the hybridoma cell cultivation process for antibody ucts, biopolymers, drugs, metabolic stud- production ies, investigation of oligosaccharides etc. were successfully performed. In the char- weis von Oligonukleotiden', Ph.D. Thesis, acterization of products of protein synthe- Bioprocess Monitoring Key requirement for a successful anal- Basel, 1995. sis MALDI now is the method of choice. ysis of compounds in complex environ- [4] 'Computer Aided Assay Design', D. Gyg- In the identification of isolated proteins ax, L. Botta, M. Ehrat, P. Graf, G. Lefevre, MALDI based methods - in combination ments using MALOI-TOF is a careful P. Oroszlan, C. Pfister, Therapeutic Drug selection of appropriate matrices and sam- with established data banks - already has Monitoring 1996, 18, 405. found wide acceptance. ple preparation methods [19][20]. Under [5] H. Towbin, J. Motz, P. Oroszlan, O. Zingel, An equally promising usage is expect- optimized conditions then even quantita- J. Immunol. Methods 1995, 181, 1676. [6] X. Meng, K. Benson, K. Chada, E.l. Huff, ed from ESI-TOF MS as far as on-line tiveresults can be obtained. Fig. 14 shows the utilization of MALDI-TOF MS for and D.C. Schwartz, Nature Genetics 1995, characterization of biomolecules and bio- 9,432. bioprocess monitoring in biotechnology. processes are concerned. With this tech- [7] D.e. Schwartz, A. Samad, Curro Opinion Profiles of insulin (cultivation additive), nology - whilst maintaining the key ad- Biotechnol. 1997,8, 70. vantages of TOF mass spectrometry - produced immunoglobulin G (IgG) and [8] I. Parra, B. Windle, Nature Generics 1993, effluents of HPLC, CE, and CEC can be light chain antibody fragment as well as 5,17. [9] M. Heiskanen, E. Hellsten, O.-P. Kalli- subjected on-line to analysis via an elec- the fate of a selected cell protein was omiemi, T.P. Makela, K. Alitalo, L. Pelto- trospray or a nano electrospray interface. monitored. Samples were taken directly out of cultivation broth with drop dialysis nen, A. Plaotie, Genomics 1995, 30, 31. In contrast to MALDI, in ESI several [10] J.W.l!i1rgenson,K.D. Lukacs, Science 1983, as the only sample preparation step. Using charges are deposited on one molecule, 222,266. the structural integrity, however, can be phospholipase 0 as an internal standard [II] B.L. Karger, A.S. Cohen, A. Guttmann,J. maintained. Mass resolution of state-of- the precision of the IgG determination was Chromatogr. 1989,492,585. the-art instrumentation is in the range of comparable to these obtained by affinity [12] A.P.J. Martin, F.M. Everaerts,Anal. Chim. Acta 1967,38,223. 5000 to 7000. HPLC. Limit of detection in raw cultivation broth was found to be ca. 160 pmol (13] S. Hjerten,J. Chromatogr.1985,347, 19l: absolute; limit of detection in pure sol- [14] S. Terabe, K. Otsuka, K. Ichikawa, A. Analysis of Complex Oligosaccharides Tsuchiya, T. Ando, Anal. Chem. 1984,56, vents was in the fmol range. Fig. 13 shows the result of an experi- 1l3. ment where a complex carbohydrate mix- [15] 1.H. Knox, I.H. Grant, Chromatographia ture derived from human secretory com- 1991,32,317. ponent was analyzed. Due to the limited [16] A. Manz, N. Graber, H.M. Widmer, Sens. Received: September 3, 1997 Actuators B 1990, 1, 244. variation of monosaccharides present in [17] e.S. Effenhauser, G.J.M. Bruin, A. Paulus, this class of oligosaccharides a direct iden- [1] G.L. Duveneck, M. Pawlak, D. Neuschafer, M. Ehrat, Anal. Chem. 1997,69, 3451. tification of the gross composition easily W. Budach, M. Ehrat, 'A Novel Generation [18] M. Karas and F. Hillenkamp, Anal.Chem. was possible. Presence or absence of sialic of Luminescence Based Biosensors: Single 1988, 60, 2299. acid in isobaric compounds (e.g. two fu- Mode Planar Waveguides', Proc. SPIE [19] U. Kallweit, K.O. Bornsen,G.M. Kresbach, cose residues have the same molecular 1996, 2928, 98. H. M. Widmer, RapidCommul1. Mass Spec- [2] G.L. Duveneck, M. Pawlak, D. Neuschafer, trom. 1996, 10, 845. weight as sialylic acid residues) could be E. Bar, W. Budach, U. Pieles, M. Ehrat, [20] K.O. Bornsen, M.A.S. Gass, G.Bruin, detected by comparing the negative and Sensors and Actuators 1997, B38-39, 88. 1.H.M. von Adrichem, M.e. Biro, G.M. positive ion profiles. [3] A. Abel, 'Faseroptische Evaneszentfeld- Kresbach, M. Ehrat, Rapid Commlm. Mass Biosensor zum Sequenzspecifischen Nach- Spectrom. 1997,11,603.