Joseph L. Glajch Central Research and Development Department E.I. du Pont de Nemours and Company Experimental Station Wilmington, Del. 19898
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One of the most dynamic areas in science today is the combination of biology, chemistry, and engineering known as biotechnology. Drawing much of their current momentum from the applications of recombinant DNA research, developments are rapidly moving from the research laboratory to industrial processes for a variety of agricultural, medical, and industrial products. Many of the advances in this field rely on the separation, detection, characterization, and possible modification of hiomolecules. These include a variety of small organic compounds, peptides, proteins, and oligonucleotides (including large fragments of DNA). Various analytical techniques are used in biotechnology, but many of them are either very traditional or so new that their proper application has not been fully realized. This REPORT will highlight five major analytical areas 0003-2700/86/0358-385ASO 1.50/0 @ 1986 American Cnemical Society
(electrophoresis, immunoassay, chromatographic separations, protein and DNA sequencing, and molecular structure determination) and discuss ' how analytical chemistry could further improve these techniques and thereby have a major impact on biotechnology.
Problems In bloanalytlcal chemisiry Although many of the analysis methods used in biotechnology are common to other chemical analyses, there are specific problems with samples of biological interest. Biological samples can have a wide molecularweight distribution, be present in a complex sample matrix (such as 88rum) in small amounts, and often have an unknown primary structure. The initial information on an interesting protein, for example, could be that a change in its concentration is the cause or result of an important hio-
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logical effect, such as increased growth rate of a plant or onset of a disease in an animal. Often the presence or absence of this protein is first detected as a band on an electrophoresis gel with picomoles or less of material available. The analytical problems of separation, detection, and identification of this biomolecule are critical to further research investigations. The solutions to these problems will govern the possible use of the biomolecule as a product of biotechnology. In the early stages of analysis, trace analysis problems and techniques are important, because picomoles of protein can take months to purify to a usable level. The techniques discussed below have already developed as major tools in biotechnology, but improvements will be required to keep up with future needs.
Electrophoresis Electrophoresis is a classical biologi-
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Figure 1. Immunoassay test (a) A known amount of labeled analyte (A’) is mixed w R an unknown amourt d lhe same unlabeledamlyte (A) and a limited number of antibodies (8). The wmpetitive equilibrlwn r e r u b in a dlswlbutlon of lhe analytes into Wm bwnd and hee forms, which are separated and ltm used to meadue thn amant d labeled analyte in each fwm. (b) k i n g a standard calibration cuve, h e analyte conCematiOn 01 lhe unknown can be determined
cal method of separation that is used in almost every laboratory. The separation of proteins and polynucleotides is done on the basis of differences in both charge and size of molecules. There is a family of related electrophoretic techniques including polyacrylamide gel electrophoresis (PAGE), isotachopboresis, gel electrofocusing, and two-dimensional electrophoresis techniques for very high resolution. Electrophoresis is especially useful for the analysis of complex protein mixtures and in DNA sequencing. Although there are many variations of electrophoresis, depending on the type of sample and the information required, a brief discussion of one common technique-SDS-PAGE-will illustrate some of the common features. In this method, PAGE is performed in the presence of sodium dodecylsulfate (SDS), an ionic detergent, because most polypeptides will bind a constant amount of SDS. The SDS imparts a .charge uniformity to all of the polypeptides, and thus the separation is based entirely on molecular weight. The separation is typically done in a slab gel of polyacrylamide freshly prepared by the investigator. The molecular-weight range of a particular gel is determined by the degree of crosslinking of the polymer (higher crosslinking, lower molecular weight range). The samples are applied in various lanes of the gel along with a few lanes reserved for molecularweight standards. Detection is accomplished after the electrophoresis is complete by staining with either Coo-
massie blue dye or silver nitrate. The latter technique has a sensitivity of nanograms per band. Although this type of electrophoresis is easy to perform and has high resolution and sensitivity, there are some disadvantages that offer challenges for improvement to the analytical chemist. Analysis time is usually overnight; it would be desirable to shorten it to hours or even minutes, if possible. Even higher resolution is needed for some very complex samples. These gels often contain many bands, and small changes in the molecular size of a protein from one sample to another (such as slight modification) cannot easily be discerned. Finally, the tecbnique is labor intensive in its present form, and instrumentation is not readily available-especially for on-line sampling and detection. Developments in these areas would greatly increase the use of electrophoresis in nonresearch environments. One possible avenue that is being explored is capillary zone electrophoresis. This technique could offer very high resolution and short analysis times because higher voltages could be used with capillaries having efficient heat transfer. Sensitive detection methods and work on pacifying capillary surfaces are needed before researchers can make further advances in this field. Another important use of electrophoresis would be as an improved micropreparative technique. Current work on electroelution from gels, hlotting samples directly off a gel onto a more usable matrix, and other methods show some promise.
Immunoassays The general technique of immnnoassay relies on the interaction between specific molecular structures, called antigens, and antibodies produced by an animal. Antibodies are normally made as part of an animal’s
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natural protection mechanism against invasion of foreign substances into its body. For analytical immunoassays, the antibodies needed are typically made by inducing an animal, such as a rabbit, to produce antibodies against a specific substance and then ‘‘harvesting” these antibodies for use as specific reagents in an analysis. Although the antigen-antibody interaction is the underlying principle for immunoassays, the specific analytical technique involves a competitive binding of the antibody to the analyte of interest and to a known quantity of a labeled version of the same analyte. One overall analysis scheme is illustrated in Figure 1for the radioimmunoassay of analyte A. In this case, antibodies raised to A are exposed to a mixture of an unknown concentration of A and a known concentration of radiolabeled A. Because the specific binding curve of A to the antibody has been previously determined (as shown in the lower corner of the figure), it is only necessary to separate the labeled antigen-antibody complex from the free labeled antigen, measure the concentration of each, and relate the relative amount to the calibration curve already determined. Immunoassays have a number of distinct advantages over other analytical methods! Due to the specific nature of the antibody-antigen interaction, they are generally very selective and can often be performed in complex matrices without significant sample cleanup. The use of radiolabeled tags for the antigens can also provide high sensitivity. Despite these obvious advantages, a number of challenges still remain for those who use or develop immunoassay techniques. There continues to be a need for extremely specific interactions, and antibodies are not always specific for only one antigen. The use of monoclonal antibodies has improved this situation over the past few
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years relative to polyclonal antibodies, hut the generation and purification of any of these antibodies still remain a major effort for those who develop immunoassays. In addition, although radiolabeled tests can be quite sensitive, the use of other methods of detection, such as enzyme-linked or enzyme-amplified detection and fluorescent or luminescent tags, is receiving increased attention. These nonradiolabeled tags would minimize some of the problems with many of the current radioimmunoassays, such as those associated with safety, stability of reagents, and the need for specific equipment to detect radiolabeled species. Two other aspects of immunoassays are receiving increased attentionautomation and reliability. A large number of immunoassays are done in the United States each year, especially in clinical labs. The growth of these analyses will depend on the continuing ability to automate these methods and obtain a high degree of reliability. Reliability is especially important in the case of disease diagnosis-for example, AIDS test kits-where an incorrect analysis could have profound consequences. Finally, although many immunoassays are currently related to medical diagnosis and drug detection, in principle an antibody can be developed against any organic molecule of interest. In fact, analysis methods based on immunoassays have recently been described for a specific herbicide residue in soil and PCBs in environmental samples. These types of techniques will represent a substantial business in the future.
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Chromatographic reparations The importance of the separations process in biotechnology cannot be overstated for work in research and development through production processes. The complexity of both natural sample mixtures and of those using recombinant technology to produce large quantities of a specific biomolecule results in many cases where most of the work involved in a process is separations intensive. The initial work in separations is for analytical purposes-to purify and then identify a biologically useful material for further study. However, once research studies have begun, the separations quickly become preparative. In biotechnology, unlike many chemical processes, the scaleup factors from the laboratory bench to the industrial process can be relatively small. For example, a typical bioactive protein could be purified on a milligram scale using analytical equipment, and total production may be only a kilogram or less per year. Traditionally, biologists and biochemists have used various forms of
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open-column chromatography involving ion exchange, size exclusion, hydrophobic interactions, and affinity for lab purification. More recently, the use of high-performance liquid chromatography (HPLC) has begun to show promise with regard to analysis speed and resolution, but many of the initial workups are still done hy traditional methods in many laboratories. There are many challenges facing researchers who use separations for analysis and preparative methods. Many separations techniques are biochemically dependent (such as affinity chromatography), and the development of optimal methods requires a good working knowledge of the biochemistry involved. A fundamental understanding of the complex mecbanisms involved in many of these separations is still needed to further improve them. Support materials are needed that are stable, nonadsorptive, selective, and rigid so that these lab separations can be easily scaled to process levels. In general, chromatography can be a powerful technique, hut often where to start and what to do first are of prime importance to the overall success of a separations scheme. Finally, specific problems still exist in the fields of very hydrophobic proteins, membrane proteins, highly glycosylated materials, and extremely large protein aggregates and DNAs.
Sequencing-proteln and DNA One of the most fundamental analysis needs in biotechnology today is for primary-structure information on the hiomolecules of interest. The basic technology for sequencing both proteins and DNA exists, and its application over the past decade has resulted in an explosion of information on the primary sequence of many biomolecules. Despite this effort, however, only a small fraction of the DNA and proteins in mammals has been sequenced, and there is an enormous amount of work remaining in primarysequence determination. The sequencing of proteins or peptides is done using Edman chemistry, which was introduced in the early
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1950s and has improved over the past few decades. The method of analysis involves the sequential degradation of one amino acid at a time from the amino terminus of the protein and its identification, usually by HPLC, as the phenylthiohydantoin derivative. Improvements in the chemistry, separations, and detection methods have now resulted in the routine sequencing of many materials at the nanomole level and even lower in some cases. However, as the technology has improved, other problems have developed, and the current limit to the sequencing of proteins and peptides is usually the purity of the sample, reagents, and chemicals. Protein sequencing has become a truly microanalytical technique that requires careful attention to many details if it is to be successful. Quantitative sequencing and reproducibility are presently goals, not realities, in most labs, and improvements will be necessary especially as sequencing is required for process quality control. DNA sequencing, on the other hand, is an analytical technique that is fairly routine in many biology laboratories and can be done with a minimum of equipment and skill with rea;onable success. The sequencing is done by either the Maxam-Gilbert chemical degradation technique (especially for shorter pieces of oligonucleotides) or the Sanger method, as illustrated in Figure 2. The latter method requires the synthesis of a primer with 3. complementary sequence to an initial portion of the unknown DNA and then relies on the formation of various lengths of labeled DNA that are comdementary in sequence to the unmown DNA. These various lengths ire separated and analyzed on a gel, @herethe sequence can be read. Often "rom 300 to 500 bases can be deternined in one set of reactions and gel separations. This can be a very rapid technique sequencing a piece of DNA 1000 3ases long could be done in less than me week by a skilled operator), but he length and total number of pieces If DNA to be sequenced still offer a lumber of challenges. In particular, ,he methodology is labor intensive and ieeds to be automated. There are a lumber of groups currently working in automated DNA-sequencing methids, and a major opportunity exists 'or someone who develops a useful and inalytically reliable instrument. Be:ause protein sequences can be deeived from the sequence of the correrponding DNA that codes for that wotein, the proper strategy of both 3NA and protein sequencing can be of yeat use in solving the primary-strucure problems facing many in the bioechnology field.
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9 labeled primer thet is complementary io he 3' end of a single strand of DNA is prepared. The 88 quence of the DNA beyond the p r i m is unknown. The primer and DNA are mixed with (1) he fau deox-
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Molecular-structuredetermlnatlon
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Once the primary structure of a biomolecule has been determined, the job has just begun. The secondary and tertiary structures play a crucial role n the function of a protein, for examile, and OUT current understanding 108s not allow us to predict protein unction based on primary-sequence nformation alone. Protein X-ray crysallography, nuclear magnetic reso. lance, and mass spectrometry (MS) .re tools whose use in the biotechnol-
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ogy field is just beginning. How do proteins fold? What interactions do they have with solvents and other proteins or DNA? What is the structure of the active site of an enzyme? These are all questions that spectroscopic analysis is beginning to solve even on these large biomolecules. Fast atom bombardment (FAB)-MS is developing into a useful tool for protein sequencing, especially for identifying modifications in structure not easily seen with Edman chemistry sequencing. However, the mechanism of FAB-
a v i i n g stable basekes even at high sensirivitieS.Even difficultto mix solventscan be ~used.In faa,the peptidesepayabone&jted .fas obtained using a solvent gradient of methanol, TFA and water!Sensitivitywas a low 0.02 AUFS while monitoring at 214nm.
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MS is still not understood, and further improvements in this technique and others in molecular structure analysis should prove extremely valuable to research efforts in biotechnology. Comluelon The five areas outlined in this REPORT illustrate some of the applications of and challenges for analytical chemistry in the field of biotechnology today. There are other general problems such as the need for specific and sensitive methods of detection for almost any type of analysis. Many of the important biological molecules still to he discovered and examined are present in suhpicomole quantities in any preparation of reasonable size. Alternatives to standard absorbance techniques are needed a t these levels, and radiolabeled tags will not be the only answer. Another key factor is that the emphasis in this field is usually on the biochemistry of the system being analyzed and not on the instrumentation. More effective communication among analytical chemists, biologists, and biochemists will be necessary to solve the difficult problems of the future. Development of standard reference materials; extremely pure solvents, reagents, and materials; and good laboratory practices are all areas in which
analytical chemists can contribute to the expansion and development of quality bioanalytical techniques. Finally, the push to move from the researchlahoratorv to the marketplace quickly places increased pressure on the development and use of good analytical methods. This rapid exoansion is. in Dart. driven hv the DOt e k a l for immehiate benefits-to aliof us in our food, health, medicine, and general quality of life. The long-term growth of biotechnology will depend strongly on how well we can analyze and quantitate the products that will he made.
Methods of Peptide and Protein Microcharacterization-A Practical Handbook;Shively, J. E., Ed.; Humana Press: Clifton, N.J.. 1986. Watson. J. D.: Tooze. J.: Kurtz. D. T.Re1 i m 6 t n o n r n.VA A Short C o u r r ~Scien; titic American Bn,ks. Freeman and Cu: h e w Sork, 1983.
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Suggested r e a d l m Allen, R. C.; Sarvais, C. A,; Maurer, H.R.
Gel Electrophoresis and Isoelectric Focusing of Proteins. Selected Techniques; Walter de Gruyter: New York, 1984. Chard. T. Laboratory Techniques in Biochemistry ond Moleeulor Biology; Work, T. S.; Work. E., Eds.; Elsevier: Amsterdam, 1982; Vol. 6, Part 2. Hancoek, W. S.; Sparrow. J. T. In HighPerformonce Liquid Chromatography, Advances and Perspectiom; Horvath, C.. Ed.: Academic Press: New York, 1983; Vol. 3. Hearn. M.T.W. In High-PerformanceLiq-
uid Chromatography, Aduonees and Perspeetiues; Horvath, C., Ed.: Academic Press: New York, 1983: Vol. 3. Jorgenson. J. W.; Lukacs, K. D.“Capillary Zone Electrophoresis.”Science 1983. 222, 266.
Joseph L. (;lojcti rwc+cxd his A.B. degree in chernistryfrom Cornell and his Ph.D. in analytical chemistry from the University of Georgia. Since 1978, he has been in the Central Research and Development Department a t Du Pont, where he is inuolued i n uarious aspects o f gas chromatography, liquid chromatography, and biological characterization. He is currently supervisor of a separotionsgroup and is doing research in liquid chromatography relating to life sciences problems.
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