The production efficiency of 10,000- and 30,000-MW cutoff hollow-fiber bioreactor cartridges using the Cellex, Inc., Acusyst Jr. and the Unisyn Technologies, Inc., CP2000 bioreactor systems was investigated. Both cartridge types produced significant quantities of mouse monoclonal antibody, but the production efficiency (milligrams of antibody produced per liter of medium consumed) was higher for the 30,000-MW cartridge. Also, the smaller pore size cartridges displayed a lengthened lag phase in antibody production and cell growth of 26 to 33 days versus 11 to 20 days for the 30,000-MW cartridge. High intracapillary medium feed rates (5.0 L/day for 30,000 MW and 8.4 L/day for 10,000 MW) may reduce overall antibody production efficiency, and lower feed rates (3.5 and 7.2 L/day, respectively) may produce more antibody per liter of medium consumed.

The periodic removal of excess cells from confluent bioreactor cartridges can augment antibody production and extend the length of the run. Batch harvesting of antibody may lead to more highly concentrated antibody product than continuous harvesting.

Bioreactor systems that use hollow-fiber cartridges as a matrix for the growth of hybridoma cells are an efficient way of producing multigram quantities of monoclonal antibody.^1­3 Hollow-fiber cartridges can produce highly concentrated monoclonal antibody relatively free from contamination by cellular or serum proteins.⁴ These cartridges are available primarily in molecular weight pore sizes of 10,000 (e.g., renal dialysis cartridges) or in larger pore size configurations manufactured exclusively in bioreactor systems.

Using the Cellex, Inc. (Minneapolis), Acusyst Jr. and the Unisyn Technologies, Inc. (Tustin, CA), CP2000 bioreactor systems, we investigated the ability of several types of hollow-fiber cartridges to sustain the growth of mouse hybridoma cells and to produce antibody. We also studied the effects of several key operating parameters, including medium feed rate, antibody harvest method, cell removal, and glucose utilization rate (GUR) on antibody production.

Materials and Methods

Cell Lines. Mouse hybridomas VA1 18D7.3.2.4 (antivalproic acid) (IgG1), MOR 9B1 (antimorphine) (IgG1), METH7-4B6 (IgA) (antimethamphetamine), and PCP1.9D10 (IgG1) (antiphencyclidine) were grown in Iscove's Modified Dulbecco's Medium (IMDM) (Mediatech, Inc., Herndon, VA) supplemented with 2 mM L-Glutamine (Gibco, Grand Island, NY) and 10% fetal bovine serum (FBS) (Hyclone Labs, Logan, UT). All hybridomas were generated using the NSO fusion partner.

Baseline static culture productivity for each cell line was established by replicate seeding of 5 x 10⁴ cells/ml in growth medium (described above) in standard T25 flasks (Corning, Corning, NY). The flasks were incubated at 37°C and 7% CO_² for 72 hours; then the medium was removed and centrifuged at 1000 rpm for 10 minutes. The resulting supernatant was assayed for total and specific immunoglobulin by ELISA (Table I), as described below in the section "Determination of Antibody Concentration." (Tables not yet available on-line.)

Bioreactor Systems and Hollow-Fiber Cartridges. The Acusyst Jr. was run with the Acucell 1100 (Cellex, Inc.) 10,000-MW cartridge with 19 sq ft of surface area and 120 ml of extracapillary space (ECS) (Table I). The cartridge uses a reverse-flow cycling process to actively transport medium back and forth between the intracapillary (ICS) and the ECS. The system was unique to the Acusyst Jr. and its cultureware and was not available for the other cartridges or systems tested.

The CP2000 was used to compare two other 10,000-MW cartridges: the Clirans T220 cuprammonium rayon kidney dialysis cartridge (19-sq-ft surface area, 100-ml ECS) (Terumo Corp., Somerset, NJ) and the BR2010 ampholytic copolymer membrane cartridge (35-sq-ft surface area, 210-ml ECS) (Unisyn Technologies, Inc., Tustin, CA). These were also compared to the BR3530 cellulose acetate 30,000-MW bioreactor cartridge from Unisyn Technologies, Inc. (Table I).

General Bioreactor Run Parameters. The mouse hybridoma cultures were grown in the IMDM formulation. Cells in log phase growth (>85% viable by trypan blue exclusion) were centrifuged at 400 x g for 5 minutes, then resuspended in fresh growth medium. Approximately 10⁹ cells were then introduced into the ECS of each bioreactor cartridge by a large syringe.

The hollow-fiber bioreactor cartridges were tested in the two bioreactor systems with IMDM as described above. The FBS concentrations of the ICS medium for all the bioreactor experiments except those using MOR 9B1 and PCP1.9D10 were 3% for the first 20 L of media and 1% for the remainder of the experiment. The FBS concentration in the ICS for the MOR 9B1 and PCP1.9D10 runs was 3% throughout the course of the experiment. The serum concentration for the MOR 9B1 and PCP1.9D10 experiments was kept at 10% throughout. The FBS concentration of the ECS for the other cell lines was gradually reduced from 10 to 1% by introducing the ICS feed medium throughout the experiment.

The operating parameters of each bioreactor run were manipulated to maximize productivity. They varied according to the requirements of the cell line, bioreactor system, and bioreactor cartridge. The initial intracapillary recirculation rates for each of the CP2000 runs ranged between 350 and 550 ml/min and were gradually increased to the maximum setting of 999 ml/min. The recirculation rate of the Acusyst Jr. was between 200 and 400 ml/min. It could not be run with a recirculation rate >400 ml/min because of pump system limitations. Temperature was automatically maintained by the two systems at 37 ±0.5°C. The 100% air and carbon dioxide flow rates were held at approximately 100 and 5­10 cm³/min, respectively, throughout the course of each CP2000 run. The pH for the runs ranged between 6.9 and 7.3.

Determination of Glucose Utilization Rate. The GUR is an indicator of cellular metabolism and cell growth.⁵ The relative ability of the cartridges to sustain the growth of cells was assessed by measuring the amount of glucose from the ICS used by the cells. Glucose concentration in the circulating bioreactor ICS medium was determined using Roche Glucose Reagent and Roche Calibrator for Serum on the COBAS BIO instrument (all from Roche Diagnostic Systems, Inc., Somerville, NJ).

A bioreactor with a low GUR probably has too few metabolically active cells to demonstrate adequate antibody production. A high GUR may lead to rapid overgrowth and premature senescence. We have, therefore, established a target GUR for the different bioreactor configurations. At the target GUR, run parameters such as medium feed rates and recirculation rates are optimized to maximize antibody production. The target GUR is determined empirically by growing each cell line at different medium feed rates; it depends on the cell line and cartridge. A target GUR of 250 mg/hr for each bioreactor system was established.

The GUR (expressed as mg/hr) was determined by the formula:

GUR = {feed rate (ml/hr) x [glucose in (mg/L)­glucose out (mg/L)]}/1000

where glucose in is the concentration of glucose in the feed medium entering the bioreactor system, and glucose out is the concentration of glucose in the recirculating medium.

Determination of Antibody Concentration. The concentrations of secreted antibody in the bioreactor systems were determined by ELISA. Ninety-six-well microtiter plates (Costar Corp., Cambridge, MA) previously coated with drug­bovine serum albumin (BSA) conjugates were blocked to prevent nonspecific binding with 1% BSA in phosphate-buffered saline (PBS)/ azide. Serial dilutions of culture supernatant drawn from the bioreactor ECS were applied to the specific plate and a standard curve for assay quantitation was generated by incorporating known concentrations of purified antibody in parallel wells on the plate.

After incubation of the samples and standards, unbound antibody was removed by washing the plates in PBS/ Tween 20 (Sigma Biochemicals, St. Louis). The bound antibody was detected by the addition of alkaline phosphatase­labeled antimouse antibody (Zymed, San Francisco). The concentration of antibody was determined spectrophotometrically at 405 nm with a CR340 plate reader (SLT Corp., Salzburg, Austria), and data analysis was performed using the ELISA version 3.0 computer software (Meddata, Inc., New York City).

Criteria for Cell Growth and Antibody Production. The rates of antibody production and cell growth in the Clirans T220, BR2010, and the Acucell 1100 hollow-fiber cartridges were compared to those of the Unisyn BR3530 cartridge. For consistency, arbitrary criteria were used for between-run comparisons. One criterion was the number of days required to reach a target GUR level of 250 mg/hr; another was the number of days required to produce 1 g of antibody. These criteria defined the lag phase in comparisons of the same cell line grown in different cartridges or different cell lines or conditions for a given cartridge.

The effect of pore size on antibody production was determined by measurement of the amount of antibody produced (in milligrams) per liter of ICS feed medium consumed over a period of time (production efficiency). Because the runs varied in duration, day 40 was chosen arbitrarily to be representative for each run. The last criterion was the total antibody produced (in grams) by day 40 of the run.

Using the VA1 18D7 cell line, we compared the rate of antibody production and cell growth in the CP2000 bioreactor system using the Clirans T220 (Terumo Corp., Somerset, NJ), the BR3530, and the BR2010 (Unisyn Technologies, Inc.) bioreactor cartridges. The MOR 9B1 cell line was used to compare the Acusyst Jr. bioreactor system and Acucell 1100 cartridge with the rates of antibody production and cell growth in the CP2000 bioreactor system using the BR3530 cartridge (Table I).

Effect of Medium Feed Rate on Antibody Production. The effect of medium feed rate on antibody production was examined in both bioreactor systems. In the CP2000 bioreactor system that effect was studied using the BR3530 cartridge inoculated with the METH7-4B6 cell line.

Cell Removal. The effect of cell removal on antibody production and on bioreactor run length was determined in the CP2000 with the BR3530 cartridge and the PCP1.9D10 cell line. Mammalian cell cultures grown in hollow-fiber cartridges have a finite life. It has been speculated that accumulating cells decrease the productivity of the system by inhibiting the transport of nutrients across the hollow-fiber membrane, causing cell death. Whether periodic removal of excess cells from the bioreactor cartridge ECS would extend run length and improve faltering run parameters such as GUR and antibody production was investigated.

Harvest Methods. Antibody was harvested from the ECS regularly beginning within two weeks after the cartridges were inoculated. Two harvest modes were used, both using the same equipment. In a continuous mode of harvest, a peristaltic pump (Cole-Parmer, Chicago) withdrew medium from the ECS at a slow, constant rate (50 to 200 ml/day). At the same time an equal volume of fresh medium was introduced into the ECS by the same pump by using a separate piece of tubing run through the pump. Harvest bottles were changed periodically, and their contents centrifuged at 1000 * g for 10 minutes to remove cells and debris. The antibody-containing supernatant was then stored at ¾­20°C before testing. The second harvest mode was a batch method: A fixed volume of medium (50­250 ml) was pumped at once into the ECS, displacing an equal volume of antibody-rich medium from the chamber. This was repeated two to three times per week. The harvests were then processed in the same way as they were with the continuous mode.

Results

Cell Growth and Antibody Production. For the MOR 9B1 cell line and the BR3530 cartridge, it was determined in two separate runs that an average of 19 days were required to produce the first gram of antibody. This same cell line in two experiments with the Acucell 1100 cartridge required 31 days to produce 1 g of antibody (Table I).

Similar experiments were performed with the VA1 18D7 cell line. The Clirans T220 cartridge needed 33 days to produce 1 g of antibody, an amount comparable to the other 10,000-MW cartridge (BR2010), which required 32 days to produce the same amount. But the same VA1 18D7 cell line grown in the BR3530 (30,000-MW) bioreactor cartridge required only 17 days to produce 1 g of antibody. Likewise, the 30,000-MW bioreactor cartridge required 17 days to reach the target GUR of 250 mg/hr, whereas the two 10,000-MW cartridges required 31 and 30 days, respectively, to reach this level.

Similar results--13 and 26 days, respectively--were obtained with the MOR 9B1 cell line in the BR3530 and Acucell 1100 cartridges. The average time a 10,000-MW-pore-size cartridge required to reach the target level (1 g of antibody production) was 32 days. This contrasted with an average of 16 days for the 30,000-MW cartridge. Similarly, the average time needed by a 10,000-MW-pore-size cartridge to reach the target GUR of 250 mg/hr was 28 days versus 18 for the BR3530 cartridge.

Antibody Production Efficiency. We compared the ability of each of the hollow-fiber cartridges to produce antibody as a function of the amount of intracapillary medium consumed by the system during the experiment. These values were then expressed as milligrams of antibody produced per liter of medium consumed over the course of the entire run.

Using the MOR 9B1 cell line, we determined the optimum production efficiency to be 50 mg/L for the BR3530 cartridge and 24 mg/L for the Acucell 1100 cartridge. The VA1 18D7 cell line grown in the 10,000-MW cartridges had a production efficiency of 35 mg/L in the Clirans T220 cartridge and 30 mg/L with the BR2010. However, when the same cell line was grown in the BR3530 (30,000-MW) cartridge, the efficiency increased to 44 mg/L of medium consumed.

Comparison of antibody production in the BR3530 hollow-fiber cartridge to that in 72-hour static tissue culture (Table I) reveals a correlation between the amount of antibody a cell line produces in 72-hour culture and the quantity produced in the 30,000-MW cartridge.

Feed Rate. The effect of feed rate on antibody production was measured in two experiments using the METH7-4B6-8C3.2.1 cell line grown in the BR3530 cartridge. In the first experiment, the intracapillary feed rate was slowly increased from 0.5 L/day to a maximum of only 3.5 L/day (Figure 1). In the second experiment, the intracapillary feed rate was increased more rapidly from 1 L/day to a maximum of 5.0 L/day. The run with the lower feed rate produced more antibody in less time than the run with the higher feed rate (Figure 2). At day 40, the production efficiency of the low-feed-rate experiment was 118 mg/L and the production efficiency of the high-feed-rate scheme was 58 mg/L (Table I).

The effect of feed rate on antibody production was measured in two other experiments using the MOR 9B1 F11 P1.1.1 cell line grown in the Acucell 1100 cartridge using the Acusyst Jr. bioreactor system. When the feed rate began at 1 L/day and was rapidly accelerated to 8.4 L/day, the efficiency of the run was increased by only 9 mg/L (Figures 3 and 4). With a lower feed rate strategy beginning at 0.6 L/day and accelerating slowly to 3.3 L/day, the production efficiency almost tripled to 24 mg/L (Table I). Total antibody production from each run was approximately the same.

Cell Removal. When the PCP1.-9D10 cell line was grown in the CP2000 bioreactor system using a BR3530 cartridge, the cells reached confluence after only 25 days. Then antibody production began to drop precipitously (Figure 5). Beginning on day 30, cells were removed from the cartridge twice weekly by attaching syringes filled with IMDM (without FBS) to the extracapillary ports and vigorously pushing and pulling the plunger of the syringes. Large numbers of cells, cell debris, and a significant amount of antibody were obtained with this procedure.

Immediately after each of the nine cell-removal procedures performed during this one bioreactor experiment, antibody production increased significantly. The GUR also had a corresponding elevation. (Figure 5).

Batch versus Continuous Harvests. How did the kind of harvest mode affect total antibody production? The batch mode of harvesting used either syringes or a faster pump rate to remove antibody. When the VA1 18D7 cell line grown in the CP2000 system was switched from continuous to batch mode harvesting, the concentration of antibody increased from an average of 413 to 3387 µg/ml (Figure 6).

Discussion

The 10,000-MW-pore-size hollow- fiber cartridges from Unisyn Technologies, Inc. (BR2010), Terumo Corp. (Clirans T220), and Cellex, Inc. (Acucell 1100), are each capable of producing significant amounts of monoclonal antibody if used in the bioreactor systems described above. However, when compared with the Unisyn Technologies, Inc., BR3530 cartridge, the BR2010 and Clirans T220 cartridges were shown to be less efficient in terms of amount of antibody produced per liter of medium consumed. Likewise, the BR3530 was also found to be more efficient than the Cellex, Inc., Acucell 1100.

All three of the lower-molecular- weight-pore-size cartridges experienced a significant lag phase (approximately 1 month) for antibody production and cell growth, as measured by GUR. The lag phase with the BR3530 cartridge was consistently about one-half that of the 10,000-MW cartridges. The cost of medium consumed during the lag phase and of the labor used to run the bioreactor for an additional two weeks may offset the savings derived from using the lower-cost Clirans T220 cartridge in a bioreactor system.

The shorter lag period for the 30,000-MW pore size cartridge may come from the ability of medium components to more easily traverse the hollow-fiber membrane. Increased mass transport associated with a larger pore size hollow-fiber membrane is well documented.¹ The larger pore size fibers have higher ultrafiltration rates, which facilitate medium component exchange.⁶ The BR3530 cartridge provides a larger surface area for the growth of cells that may enhance cell growth and antibody production.

When low- and high-medium feed-rate strategies were compared, both the 10,000- and 30,000-MW cartridges showed that it was possible to overfeed cells in these bioreactor systems. High feed rates led to poorer cell growth and antibody production. By gradually increasing the feed rate throughout the run, more antibody was produced at a more efficient rate.

The high feed rates may dilute soluble factors that these cells require for growth. Some cell lines of the B cell lineage can stimulate autocrine growth.^7,8 The factors secreted from cells may be small enough to pass through the hollow fibers into the ICS. Some manufacturers of hollow-fiber bioreactor equipment propose high feed rate strategies to maximize antibody production,^4,6,9,10 but experience has shown that these strategies may not work well with all hybridoma cell lines.

The removal of cells from confluent hollow-fiber cartridges is useful to maintain the antibody production level of the bioreactor run. The cell-removal procedure rids the cartridge of large numbers of dead cells and cellular debris. This gives space for additional cell growth and clears the semipermeable fibers of cell components that may interfere with the efficient exchange of nutrients.

Harvesting the antibody in batch rather than in a continuous mode can increase the concentration of the antibody product if the batch harvests use small volumes of medium. This provides a reasonable alternative to continuous harvesting, especially for cell lines that do not secrete much antibody or if there is demand for a more highly concentrated product.

In the authors' experience, rapidly accelerating the medium feed rate in hollow-fiber bioreactor experiments may have an adverse effect on antibody production. It is recommended that once the GUR has begun to decrease, cells be periodically removed from the bioreactor cartridge to enhance antibody production. Inexpensive hollow-fiber cartridges, for kidney dialysis, can be a substitute for hollow-fiber cartridges used in antibody production with these instruments.

References

1. Evans TL, and Miller RA, "Large Scale Production of Murine Monoclonal Antibodies Using Hollow Fiber Bioreactors." Biotechniques, 6:762­767, 1985.

2. Lowrey D, Murphy S, and Goffe RA, "A Comparison of Monoclonal Antibody Production in Different Hollow Fiber Bioreactors," J Biotech, 1994, in press.

3. Lowrey DM, Meslovich K, Murphy S, et al., "Small Scale in Vitro Antibody Production Using a Disposable Hollow Fiber Device," presented to the 12th Annual Meeting of the European Society for Animal Cell Technology (ESACT), Wurzberg, Germany, May 1993.

4. Muragachi A, Nishimoto H, Kawamura N, et al., "Cell Derived BCGF Functions as an Autocrine Growth Factor in Normal and Transformed Lymphocytes," J Immunol, 137:179, 1986.

5. Knazek R, Gullino PM, Kohler PO, et al., "Cell Culture on Artificial Capillaries: An Approach to Tissue Culture Growth in Vitro," Science, 178: 65­67, 1972.

6. Gordon J, Ley SC, Melamed MD, et al., "Soluble Factor Requirements for the Autostimulatory Growth of B Lymphoblasts Immortalized by Epstein Barr Virus," J Exper Med, 159:154, 1984.

7. Hiefetz AH, Brantz JA, Wolfe RA, et al., "Monoclonal Antibody Production in Hollow Fiber Bioreactors Using Serum Free Medium," Biotechniques, 7:192­199, 1989.

8. Lowrey D, Murphy S, and Goffe RA, "The Effect of Intracapillary Media Feed Protocols on Hollow Fiber Cell Culture," Biotech Lett, 15:1025­1030, 1993.

9. Caple MV, Fletcher TR, Owens WJ, , et al., "A Dual Formulation Serum Free Media System," Biopharm, 5:52­60, 1992.

10. Hirschel M. and Keznoff S, "The Effect of Perfusion Rates on Hollow Fiber Bioreactors," presented to the Federation of Applied Scientists and Experimental Biologists, New Orleans, 1990.

Rudolph J. Czirbik, PhD, and Steven M. Rosen, PhD, are senior scientists; Diane M. Trunfio is an associate scientist; Ellyn W. Fischberg-Bender is research group manager; and Stuart M. Palmer, PhD, is director of research and development for the Therapeutic Drug Monitoring Business Unit of Roche Diagnostic Systems, Inc. (Somerville, NJ).

Molecular imprinting is becoming increasingly recognized as a technique for the ready preparation of polymeric materials containing recognition sites of predetermined specificity. The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules by the use of templates. Molecularly imprinted polymers (MIPs) have been used in an increasing number of applications. Several useful reviews of the molecular imprinting field have recently been published.^1­3 Of special interest to the developers of diagnostic assays is the potential use of MIPs in sample preparation as antibody or receptor binding site mimics in recognition and assay systems, and as recognition elements in biosensors.

History of Molecular Imprinting

The concept of molecular imprinting has its roots in Linus Pauling's early theory of the formation of antibodies. Pauling suggested that antibodies were formed when serum proteins assembled around template antigen molecules. The assembled antibodies were thought to have specificity-endowing binding pockets complementary in shape to the antigens. Furthermore, strong antibody-antigen binding energy would result from multiple noncovalent binding interactions including hydrogen bonds, ionic bonds, and van der Waals forces. This theory led to the hypothesis by Pauling and Campbell that artificial antibodies could be assembled using these basic principles.⁴ Although Pauling's theory of antibody formation was later disproved, several groups subsequently tried to apply it to synthetic systems. Pauling's student, Frank Dickey, attempted to form specific absorbents using silica.⁵ In the 1970s Wulff, at the University of Düsseldorf (Germany), formed covalent bonds between a monomer and the template molecule, followed by polymerization and template cleavage to yield a specific binding site. This method is limited by the synthetic necessities of first preparing a monomer-template molecule conjugate, and later chemically cleaving the template molecule from the polymer.⁶

Pauling's original concept was finally applied to the synthesis of artificial antibodies with the development of molecular imprinting by Klaus Mosbach's group at the University of Lund (Sweden).⁷ Mosbach's approach of preassembling a noncovalently associated monomer-template complex in solution prior to polymer formation was the breakthrough that enabled molecular imprinting to be used in a variety of applications. Mosbach's group continues to lead in new developments including studies on various polymer systems, classes of template molecules, aqueous imprinting systems, and novel physical formats, and has extended the potential usefulness of molecular imprinting. Imprinting has now reached a high level of sophistication, and patent coverage in the field is extensive.

MIPs are achieving commercialization through a collaboration between Mosbach and IGEN, Inc. (Gaithersburg, MD). IGEN's Separations Business Group is researching new applications, developing molecular-imprinted products, and offering molecular imprinting services for use by diagnostic and drug manufacturers. IGEN holds the rights to numerous patents and patent applications in molecular imprinting.

Making an Imprint

The concept of molecular imprinting is shown in Figure 1. To make the imprinted polymer, the molecule to be imprinted is dissolved in an organic solvent, such as chloroform, with a functional monomer, a cross-linking monomer, and a polymerization initiator. The functional monomer is chosen to have a chemical functional group that will interact and preassociate with the imprint molecule (Figure 1A­B). Ionic, hydrogen bonds, ¼-¼, hydrophobic, metal coordination, and covalent bond interactions are typical. Functional monomers that have been used for noncovalent interactions are shown in Table I. Following preassociation, polymerization occurs by UV irradiation or mild heating (Figure 1C). Once the solid polymer has formed, it is ground in a mortar and pestle and sieved to obtain a desired size, and the print molecule is extracted by incubation in a solvent capable of disrupting the specific interactions between the imprint molecule and the polymer (Figure 1D). This extraction step often involves including an acid or base in the solvent. What remains are rigid stable polymer particles that have pockets complementary in shape and electron density to the imprint molecule (Figure 1E). Shape complementarity results in high specificity while multiple interactions between the polymer and individual imprint molecules yield high affinity. (Figures and tables not yet available on-line.)

Several advantages make noncovalent imprinting the usual choice when a new method is being designed. For covalent imprinting, chemical modification of the print molecule with, for example, a vinyl group is necessary before forming the imprint. A wide range of monomers are available for noncovalent imprinting. In some cases the monomers can be combined to increase the strength of binding. In noncovalent interactions, association and dissociation of the imprint molecule to the imprinted polymer occur by the imprint molecule simply diffusing in and out of the complementary sites.

Recent developments in the formation of MIPs include imprinting of beaded polymers⁸ or silica resins³ and surface imprinting of membranes,⁹ which open additional methods of use, especially in diagnostic analysis.

Benefits and Current Limitations of Molecular Imprinting

Although molecular imprinting has some limitations, MIPs provide a combination of mechanical and chemical robustness with highly selective molecular recognition. They can be stored in the dry state at ambient temperatures for several years without loss of recognition capabilities, and are inexpensive, simple, and easily prepared. Generation of molecular imprints does not involve the use of laboratory animals or any material of biological origin other than the imprint molecule. MIPs are much more resistant to matrix effects than are biological antibodies. Highly lipophilic analytes can be assayed in organic extraction solvents using MIPs, which are formed in those solvents (Table II). Also, imprinted polymers can be made against print molecules that are too toxic for immunization in animals to raise antibodies. MIPs can also be made against molecules that are difficult to raise antibodies against--for example, short peptides. The preparation of antibodies against small organic recognition elements (haptens) requires hapten conjugation to a carrier protein before immunization. Preparing a molecular imprint avoids the need for derivatization of haptens.

Molecular imprints have been demonstrated against many classes of molecules. These include drugs,¹⁰ hormones,¹¹ pesticides,^2,12 proteins,¹³ amino acids,¹⁴ peptides,¹⁵ carbohydrates,¹⁶ coenzymes,¹⁷ nucleotides,¹⁸ nucleotide bases,¹⁹ steroids,²⁰ dyes,²¹ and metal ions.²² Some examples of interest for developers of in vitro diagnostics are shown in Table III.

Because noncovalent interactions are strongly dependent on the polarity of the solvent, the best imprints are made in organic solvents such as chloroform or toluene. Subsequent specific recognition of the imprint molecule by the imprint polymer is strongest under conditions that most closely resemble the cocktail used for polymer synthesis. If the MIP is transferred to aqueous solution, binding strength can be reduced significantly. Mosbach and others are currently working to improve aqueous imprints. As with polyclonal antibodies, the individual imprint cavities have varying degrees of selectivity. The distribution is usually around an average figure of high selectivity but with some sites of low selectivity. In chromatography, the low-selectivity sites do not influence the separation as long as the column is not overloaded with sample. In an assay format, specificity depends on the high- selectivity sites.

Examples of Molecular Imprints

MIPs have the potential for use in assay formats in a manner similar to the use of antibody-conjugated microspheres. For example, a radiolabeled ligand-binding assay, called the molecularly imprinted sorbent assay (MIA), was reported by Mosbach's group.¹⁰ Molecular imprints were made against two chemically unrelated drugs, theophylline and diazepam. Theophylline is a bronchodilating drug commonly used in the prevention and treatment of asthma. The assay accurately measured drug levels in human serum, with results comparable to those obtained using the established enzyme-multiplied immunoassay (EMIT). Specifically, the MIAs for theophylline and diazepam were linear over the ranges of 14­224 and 0.44­28 µm with detection limits of 3.6 and 0.2 µm, respectively, which are both satisfactory for therapeutic monitoring of the drugs. The specificity of the imprints was tested by determination of the cross-reactivity between major metabolites and of structurally related drugs. The imprints were shown to be highly selective, similar to those reported using commercial antibody­ based immunoassays (Table IV).

MIPs for leu-enkephalin and morphine have been made.¹¹ Because it is a closely related structure, codeine is a difficult cross-reactant for antimorphine antibodies. Cross-reactivity of the MIP with codeine is less than with most of the antimorphine antibodies (including monoclonal antibodies) reported to date. Although prepared in organic solvents, these MIPs were also efficient in aqueous solution with a performance sufficient for screening assays for drugs of abuse.

A molecularly imprinted membrane was recently developed that specifically bound theophylline over other purine bases.⁹ Such artificial affinity membranes should have applications for rapid analytical method development or for sample preparation methods. An additional benefit could be the combination of sample preparation and detection on the membrane.

MIPs that specifically bind particular metal ions (such as calcium and magnesium) have been produced using polymerizable metal-binding complexes. These have been used to make ion- selective electrodes.³ Imprints against pesticides and industrial pollutants have been reported by several groups.^2,12,16 Since many of these compounds are only soluble in organic solution, a solvent extraction step is necessary before the assay. Because MIPs can act as artificial antibodies in organic solution, they provide the possibility for developing formats similar to immunoassays, simplifying the current procedures.

Assays using molecular imprints with dissociation constants in the order of 10^­7­10^­8 M for cortisol and corticosterone were recently demonstrated.¹³ When the imprints were used in water systems, the interactions were disrupted because of the strong hydrogen bonding capacity of the water molecules, and the selectivity was severely reduced. Although many antibody-based steroid analytical methods have been developed for immediate use in aqueous environments such as plasma or urinary samples, several techniques use an extraction step for the steroids with organic solvents to avoid interferences with native binding proteins. In this perspective, anticorticosteroid MIPs may be an alternative for analytical applications, either by direct analysis or as a sample preparation step.

In some studies by Mosbach's group, target molecules have been chosen against which it is difficult or expensive to prepare natural antibodies. One example involves imprints against various macrolide antibiotics, such as erythromycin. Another example is imprints against immunosuppressants, such as cyclosporin.

Besides the many studies on resolution of the enantiomers of amino acids and sugars, molecular imprints against ß-blockers²⁴ and antiinflammatories²⁵ have also been reported.

Future Directions

The development of sample preparation methods and assays for small organic molecules using molecular imprints is expected to be completed in the near future. The preparation of MIPs for the isolation and detection of biomolecules is under development. Preparing an artificial antibody by molecular imprinting may be far more efficient in time and effort than is raising and producing a new antibody when none exists. Still needed are improvement of the binding strength of molecular imprints in aqueous solution and further development of surface imprinting and beaded imprints for higher-molecular-weight biopolymers such as proteins.

Molecular imprints have been included in several experimental biosensor designs.^1,3 Special advantages of MIPs in this application is their long-term stability at ambient conditions and in harsh environments. However, one obstacle to such biosensors is the current lack of a suitable interface between the MIP and the sensor element. Further developments in molecular-imprinted membranes may help in this area.

The creation of artificial antibodies through the combination of polymer chemistry and biochemistry is an ambitious goal, and one that promises considerable benefits. After many years of experimental development, MIPs are examples of progress toward this goal.

References

1. Mosbach K, and Ramstrom O, "The Emerging Technique of Molecular Imprinting and Its Future Impact on Biotechnology," Bio/Technol, 14:163­170, 1996.

2. Muldoon M, and Stanker L, "Plastic Antibodies: Molecularly-Imprinted Polymers," Chem Indust, pp 204­207, 1996.

3. Wulff G, "Molecular Imprinting in Cross-Linking Materials with the Aid of Molecular Templates--A Way Towards Artificial Antibodies," Angew. Chem Indust Ed Engl, 19:9­14, 1995.

4. Pauling L, and Campbell D, "The Manufacture of Antibodies in Vitro," J Exper Med, 76:211­220, 1942.

5. Dickey FH, "Specific Adsorption," J Phys Chem, 59:695­707, 1955.

6. Wulff G, Sarhan A, and Zabrocki K, "Enzyme-Analogue Built Polymers and Their Use for the Resolution of Racemates," Tetrahedron Lett, 44:4329­4332, 1973.

7. Andersson L, Sellergren G, and Mosbach K, "Imprinting of Amino Acid Derivatives in Macroporous Polymers," Tetrahedron Lett, 25:5211­5214, 1984.

8. Mayes A, and Mosbach K, "Molecularly Imprinted Beads: Suspension Polymerization Using a Liquid Perfluorocarbon as the Dispersing Phase," J Molec Recogn, in press.

9. Kobayashi T, Wang HY, and Fujii N, "Molecular Imprinting of Theophylline in Acrylonitrile-Acrylic Acid Copolymer Membrane," Chem Lett, 10:927­ 928, 1995.

10. Vlatakis G, Andersson L, Müller R, et al., "Drug Assay Using Antibody Mimics Made by Molecular Imprinting," Nature, 361:645­647, 1993.

11. Andersson L, Müller R, Vlatakis G, et al., "Mimics of the Binding Sites of Opioid Receptors Obtained by Molecular Imprinting of Enkephalin and Morphine," Proc Natl Acad Sci, 92:4788­4792, 1995.

12. Siemann M, Andersson L, and Mosbach K, "Selective Recognition of the Herbicide Atrazine by Noncovalent Molecularly Imprinted Polymers," J Ag Food Chem, 44: 141­145, 1996.

13. Kempe M, Glad M, and Mosbach K, "An Approach towards Surface Imprinting Using the Enzyme Ribonuclease A," J Molec Recogn, 8:35­39, 1995.

14. Kriz D, Ramström O, Svensson A, et al., "Introducing Biomimetic Sensors Based on Molecularly Imprinted Polymers as Recognition Elements," Anal Chem, 67:2142­2144, 1995.

15. Ramström O, Nicholls IA, and Mosbach K, "Synthetic Peptide Receptor Mimics: Highly Stereoselective Recognition in Nonconvalent Molecularly Imprinted Polymers, Tetrahedron: Assymetry, 5(4):649­656, 1994.

16. Mayes AG, Andersson LI, and Mosbach K, "Sugar Binding Polymers Showing High Anomeric and Epimeric Discrimination by Noncovalent Molecular Imprinting," Anal Biochem, 222:483­488, 1994.

17. Andersson LI, and Mosbach K, "Molecular Imprinting of the Coenzymer Substrate Analogue N-Pyridoxyl-L-Phenylalaninanalide," Makromolec Chem, Rapid Commun, 10:491­495, 1989.

18. Norrlöw O, Mänsson MO, and Mosbach K, "Improved Chromatography: Prearranged Distances between Boronate Groups by the Molecular Imprinting Approach," J Chromatog, 396:374­377, 1987.

19. Shea KJ, Spivak DA, and Sellergren B, "Polymer Complements to Nucleotide Bases: Selective Binding of Adenine Derivatives to Imprinted Polymers," J Am Chem Soc, 115:3368­3369, 1993.

20. Ramström O, Ye L, and Mosbach K, "Artificial Antibodies to Corticosteroids Prepared by Molecular Imprinting," Chem Biol, in press.

21. Norrlöw O, Glad M, and Mosbach K, "Acrylic Polymer Preparations Containing Recognition Sites Obtained by Imprinting with Substrates," J Chromatog, 299:29­ 41, 1984.

22. Rosatzin T, Andersson LI, and Mosbach K, "Preparation of Ca²⁺ Selective Sorbents by Molecular Imprinting Using Polymerisable Ionophores," J Chem Soc, Perkin Trans, 2:1261­1265, 1990.

23. Andersson L, Nicholls I, and Mosbach K, "Antibody Mimics Obtained by Noncovalent Molecular Imprinting," in Immunoanalysis of Agrochemicals, Nelson J, Karu A, and Wong R (eds), Washington, DC, American Chemical Society, pp 89­96, 1995.

24. Fisher L, Müller R, Ekberg B, et al., "Direct Enantioseparation of ß-Adrenergic Blockers Using a Chiral Stationary Phase Prepared by Molecular Imprinting," J Am Chem Soc, 113:9358­9360, 1991.

25. Kempe M, and Mosbach K, "Direct Resolution of Naproxen on a Non-Covalently Molecularly Imprinted Chiral Stationary Phase," J Chromatog A, 664:276­279, 1994.

Gary P. Henricksen is vice president, Separations Business Group, and Mark T. Martin, PhD, is director, Discovery Research Group, IGEN, Inc. (Gaithersburg, MD).

A company that can produce instruments faster than its competitors will not just have the first product on the market. It will also have the best product because it can receive customer feedback and produce second- or even third-generation products in the time it takes competitors to get their first-generation product out.

This article describes a system developed by LJL BioSystems, Inc. (Sunnyvale, CA), for completing instrument development projects in half the usual time and with a reliability rate well above the industry average. For example, the automated chemiluminescence reader for DNA probe assays that LJL developed with Chiron Corp. (Emeryville, CA) took just 13 months from start to full production and worldwide shipment. Similar projects typically require 24 to 26 months at other facilities. The reader's mean time between failures is well above the industry average of 10,000 to 15,000 hours for this type of instrument. In fact, it has been so reliable that, unfortunately for LJL, Chiron has had no incentive to buy a maintenance contract at the end of the instrument's warranty period.

Most of LJL's secrets for rapid but thorough instrument development are not so much secrets as basic processes that could be applied by other instrument manufacturers as well. This article presents a few of these basic processes as LJL practices them.

Program Management

One of the keys to rapid instrument development is having a program management system that is both clearly defined and strictly enforced. Program management should be thought of as a discipline that is every bit as important as the various engineering disciplines that are required for instrument development.

The development process should be comprehensively documented, and the document should be provided to all employees. In addition, LJL presents a customized version of this document to every manufacturing partner (the term LJL prefers to use instead of customer). It provides a detailed outline of all the activities and milestones involved in the development process, broken down into four clearly defined phases: product definition, concept and feasibility, prototype development, and manufacturing transfer and production.

During phase one, product definition, the instrument concept is defined in terms of instrument attributes and engineering specifications. This instrument concept is then used to evaluate and define key instrument functions, human factors issues, industrial design issues, and an estimated development schedule and budget.

A clear product definition is vital to delivering a high- quality instrument. Specifications must be agreed upon and approved by all functional groups involved in the project (including marketing, development engineers and chemists, and manufacturing) before proceeding to phase two.

During phase two, concept and feasibility, LJL develops detailed concepts for the package industrial design, mechanical subsystems, user interface, electrical subsystems, and firmware/ software components. It builds an instrument that is functionally similar to the final production instrument but not necessarily identical in form. If the form is substantially different from that of the planned production instrument, a nonfunctional model of the packaging may also be created during this phase to obtain user feedback on human factors and the industrial design.

In phase three, prototype development, functional prototypes are built that look and perform like the final production instrument and may be used for preclinical or beta trials. The engineering efforts during this phase focus on achievement of the reliability, manufacturability, and cost targets of the instrument.

The goal of phase four is to transfer a quality product into production. Pilot production instruments are built during this phase, the manufacturing transfer process is completed, and final production instruments are manufactured. The final production instruments completed during this phase are built by manufacturing rather than by the engineering team. All engineering hardware, firmware, and software designs, and all fabrication, assembly, and test documentation are upgraded at this time to meet production and good manufacturing practices (GMP) requirements.

By having a defined program management process that is broken down into clear phases, we not only know exactly what the project involves and have a schedule that we follow meticulously, but we can also maintain momentum by completing the project in short bursts rather than in one long haul. Each phase of the project has a predefined and strictly enforced start and stop time, so team members know they have to accomplish a specific task in a set time. This allows the team to focus on an immediate goal rather than try to do everything at once. By meeting a series of interim goals, team members not only keep the project moving along rapidly, they also have the reward of a continuous string of short-term successes to keep them motivated. LJL offers additional motivation in the form of a bonus for on-time completion of all tasks. If any interim goal is not met on time, all team members forfeit a portion of their bonus. This provides an incentive for team members to cooperate, be creative, work hard--to do whatever it takes to get the task done on time.

Concurrent Product Development

Beginning with phase one, our project teams include staff from all functional groups: marketing, research and development, product design, manufacturing engineering, and quality control. These permanent project teams include experts in each critical engineering technology and are led by a project manager with a technical background and project management experience. On average, teams contain between 5 and 10 members. We also consider our customer to be a part of the team. All functional groups have input at all stages of the project, providing concurrent product development. This avoids the possibility that marketing will decide that a product needs significant modification during research and development or, even more costly and time-consuming, during scale-up for manufacturing. With this method, instead of passing the project from team to team, one integrated team moves from milestone to milestone.

The entire team is required to attend all design review meetings, which are held weekly during phase one and less frequently during subsequent phases, with a minimum of three to four meetings per phase. Meetings may take all day, but they save weeks over the course of the project because issues are resolved and specifications updated and agreed on before the meeting ends. We bring a computer attached to an overhead projector and printer into all design review meetings. Team members can clearly see what is being discussed and, when they leave, always take with them a printout of the agreed-upon design specifications and action items.

An additional value to having all team members present at all meetings is that it enforces communication. People who tend to be task rather than people oriented sometimes need this inducement to discuss their individual projects in the context of the completed instrument. One of the biggest challenges in instrument development is to integrate the different engineering disciplines and systems with the chemistry requirements. Through the scheduling of frequent design review meetings, communication and preintegration are automatically built into the project. This ensures that the various elements that have been designed separately (e.g., packaging, mechanical, electrical, optical, software) will fit together more elegantly and integrate more easily with the chemistry.

The Right People

We have found that smaller teams work best because they generally require less management, which means less red tape, which means faster movement of the project. In addition, small permanent teams have a greater sense of ownership of the project, and all members share their expertise to solve whatever problems arise.

Basic to having a small team is hiring the right people. We prefer to hire high-end, experienced people and have fewer of them. Although well-seasoned people are expensive, they can make the initial product definition much easier and contribute significantly to the thought process that avoids last-minute issues. An additional value is that, if last-minute issues do arise, well-seasoned employees are much better equipped to resolve them with minimum risk because they know from experience what the risk parameters are. Thus, the price for these employees may be higher, but the overall costs to the company are actually lower. We prefer employees who have been through some product development scrapes, and we always ask when interviewing prospective employees whether they have been through a full product launch, all the way to market, dealing with the various issues that turn up during each separate phase of the process.

The right people are also versatile. An engineer who understands both hardware and software is invaluable because there is never any need for an outside arbitrator to help decide whether the instrument in development has a software or a hardware problem. If the same person is responsible for solving either problem, problem ownership is clear. Also, having a mechanical engineer with experience in design avoids the common situation of having the designer say, "It should look like this," and the engineer replying, "Well, it won't fit in that box." With these two disciplines combined, form will always follow function.

Inside/Outside

The right people do not always have to be in-house (see sidebar, "When and how to outsource"). Having a network of consultants can be far more cost-effective than hiring specialists who may not be needed for every project. Here it is critical to locate and maintain contact with technology centers that are a rich source of technical expertise. For example, LJL is fortunate to be located in the heart of Silicon Valley, one of the premier technology centers in the United States. We benefit greatly from regular contact with the many vital, innovative technology leaders in the area.

In addition, not every part needs to be or even should be manufactured in house. A good balance must be maintained between what is made in house and what is bought from a network of specialized and cost-effective vendors who supply both custom and commercial parts.

Deciding what should be made and what should be bought can be difficult. During phase one, we involve all of our functional groups in defining which components are high-risk, low-risk, high-return, and low-return, and then give each component a make or buy designation. We consider our own in-house capabilities when making these decisions. If a vendor can provide a component more quickly, less expensively, and with better quality than we can make it in house (all three criteria must be met), we buy it rather than make it.

It is critical to decide at the outset of the project who will make what, in order to know how much each component will cost and what the lead times will be. Plus, by making these decisions in advance, we can include our vendors in the project team. They are often involved in the design of the instrument and always in its transfer to manufacturing. This way, manufacturing knows whom to go to with questions, and the vendor knows exactly what manufacturing requires. As a part of the team, vendors see the instrument as it is being created and can better integrate their efforts with those of the in-house staff.

Using vendors for standard components also leaves in-house staff free to focus on elements of the instrument design that can make a difference in the marketplace. Tough projects are good because they provide a motivating challenge; out of them come new solutions and technologies. For example, given the challenge of designing a low-cost, highly reliable controller for one instrument, we used PIC (programmable integrated circuit) technology to create an electronic brain for this instrument that we can modify and transplant into any instrument we design in the future.

State-of-the-Art Equipment

To develop technological innovations rapidly, in-house staff needs the right equipment. Powerful advanced mechanical and electrical computer-aided design (CAD) packages allow sophisticated computer modeling that reduces the number of design iterations and the duration of the engineering programs. For example, we have nine CAD workstations for mechanical and package design: five Sun systems and four Silicon Graphics systems. All of these systems currently use the Pro-E software package from Parametric Technology Corp. (Waltham, MA), all are connected to the same network, and so all are able to share the same files. File sharing allows one engineer to access another's design, ensuring better integration of separate components.

These CAD systems also offer paperless prototyping. We have a direct link via modem to several vendors that produce models or even finished parts directly from our CAD files within 48 hours. All electronics design is done on CAD systems as well. We have an in-house system, Quick Circuit, linked to our CAD network that makes prototype circuit boards right from our CAD files, so we can go directly from the design of mechanical or electrical systems to prototypes without the need for any intermediate drawings or schematics (Figure 1). This allows production of prototypes in a matter of hours or days, not weeks or months. Further, building tooling directly from CAD databases is a significant money saver.

Modular Design Approach

Perhaps most important to LJL's rapid development capability is that when we develop a subsystem (such as a robotic arm, or a printed circuit board, or an optics system, or a piece of software), we think ahead to other applications for it in other markets. By doing this, we are developing a subsystem not only for the current customer but for the next one as well. LJL makes it a condition that we, rather than the customer, retain the manufacturing rights to these subsystems, which we can then modify for use in a wide range of instruments for various markets. This modular design approach is of value to any instrument manufacturer because it not only accelerates the design process but also improves reliability by allowing subsystems to be pretested independently of the completed instrument. Figure 2 illustrates how technology developed by LJL for one product was adapted for use in several others. Having a base of these core subsystems or technologies gives us an edge in getting an instrument to market quickly.

Conclusion

There are no real secrets to rapid instrument development. All it takes is small, focused, sharing, highly competent, well-equipped technical teams and a clear, proven process. Having a clearly defined process speeds development by reducing the amount of change: change of mind, change of personnel, change of project. Although a company must always accommodate some level of change, the rate of change should never exceed the rate of progress. Controlling change by having and enforcing a well-defined process reduces the risk of running out of time and ensures being able do all that was planned and more.

Lev J. Leytes is president and chief executive officer of LJL BioSystems, Inc. (Sunnyvale, CA). Amer El-Hage is director of program management for LJL. Ann C. Petersen is a technical writer specializing in the diagnostics industry.

When and how to outsource

Sometimes the quickest way to get something done is to give it to an outside vendor. The engineering teams of OEM (original equipment manufacturer) companies are experienced with a wide variety of projects, are usually in tune with the latest engineering technologies, and get a great deal of practice in the rapid development process. A vendor that has kept abreast of the new technologies and business processes can often provide cost-effective and technologically advanced products quite rapidly.

In many cases, the decision to outsource can be based solely on the amount of practice your company has or wants to have in a particular field. If you have a great deal of experience producing certain components, they should be manufactured in house. However, if expertise in a particular area does not reside in house and is not needed on a continuing basis, the project should be outsourced.

When choosing a vendor, size should not be a ruling factor. The largest vendor candidate may not automatically guarantee dependable service, because your project may be less important to a larger company with multiple projects than to a smaller one focused on a few important projects. Concentrate on the quality of the company's people and its track record with projects similar to yours.

Keep in mind, too, that the lowest bidder is not a bargain if it can't deliver to your quality standards and you have to change vendors. Check the prospective vendor's record of on-time deliveries with other customers. If a vendor misses shipments to you, your revenues from new instrument installations will be delayed and may be lost. Also, look beyond the quoted prices and evaluate the total costs over the lifetime of the program. Consider the costs of maintenance, repair, and downtime. Make sure that the quoted price includes adequate testing of the instrument during the development phases. Correcting problems that arise in the field because they were not tested for and corrected during development can be extremely costly in both dollars and reputation. Check for built-in loops that allow the vendor to come back for more money.

Choose vendors that can demonstrate a commitment to both price stability and quality. Look for those that will link their success with yours rather than merely play back your requirements to get your order. You do not want an outside contractor to come back and tell you that the program has failed because they took direction from you.

Consider offering the supplier exclusive rights to specific technologies it develops for your project but may want to use in future projects for other customers. This can motivate a supplier to invest its full creativity into the project and also allow you to get better pricing.

By outsourcing, you can dedicate precious internal assets to those activities that generate the best return and the highest shareholder value, while distributing functions that are peripheral to or do not match your company's core competence to other companies that specialize in those functions.

We learned at an early age that the first-place finisher gets the gold medal. In the IVD industry, the winners are those that are first to market. These competitors are rewarded not only with profits but also with increased chances of long-term survival. Miles Laboratories with its dipstick technology, Ortho Diagnostics with its HCV ELISA technology, and Perkin-Elmer with its PCR technology are a few examples of companies that have achieved market dominance by being first.

The time required to develop a new product affects its profitability more than do its initial costs. A McKinsey study reports that, on average, companies lose 33% of after-tax profits when they ship products six months late, compared with 3.5% when they overspend 50% on product development.

Many companies try to streamline the new product introduction process by improving their business organizations. Toward this end, they often establish cross-functional development teams and specific company guidelines on departmental interrelationships. Collaboration and communication are effective ways to improve the development process, but only if the product or project has been chosen carefully. A company may spend a lot of time developing the perfect organization and never launch a new product in time to capture the market.

Even the best-run company can improve its product introduction time by choosing a new technology or product carefully. Indeed, this is the most important factor in becoming the first to market.

The industry in recent years has had to weigh the appeal of new technologies against the inevitability of regulatory roadblocks. Assay formats with no historical precedent require more than the average product development time, for both in-house quality assurance and regulatory licensure. Assays that are similar to previously marketed products, however, have limited market potential if they offer no advantage over those existing products. In the blood glucose monitoring field, assay formats were predictable for many years. Newcomers in the marketplace could expect a 1 to 2% market share and a predictable lackluster product lifespan. Johnson & Johnson's introduction of LifeScan One Touch Glucose Monitor and Test Strips changed the rules and found high customer acceptance. The product development process unifying reagents and handheld instrumentation required longer-than-average product development time but in the long term is increasing the company's market share and market stability.

Having a clear mission is one key to timely product launch. The mission should be matched to a specific need of the intended customer. "Mission creep"--brought on by new marketing data, management redirection, or attractive technological advances--can bog down new product development. The development team must stay focused on the goal while still being able to react to the dynamic product development en-vironment. Project leaders may find themselves in the role of "coaching" when strategies must change to meet the desired goal.

After choosing the product or technology, the investigation or feasibility stage comes next. Within a predetermined time, the company should decide whether to commit to developing the product. During this phase, the company should outline the product's desirable features, demonstrate its feasibility, and estimate its cost and price. Marketing and R&D should collaborate to determine what features the customers want and how to include them in the product. New product ideas may be generated at any level within or even outside an organization, but the allocation of funds should be approved by the highest levels of management. Formal presentations and documentation by the designated product development groups are recommended. The technology should be understood and agreed upon before any money is allocated. Early allocations for research feasibilities are usually made after idea generation and before a formal contract or proposal.

The development phase begins only after a thorough investigation of feasibility. The developed product must be made correctly the first time to ensure a timely introduction and long life cycle. At this point, the technology transfer and manufacturing personnel carry the ball. The development phase should include all the stages of research and development including scale-up and product stability and clinical studies. The main focus should be to determine how to make the product at the desired price. Strategic project staffing at this time is critical. Management must provide adequate staff to meet deadlines and keep development costs down.

Processes and test methods that become carved in stone at this stage must be easily scaled up for manufacturing and must be accepted by FDA. Reengineering of licensed products is not cost-effective in the 1990s IVD market. Any process changes made to an existing product require that the manufacturer reevaluate validated standard operating procedures, specifications, stabilities, or clinical studies. In "Changes to Be Reported for Product and Establishment License Applications," published on April 6, 1995, in the Federal Register (60 FR:17535­17538), FDA provides guidance to manufacturers who are attempting to correct problems or address process changes with minimal expense and without unnecessary waste. Although this document has sorted process changes into distinct categories requiring different levels of compliance, the marketplace and the time factor control the actuality of manufacturing process changes. As technology has advanced and new antigens have been discovered, for example, infectious disease ELISA manufacturers have found it easier to license new versions of their assays rather than try to improve their earlier formats.

The manufacturing phase is the last step where new product introduction can proceed quickly or become snarled. Successful manufacturing is the result of training and collaboration between technical personnel and manufacturing personnel.

New technologies may be moved into the market faster if the pro-cesses used to make the reagents or the instrumentation are tried and true. Technology transfer professionals must determine not only the best technical processes and test methods to use in manufacturing a product but also whether FDA has previously cleared products made with those processes. Raw materials, chromatography resins, viral clearance techniques, and cleaning procedures are a few examples of reagents and processes regarding which novelty should be avoided if possible. All FDA-licensed intravenous antibodies marketed today are purified by ion-exchange chromatography. There are other techniques for purifying antibodies, but they require extra time and money. Unless a company really wishes to be a biotech pioneer, it should follow accepted practice.

At this stage, validation of the manufacturing process should occur. A successful validation should be a sure thing, since so much of the investigational work was performed at the development phase. First-place finishers in the marketplace know that validation is not experimentation, and they are confident of success by the time the product reaches manufacturing.

Long-term survival of an IVD company depends on its ability to beat competitors to market with new products. Improved profits, prestige, legal position, and regulatory acceptance are the prizes awarded to the winner in the IVD marketplace.

Alfred W. Schweikert, PhD, is a member of IVD Technology's editorial advisory board and manager of process development at Ortho Diagnostics Systems, Inc., in Raritan, NJ.

Conventional medical wisdom holds that finding cancer early is the next best thing to a cure. But the U.S. government has decreed that when it comes to bladder cancer, this doesn't apply. The Guide to Clinical Preventive Service, 2nd Edition issued in 1995 by the U.S. Preventive Services Task Force specifically recommends against routine testing for early detection of bladder cancer. The guidelines are available from the US Government Printing office.

Where does that leave two new bladder-cancer diagnostic tests? It keeps them confined to checks for recurrence of disease, a realm only a fraction as effective as more widespread screening.

As the 10-member task force was formulating its new guide, two diagnostics were getting ready to hit the market: the Bard BTA bladder test (Bard Diagnostic Sciences, Redmond, WA), currently available and Matritech's nuclear-matrix protein test (Matritech, Inc., Newton, MA).

But physicians on the task force found little evidence to support urine testing as a routine screen. The reason: like the prostate-specific antigen test, the most reliable marker for prostate cancer, bladder-cancer diagnostic screening doesn't have a strong track record of documented benefit. To be included in the recommendations, such tests need proof of improved morbidity and mortality.

Bard launched its product this spring after the final go-ahead from FDA. The dipstick like test is the "world's first three-minute bladder-cancer test" and the assay "requires no special training to carry out and interpret," according to company descriptions. The test relies on detection of proteins associated with developing cancer; a yellow color-change indicates positive green indicates negative.

Matritech, with approval pending, also has a urine test, also based on a protein marker. It includes antibodies that recognize nuclear-matrix proteins (NMPs). The advantage, say scientists who have worked with the assay, is that NMPs are specific to cancer cells -- they don't seem to appear without the presence of the carcinoma. Scientific literature shows that bladder cancer has increased by about a third over the past decade. The reasons probably are multifactorial, explains Donald Lamm, MD, professor and chair of urology at the Robert C. Byrd Health Sciences Center of West Virginia University in Morgantown.

One reason for the increase is the influence of environmental factors, such as smoking and exposure to chemicals, particularly in industries as varied as truck driving and hair dressing. But another reason is that more than a quarter of patients with bladder cancer -- which generally shows up first as blood in urine -- may yield a negative cytologic result during cytoscopy, the traditional confirmatory measure.

Clearly a better screening tool is needed, Lamm says. Although neither the Matritech nor Bard test is to be used for initial detection, that is precisely what is needed to find cancer early, when intervention is most likely to pay off, he adds.

Lamm and his colleagues are advocating a selective screening process, in which those at risk are carefully followed. A family history of disease, exposure to workplace chemicals, and smoking all fit into that category, Lamm says.

But, to treat this population, "we really need a precise way of detecting the cancer," he stresses.

An even newer, experimental immunoassay, Aura-Tek FDP by Perimmune, Inc. (Rockville, MD), is showing heightened sensitivity in detecting the cancer, when compared to both hemoglobin-based dipsticks and cytologic evaluations, Lamm says. At the University of Southern California School of Medicine, David Esrig, MD, Richard Cote, MD, and Peter Jones, PhD -- along with others -- have developed a histochemical stain that shows promise in predicting a recurrence, which could also aid in narrowing the patient population in need of close monitoring.

A mutation in the p53 gene, which leads to an increase in a related protein, occurs in significantly more patients who experience a recurrence. "These people are twice as likely to recur," Jones, who is director of the university's Norris Comprehensive Cancer Center.

In the future, genetic testing may be used to further divide that pool of higher-risk people, predicts Lance Willsey, MD, a urologist at Massachusetts General Hospital in Boston. Then one of these more-sensitive assays may be applied to actual screening.

This kind of "second-layer selection" is likely to be the first wave of a true clinical application of gene testing, a fit that may go hand-in-glove with in vitro diagnostics down the road.