Trends in Biotechnology, VoL 3, No. 9, 1985

Callus conclusion

We agree with Brodelius and Rosevear's conclusion (Trends in Bio- technology, Aug (1985) p. 195) that present practice of plant cell immobili- zation by entrapment in macroporous hydrogels is the best technique cur- rently available. However the crux of our argument is that claims for 'superior' characteristics (e.g. longev- ity, productivity) of immobilized plant

cells are usually based on a comparison with cells grown in suspension culture, despite the observation that, morpho- logically, immobilized cells resemble callus, not suspension cultures. Thus, the usual conclusion from such studies (that immobilization enhances the use- ful characteristics of plant cells) could well be invalid.

Our implication was that immobiliza- tion is merely a convenient way to return plant cell suspensions to a callus-

219

like morphology, with a concomitant return to callus-like productivity, long- evity etc. It is not claimed that self- immobilization in aggregates is superior to entrapment ofceUs in a gel matrix, but that it could offer an alter- native method to achieve the same end, and therefore justifies full investi- gation.

A. L. DAINTY, K. H. GOULDING, P. K. ROBINSON, I. SIMPKINS

and M. D. TREVAN

Division of Biological Sciences, Hatfield Polytechnic, PO Box 109, Hatfield, Herts, UK.

On the emergence of commercial products from biotechnology

Martin Nash

For s o m e years students o f sc ience and industry have been predict ing the pervas ive impact ofb iotechnology on the health care industry. Much has been discussed about the role b iotechnology will play in industrial pro- cess , diagnostics, and pharmaceut ica l products . This rev iew examines two branches o f b iotechnology which are emerg ing in the in-vitro diag- nostics arena which are l ikely to bear edible fruit in the current decade -

monoc lona l antibodies and D N A probes.

Monoclonal antibodies (Mabs) and DNA probes have been under investi- gation for several years in both the public sector and industry with a view to developing useful reagents for either monitoring clinical outcomes or eluci- dating clinical conditions. At times, it has appeared that exploitation of these new technologies has proceeded for reasons remote from the merits of the project itself. At other times, suitable broad applications have been tantaliz- ingly close. Today it is possible to discern areas in which diagnosis using these approaches may be possible; they provide clinicians with information which has genuine benefit in and impact on patient management. This paper will look broadly at the areas in

Martin Nash is a biotechnology healthcare consultant at 2739 Inverness Drive, La Jolla, CA92037, USA.

which the products of the new tech- nology will be employed in diagnostics, assess their advantages (both absolute and relative) and, finally, examine, for Mabs and nucleic acid cellular probes, some of the criteria for commercial success which would apply to any diag- nostic reagent.

There are questions which can be decided, if not answered, with pre- cision; if one asks whether products derived from biotechnology will contribute to health care and company profits, the answer is undoubtedly - yes - even if the precise products cannot be named. It is also possible to identify laboratory uses of such pro- ducts. Figure 1 describes, in general terms, some of the areas where the first in-vitro tools of the new technologies are likely to be used and also indicates which areas seem best suited to each tool; it also shows that there are areas where Mabs or probes have procedural

or technical advantage, and an area of overlap between them.

It is a dictum of capitalism that in the main (and in the long term) markets will operate efficiently and therefore advantages of a particular product type will eventually come to the fore. Some market segments will clearly fall within the area of advantage for Mabs because the products being tested for lack DNA, or are characterized by plentiful and unique surface proteins against which the antibodies can be targeted to provide very high sensi- tivity in the immunoassay format. On the other hand, testing for genetic abnormalities or disease susceptibility at the gene level certainly is feasible with probe technology.

The more challenging and perhaps interesting area to contemplate is the center of Fig. 1 where both probes and Mabs have contributions to make. The question is, if both function on the technical level, which are clinicians and investigators likely to favor in the coming years?

Preparat ion of D N A probes and Mabs

All probes are not created equal, or in fact in the same way. 'Long' probes are the more traditional entity, while 'short' probes are the more recent development produced synthetically, often on DNA synthesizers currently manufactured by some eight com- panies.

In 'long' probe production (Fig. 2), DNA from a target of interest (for example, a virus or pathogenic bac- terium) is isolated using restriction enzymes. The broth is purified to obtain DNA free of debris, which is then inserted into a suitable vector

© 1985, Elsevier Science Publishers B.V., Amsterdam 0166 9430•85/$02.00

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Immunoassay J Cancer replacement " _ ~ Genetic screening

- Hepatitis

Therapeutic Sexually trans-mitted diseases Drug

drug ~ resistance monitoring testing " Industrial purification

- Microbiology

HLA Disease susceptibility

Virology testing

Fig. I. Applications of monoclonal antibodies and DNA probes.

molecule (e.g. plasmid, bacterio- phage). These molecules are then cloned thereby replicating the inserted DNA and, when an appropriate amount is obtained, the DNA of interest is removed by restriction enzymes. The resulting material must be purified on a gel and then labelled with signal-generating reporter groups (or their precursors) using poly- merases, reverse transcriptases, or other appropriate enzymes. In some cases, probe populations are further purified.

To produce 'short' probes using

Isolation of DNA fragment from target molecule

Selective restriction, isolation and testing of target fragment

Insertion of DNA into a vector molecule, e.g. plasmid

Propagation of host molecule

Removal and isolation of DNA

Enzymatic restriction of DNA

Purification (gel, sedimentation)

Identification and isolation of desired probe fragment

Enzymatic incorporation of reporter group into probe

Isolation of labelled probe

Fig. 2. Production of "long" probes.

chemical DNA synthesis (Fig. 3), one must begin by identifying a desired sequence; the chemists must know the sequence desired before they can pro- duce the probes (this requirement does not exist for long probes). A single synthesis can typically produce enough probe for millions of tests, often in a single afternoon. The resulting material is then purified by HPLC or gel electrophoresis. Signal- generating groups (if not already present) are then attached and the final probe purified.

The method for obtaining Mabs is sufficiently well-known to readers of this publication that it is not discussed here, but is outlined in Fig. 4.

Technical and production aspects do not in themselves determine the desir- ability of a technology to industry. They must be viewed through the filter of characteristics which apply to all in- vitro diagnostic tests and determine their suitability for development into products. These are not the only elements which enter into a product decision, but they do enter into all deci- sions: • cost of production • ease of production • procedural difficulty • sensitivity • specificity • technical familiarity • time of development

Cost of production As used here, this refers to the cur-

rent production method. It does not encompass acquiring capital equip- ment since the impact oftax/deprecia-

Trends in Biotechnology, VoL 3, No. 9, 1985

tion varies widely among companies for the same hardware. Additionally, the product volume against which the equipment could be written off is con- jecture at present. In the case of Mabs the term presumes murine production yielding some 5 ml ofascites fluid per mouse and 5-10 mg/ml of antibody.

Ease of production While this influences the cost of

production and the time of develop- ment (discussed below), it is con- sidered in its own right by manu- facturers because of the ubiquitous Murphy's Law and because of the very substantial impact of high failure rates :and labor overtime charges on profit- ability. Ease of production skews the mix of desirable products toward the better known of the technologies in question (Mabs) and the more auto- mated (short probe). Anything which involves a great deal of 'art ' or extreme complexity will be relatively disad- vantaged. Art particularly is anathema to rational production decisions and to the regulatory and supervisory mechanisms in the health care industry of most countries.

Procedural difficulty This aspect eases the discussion into

the arena which companies call the market-place and consumers call the laboratory. The term could also be called laboratory procedural ease. It is common for tests which are complex to be run less successfully than those which are straightforward. Techni- cians in the diagnostic laboratory are usually not interested in blazing new research trails nor in creating unique diagnostic events; indeed this would be counter-productive in the preparation of routine diagnoses. Perhaps the most overwhelming advantage of Mabs over any type of probe assay is that they are used in familiar assay formats, com- patible with existing instrumentation. Especially in that segment of the market-place where Mabs can simply

Obtain target sequence

Chemically synthesize modified probe

Purify (HPLC, electrophoresis)

Attach reporter groups

Fig. 3. Production of "short' probes.

Trends in Biotechnology, VoL 3, No. 9, 1985

be substituted for polyvalent antisera, the use of Mabs causes virtually no increase in procedural difficulty. On the other hand, DNA probe tech- nology involves rupturing cells, fixing target DNA to support matrices and then using this as a target for the interactions of reagent and amplifica- tion/detection systems. Since many of these steps differ from those used in the familiar immunoassays, and since in the early days of probe use, automation will not be prevalent, the penetration of probes will be retarded. Those with long memories will recall that the same factors applied during the early days of immunoassay tests: as the assays grew in popularity their complexities were overcome by technical advance. The presumption that DNA probes will provide useful information gives rise to the conviction that convenient ways will be found to extract this infor- mation, but the task is yet to be accom- plished.

Sensitivity This is commonly, i f incompletely,

defined as positivity in the disease state. At the simplest level it is a ratio between signal and noise. The most common way to improve the signal/ noise ratio is by increasing signal. In this respect Mabs have the advantage of the numerous surface antigens

Isolate antigenic determinant

Immunize mice

Harvest antibody-producing lymphocytes

Harvest cultured myeloma cells

Mix

Fuse

Screen immunoglobulin-producing hybridomas

Inject in host mice or grow in tissue culture

Harvest

Purify from ascites

Attach reporter groups

Fig. 4. Production of monoclonal antibodies.

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Table I. Characteristics of in-vitro diagnostic tests using monoclonal antibodies and DNA probes

DNA probes

Mabs Long Short

Cost of production (one million tests) r~S$3300 US$30 000 US$500 Ease of production fair fair good Procedural difficulty of test low fair good Sensitivity good high fair General specificity fair high high Technical familiarity high low low Time of development 7 months 6 months 2 months

against which they can target the labelled antibody. Probes very pos- sibly have fewer targets per sample, but should have lower noise and thus a favorable ratio due to their inherently high specificity.

S p e c ~ Defined as negativity in non-disease,

this is a critical but complex issue. Probes are inherently specific for the target of interest since they look directly at the DNA, hopefully at the region which codes for the identity of the organism in question or the stretch which defines the target of interest. For probes of infectious disease, the underlying assumption is that varia- tions in structure are restricted to dis- crete fragments; thus it is possible to direct a probe to a region conserved within a species so that all strains will react with the probe.

Specificity is more complex in the case of Mabs detecting a variety of serotypes. The technology depends upon a unique surface-marker being accessible as an immunogen and a tar- get. The immunologist, therefore, depends on nature to provide such an antigen. Flow cytometry or even cytotoxic sorting make it possible to construct hundreds or thousands of hybridomas and screen them effi- ciently to identify the one (or few) which, used collectively, can detect all relevant strains but no others. Nature has generally been kind to immuno- logists, and companies producing Mabs must presume this situation will hold in the future.

Two additional points concerning specificity may be relevant to the dis- cussion. Firstly, it may be that probes will prove particularly useful in diag- noses of viral or bacterial infections. The presence of most viral proteins (or antibodies to viral antigens) correlates poorly with infectivity because the protein half-lives are not the same as

those of viral DNA or intact viral par- ticles. Secondly, there may be dif- ferences in specificity between long probes and short probes. A long probe can have 200 to several hundred bases. I f one is probing a target genome to dis- tinguish a 'normal' genome from a closely related one, a long probe cannot currently be used stringently enough to detect a one or two base-pair mis- match. Short probes, however, can be used under conditions in which hybridization will not occur in the pre- sence of single base-pair mismatches.

Technical familiarity This refers to the familiarity of the

operator with a given test protocol. Many existing diagnostic techniques (e.g. ELISA) are difficult procedures and yet are practiced routinely. Pro- cedural difficulty is not, therefore, in itselfa barrier to the entrance of a pro- duct into the diagnostic market if the test is configured in a familiar format. In this respect Mabs hold a clear advan- tage over probes because of the wide range of existing diagnostics based on antibodies.

Time of development For Mabs this is defined as the

period from the identification of the immunogen and its purification to release of the product; for the long probes it is the period from availability

Table 2. Comparison of in-vitro diagnostic tests using monoclonal antibodies and DNA probes

Basis of competition Advantage

Cost of production Probes (short) Ease of production Probes (short) Procedural ease Mabs Sensitivity Mabs and probes

(long) Specificity Probes (particularly

short) Technical familiarity Mabs Time of product development Probes (short)

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ofa DNA fragment to the product; for short probes it is the time from which the target sequence is known to the product. Here, regulatory complica- tions are not considered since they are so variable.

Table 1 summarizes the particular technologies with reference to charac- teristics discussed above and Table 2 gives a subjective analysis of where the advantage lies at present.

The future A difficulty in this analysis is that it is

much easier to specify what the ideal situation will require than to actually arrange the circumstances. The aspects described above are inherently in flux as researchers within industry

and outside grapple with removing the limitations. In the near future cost of production will presumably decline in all cases and ease of production will pre- sumably improve. The most striking areas of Mabs advantage are procedural difficulty and technical familiarity. In all likelihood the Mabs advantage will be diminished with further work on gene probes. Improved assay tech- niques, particularly those which give rise to new families of fluorophores and new methods of reducing the noise currently associated with support mat- rices, will probably bring probes some- what closer to Mabs in sensitivity. Specificity is a biological function and it will not change in the timeframe any reader of this article is likely to be interested in, while time of development

Trends in Biotechnology, VoL 8, No. 9, 1985

is likely to improve for all methods and so remain relatively much the same.

So we come around to the question of whether any or all these technologies will contribute to the emerging diag- nostic laboratory. This article has em- ployed the device of the tension bet- ween the technologies and probably the most important message to come from the analysis is that most of the tech- nologies are already acceptable in most aspects. The conclusion from this is that we shall all be seeing much more of all these technologies in the years to come because (1) they clearly provide information which is useful in the examination of disease processes; and (2) none fails utterly when measured against the seven yardsticks described above.