Stephen J. Ubl is president and chief executive officer at AdvaMed (Washington, DC). He is recognized as a top healthcare advocate and policy expert with considerable experience across multiple health policy sectors. He can be reached at president@ advamed.org.

All too often, when it comes to healthcare costs, policymakers in Washington are too focused on making short-term cost savings rather than providing long-term value to patients and the healthcare system as a whole. That can be seen once again this year in Medicare reimbursement policies for two types of critical diagnostic technologies: laboratory testing and medical imaging.

The Bush administration's budget proposal for fiscal year 2008 includes a competitive bidding program for laboratory tests, which could drastically cut payments for such critical medical tests. Competitive bidding could reduce beneficiary access to tests and undermine incentives for developing the new generation of molecular diagnostic tests that will revolutionize the practice of medicine.

Fortunately, more-forward-thinking proposals are on the table to establish a payment system that recognizes the full value of lab testing. In early March, Congressmen Bobby Rush (D–IL), Michael Ferguson (R–NJ), Mike Thompson (D–IL), and Phil English (R–PA) introduced the Medicare Advanced Laboratory Diagnostics Act. The following are the particular provisions of the bill and the problems they target.

The current 23-year-old lab fee schedule provides little incentive for developing and adopting new molecular diagnostics tests. The legislation would establish a demonstration project to evaluate an entirely new payment system for molecular diagnostic tests, which are the future of the IVD industry. This system would be structured so that the payments more appropriately reflect the value of such technologies by creating a panel of experts who know and understand diagnostics to recommend payment rates.

Today's payment levels bear little relationship to the value of diagnostic tests, and the system for determining new rates is confusing and not transparent. The legislation would require CMS to establish and publish the procedures and criteria it uses to set payment levels for tests.

One of the most serious problems with Medicare's current system is that it operates in a black box, with virtually no input from stakeholders. The legislation would create more interactions among patients, physicians, laboratory groups, IVD manufacturers, and CMS at an annual public meeting on new test payments. It would also require CMS to give advance notice and allow public comment when a test is considered for inherent reasonableness adjustment.

Finally, despite all the problems with the current fee schedule, CMS has limited official authority to correct the gross payment errors that occur. The legislation would provide mechanisms for interested stakeholders to pursue a correction of many long-standing errors.

Connecting the Dots

AdvaMed is working on many fronts to support this legislation. The association is working with members of Congress and their staffs, medical specialty groups, patient organizations, and various laboratory and diagnostics groups to build support for this new bill. The association has also been in touch with officials at CMS and the Department of Health and Human Services. The hope is that this is the year when Medicare's diagnostic payment system will finally be modernized.

However, there is similar shortsightedness in Washington toward another critical diagnostic technology, that is, medical imaging. Last year, Congress cut Medicare funds for medical imaging by $8 billion during the next 10 years.

Here again, AdvaMed will be making efforts to reverse those cuts and prevent future cuts. But much like the competitive-bidding proposal for lab tests, these imaging cuts clearly show that policymakers either cannot or will not see the link between diagnosis on the one hand, and healthcare quality improvement and cost savings on the other. This is especially discouraging as genomics-based diagnostics and molecular imaging are increasingly teaming up to open the vast potential of personalized medicine, including its potential to prevent disease and dramatically save costs.

As AdvaMed pushes to modernize payment for lab testing and prevent massive cuts in medical imaging, a primary goal is to help policymakers in Washington and elsewhere finally connect the dots between diagnostic technologies and the tremendous savings in costs and lives they create.

Danny Levenson is president and chief executive officer at Millenia Diagnostics Inc. (San Diego). Millenia Diagnostics is a biotechnology company that is focused on the supply of research and manufacturing products and services in the field of diagnostics. Levenson oversees development and manufacturing of IVD products and reagents in a variety of technology platforms. He can be reached at danny@ milleniadiagnostics.com.

In the face of a growing global IVD market, U.S. diagnostics companies need to keep product prices low to remain competitive. One way they can accomplish this without sacrificing quality is by improving manufacturing efficiency—for example, by using equipment that is automated and more reliable. Another way is by outsourcing certain production tasks, allowing them to dedicate more time to their areas of specialty.

However, in taking steps to lower costs, IVD companies must remain mindful of the rapid advances in diagnostic technologies. Any new processes introduced should be flexible enough to accommodate new technologies as they are developed. In addition, companies need to ensure they are meeting the requirements of both domestic and international regulations.

To find out more about the manufacturing and processing issues that diagnostics companies face, IVD Technology editor Richard Park spoke with Danny Levenson, chief executive officer at Millenia Diagnostics Inc. (San Diego). In this interview, Levenson discusses the different strategies IVD companies can use to run more efficiently and meet user demands. He also talks about the realities of FDA regulations and inspections, trends toward multiplexing, and how diagnostics companies should be structured to encourage collaboration and innovation.

IVD Technology: Does Millenia Diagnostics consider itself more of an IVD manufacturer or a supplier to diagnostics companies?

Danny Levenson: We do both. Millenia Diagnostics is a San Diego–based biotech company focused on the supply of research and manufacturing products and services in the field of diagnostics. Our products and services are used by universities, government agencies, publicly traded companies, small private companies, and international businesses. We're good manufacturing practices (GMP) compliant and recently obtained our medical device manufacturing license. We produce both Class I and Class II medical devices.

What have been the most significant advances and trends in the area of diagnostics manufacturing and processing technologies over the past few years?

Although the idea of lean manufacturing systems is not new to IVD producers, they continue to be employed and refined. In addition to reducing waste, these systems help improve processes. By reducing material cost, streamlining design and manufacturing processes, and improving quality, the domestic IVD industry can stay globally competitive.

Many companies are focusing on their manufacturing equipment to reduce costs and increase quality. Manufacturing personnel are calling out for robust equipment that enhances efficiencies by virtue of its reliability on the production floor.

In addition, there's been a movement toward custom and automated equipment. The benefits of this include improved reproducibility and the reduction or elimination of labor-intensive processes.

One such company, Kinematic Automation Inc. (Sonora, CA), is an industry leader in equipment for the development and manufacture of rapid diagnostics.

Quality has always been the main attribute of U.S.-based diagnostics companies, but as we're faced with a global market, pricing is becoming more and more important. Lean manufacturing systems help our pricing to stay globally competitive.

Do increased quality requirements and demands place pressure on the development of manufacturing and processing technologies?

Certainly the increased quality needs impact the manufacturing processes by requiring quality systems such as corrective and preventive action, process control, and validations. These are not just requirements, but are essential to producing consistent, high-quality products. In addition, product development is affected by the implementation of stricter design controls and risk analysis.

Looking at shared technical complexity, what are the most troublesome areas involved in manufacturing IVD products today?

One troublesome area is in using highly variable raw material to manufacture a finished product with low variability. There's a natural inherent variability in biological reagents as well as in many other materials used in the manufacture of IVDs. Thus, one trend is the movement toward raw materials with greater reproducibility.

For example, polyclonal antibodies, which can have a high degree of variability, are undergoing improved purification processes or are being replaced with monoclonal antibodies, which can be produced with a low degree of variability from one batch to another. Another example is the use of recombinant proteins in place of native proteins. In some cases, natural materials are being replaced with synthetic material.

Controlling raw-material variability leads not only to more readily reproducible end-products but means that less time is spent evaluating materials and making changes to compensate for their variability.

There's also a movement toward multiplex diagnostic devices that are able to detect numerous agents in a single device. Determination of required reagent concentration becomes more difficult as the number of markers increases.

There's a risk that higher production costs would arise from rejected components containing numerous high-cost reagents, but this can be controlled with proper preproduction quality control measures.

Multiplexing has been received favorably by the market because of the advantages it offers. From a manufacturing and processing standpoint, what sorts of challenges and complexities are involved in multiplexing?

One challenge is to calibrate all the markers once they've been combined. There's a lot of preproduction work that's needed to qualify and characterize the raw materials so that when they are manufactured as a multiplex device, all the reagents are calibrated properly.

As the number of analytes the device is intended to detect is increased, calibrating the device becomes even more complex.

You noted that in many cases, natural materials are being replaced by synthetic ones. Are companies able to replicate materials well enough that they exhibit the same properties as their natural counterparts?

Yes. In some cases, they are quite adept at matching—and even exceeding—the performance of natural materials due to their sheer reproducibility.

One example is sample-application pads, which are typically made from a composite of natural materials. There are new synthetic materials in development that are sure to capture the attention of many IVD producers.

Are there different challenges for the chemistry and the instrument sides of IVD manufacturing?

There are definite differences in the challenges faced by the two sides. One such difference is the regulatory challenges faced by instrument makers.

The validation procedures for electronics and software can be cumbersome. On the chemistry side, there's a greater flexibility in design changes; however, instrumentation makers are faced with more-rigorous validation procedures. The instrument makers have a tougher job of dealing with user interface, complexity of design, and the never-ending demand for a smaller instrument.

Although the obstacles are not as great on the chemistry side, it is still a design challenge to get the reaction chemistries to work as quickly as the end-user and the market demand. The older IVDs were considered rapid if they gave you a result in two hours. Today, point-of-care diagnostics are expected to return results in 10 or 15 minutes.

Facing Regulatory Challenges

According to the quality system regulation (QSR), FDA is obligated to conduct inspections of manufacturing facilities every two years. Have you found this to be the case, and what do these inspections typically entail?

In our experience, FDA absolutely has lived up to its obligations. Millenia Diagnostics has been inspected twice in the last three years.

Typically, these are inspections of three or four of a company's quality systems. The inspectors choose one or two quality systems that they can look at in depth. Inspectors commonly look at batch records of finished products and trace all of the components that went into them to make sure all the quality systems were used.

How can IVD companies prepare for an inspection?

IVD companies should be prepared for an inspection at any time. Regular internal audits are required, and I highly recommend using the quality system inspection technique (QSIT). This is a guide that's used to train FDA inspectors on performing their audits. It gives manufacturers a good perspective of what FDA will be looking for and which systems inspectors will want to see.

Can you recall your reaction when you were first confronted with the need to meet FDA and similar regulatory requirements?

At the time, I was in charge of the manufacturing department of a company that was GMP compliant. When the new QSR was put into place, I had the task of taking the company's quality systems and updating them.

Initially, it felt like an overwhelming task. However, once I made use of the many resources available—both private and through FDA—that feeling quickly faded.

FDA has made many tools available to help companies understand its requirements. Tools such as the QSIT guide provide a great understanding of which systems will be inspected and give manufacturers insights into an inspector's point of view.

When performing internal audits using the QSIT, companies can easily identify noncompliance and take actions to correct and prevent them. This is a valuable tool that should be used for training all IVD manufacturing personnel.

Another useful tool is The FDA and Worldwide Quality System Requirements Guidebook for Medical Devices. This book provides a great overview of the QSR as well as a comparison chart with GMP and international standards. Taken together, these tools help lead manufacturers toward better compliance.

What other sorts of private resources did you tap into and use?

There are numerous consultants that provide private on-site training. In addition, there are many GMP and quality systems seminars given, to which I routinely send my employees.

Of course, FDA's Web site is also a good resource and offers a wealth of compliance information.

The regulatory requirements and international standards you mentioned are intended to keep companies from designing and producing bad products. In what ways do these requirements help make technically complex processes simpler for IVD manufacturers?

One requirement—design controls—is an essential part of the design process of IVDs. Regulatory agencies have recognized a lack of design control as one of the major causes of device failures and recall.

These standards helps simplify the complex process of IVD development by calling for the identification and assessment of design requirements that are necessary for developing safe and effective medical devices.

Although IVD development is a complicated process, it is simplified by the establishment of design controls that spell out the starting specifications, as well as the labeling, user, and performance requirements. In addition, these regulations help with the validation of design, the transfer to manufacturing, and the creation of a design history file.

The regulatory requirements guide the production of medical devices from design through release for distribution. Imagine if every company had to come up with its own quality regulations. FDA has done a great job simplifying and standardizing the process by which medical devices are developed and manufactured.

Based on your experience, does FDA change its requirements in order to maintain a level of quality?

In my experience, FDA changes its requirements to maintain product safety and effectiveness. The pace of change allows a company to stay up to date with the GMPs and current regulation. As these regulations move forward, they're actually becoming less complex. FDA is doing its best to help us understand, as IVD producers, what quality systems we need. As FDA revises its quality system requirements, the tools for complying with them are becoming more useful.

Are regulatory requirements and international standards equally useful when dealing with cutting-edge products?

Sure. Manufacturers are tasked with establishing and complying with quality systems to ensure that their products continually meet specifications. Systems such as corrective and preventive action (CAPA) are valuable tools in high-tech manufacturing environments.

CAPA is used to identify the root cause of nonconformances. In some cases, this may be poor design. The system calls for preventive measures, as well as for validation that the measures are effective.
A good program includes regular follow-up investigations and meetings to discuss CAPA issues and encourages trend analysis to prevent further nonconformity.

CAPA is just one component of the quality system. Quality system components such as production and process controls, facility controls, document controls, and purchase controls are not just requirements, but are necessary for producing high-quality products.

New manufacturing processes will have to be developed as new technologies are born.

To eliminate redundancies, how can IVD manufacturers streamline their compliance activities for FDA and for international standards? Are there still areas where independent efforts are required?

There will always be some independent effort required for compliance between domestic and international standards. Still, IVD manufacturers that are FDA compliant need to make only minor modifications to meet international standards.

Many domestic companies whose products are selling overseas are becoming certified as compliant with the International Organization for Standardization (ISO; Geneva). Registration of a device in each country is done independently and may vary from one country to the next. So, there is still some independent effort required, but in general, the requirements overlap fairly well.

What roles do the ISO standards and other requirements play in an IVD manufacturer's manufacturing and processing technology?

If a manufacturer is selling products overseas, international requirements play a big role. The main differences between the international standards and FDA requirements are in the labeling, language, and approval process.

How does an IVD company's organizational structure make the manufacture of a product line easier or more complicated?

I'd say that integration of departments is essential. The trend is to have entire companies committed to high quality, reduced cost, and continued process improvement.

It is not enough to have just the manufacturing department on board. There needs to be cohesion between each department, including research and development, where new products begin to take shape, and the manufacturing team.

The purchasing department has the task of getting better pricing on raw materials, as well as ensuring that suppliers are able to meet in-house specifications. The role of the quality control groups should be not only to pass or reject products, but to advise and educate the manufacturing group about improvements that can be made at each step in the manufacturing process. Finally, the research and development team should consider the ability of the manufacturing group in its design process; this could lead to simpler designs and more manufacturer-ready products.

Deciding to Outsource

Do IVD manufacturers outsource any of their product development or manufacturing activities? What would be the business rationale for such a decision?

IVD manufacturers are increasing their outsourcing activity. Economics, quality, regulatory compliance, and in-house capability are a few key factors to consider in deciding whether or not to outsource.

In addition to its lower cost, outsourcing allows companies to focus on their core competencies such as marketing, business development, intellectual property, discovery and licensing, and distribution.

The cost of outsourcing IVD development and manufacturing can be tremendously lower than performing these activities in-house. To develop IVD products in-house, a company must assemble a knowledgeable team and be prepared to take the time to develop quality systems, to obtain manufacturing equipment, to develop manufacturing processes, to validate processes, to obtain the proper permits and licenses, et cetera.

Contract and research and development organizations can perform IVD development at a fraction of the in-house cost and, just as important, can shorten the time to a commercial-ready product.
It could take a relatively long time and be costly for a company to set up an FDA-compliant manufacturing facility and quality system. However, contract manufacturers are already compliant and have experience navigating through the regulatory environment.

What types of IVD manufacturers tend to outsource their manufacturing activities? Are they typically larger companies or smaller companies?

I'd say that a majority of companies are outsourcing at least one or more components of their IVD product. They tend to be larger companies, but small companies have limited resources and tend to outsource as well.

It seems logical that a smaller company, which may have a great product but lacks the capital to set up its own manufacturing facility, would gain benefits from outsourcing. What is the rationale for larger IVD companies outsourcing their manufacturing activities?

IVD companies look for teams that specialize in areas where they may lack expertise. So, several small companies that specialize in diagnostic development may serve a niche, whereas some large companies are more in tune with marketing, distribution, and intellectual property discovery and licensing. But when it comes to outside development and manufacturing, they look for outsourcing partners.

Outsourcing of manufacturing has been increasing quite a bit. I see this trend continuing.

What experience does Millenia Diagnostics have with contract manufacturing?

We are an outsource for contract development and manufacturing of critical components and finished devices. Some IVD manufacturers outsource many of their labor-intensive tasks overseas, but they tend to keep their most critical components close to home if they have to outsource them. Because IVD companies are more concerned about not losing quality for these critical components, they look for FDA-compliant companies they can visit more frequently.

I would guess that more-complex IVDs require certain complex manufacturing processes. As the trend toward these types of tests continues, what sort of outsourcing challenges will IVD companies encounter? Will it be difficult for them to find the right contract manufacturing partner?

Certainly the outsource partner and the company need to be a good match. But for contract manufacturers and developers, it's important to stay at the cutting edge of technology. That's just more of a reason for other companies to outsource their development to your company.

Contract manufacturers must dedicate resources to learning about the technologies that are out there, what the current trends are, and how can they be improved upon.

The Future of IVD Manufacturing

What trends do you think will emerge in the area of diagnostic manufacturing and processing technologies?

I see a trend toward a point-of-care multiplex device—in other words, a point-of-care device that can measure multiple analytes, detect multiple infectious agents, and give more information to the clinician with just one single test.

The challenge for IVD manufacturers and producers will be to generate processes along with these new multiplex diagnostic technologies. The big challenge will be in the calibration of all the markers once they are combined into a single diagnostic test.

The demand for the IVD industry is for more information and for maintaining ease of use.

What types of manufacturing challenges would calibrating multiple markers on a single device present?

Each analyte in a multiplex assay has to be calibrated individually and then combined into a single assay. Then, further calibration may be required due to the influence of the reagents for the other biomarkers.

So, lots of preproduction work and prequalification of reagent processes will have to be completed to ensure that calibration is correct for the end-product.

Timothy B. McBride, JD, is an attorney at Senniger Powers (St. Louis), an intellectual property law firm specializing in the acquisition, management, and enforcement of intellectual assets. He can be reached via e-mail at tmcbride@senniger.com.

Intellectual property rights provide companies and educational institutions with powerful revenue-earning tools. In particular, companies conducting pioneering research can protect and take advantage of their intellectual property by obtaining patents that prevent unauthorized parties from exploiting their patented technologies.¹

However, companies that are not necessarily technological pioneers can still use the rights provided in the patent laws by licensing technologies from patent owners. Patent licenses are contracts between a patent holder (licensor) and a company that wants to commercialize the patented subject matter (licensee). Such licenses allow the licensee to use the patented technology within the scope of the patent.

Patent licenses offer numerous advantages. For example, entities unable to market their patented technologies (e.g., universities) can gain financial rewards by licensing their technologies to companies that can commercialize them.

Patent licenses also provide benefits to the public. A patent holder can exclude others from performing activities with the patented technology that fall within the scope of the patent, regardless of whether it has been commercialized. But without the ability to license such technology, it would remain unavailable to the public for the duration of the patent term.1 Patent licenses play a significant role in utilizing and sharing patented technol-ogies, maintaining commerce, and providing the public with the benefits associated with licensed technologies.

The Supreme Court's decision in Laboratory Corporation of America Holdings v. Metabolite Laboratories Inc. fueled significant discussion in the IVD industry, and the recent decision in KSR International Co. v. Teleflex Inc. promises to do the same.^2-3 However, the Court's decision in MedImmune Inc. v. Genentech Inc. may have the greatest practical impact due to the importance of patent licenses to the diagnostics industry.^2-4 Understanding how this decision may affect not only the enforcement of current patent licenses but also the preparation and enforcement of future licenses is vital.

Prior Rulings Affecting Patent Licenses

Before the Supreme Court's MedImmune decision, several other key decisions dealt with patent licenses and a licensee's ability to invalidate a licensed patent. In Lear v. Adkins, the Court established that a licensee cannot be prevented, or estopped, from challenging a patent's validity.⁵ The Court also held that once a licensee breaches a license agreement and discontinues royalty payments, it cannot be forced to pay royalties during a challenge. Lear unequivocally established a licensee's right to challenge a licensed patent's validity and to forgo royalty payments during the challenge period.

Thirty-five years later in Gen-Probe Inc. v. Vysis Inc., the Federal Circuit court held that a licensee cannot bring a declaratory judgment action seeking to invalidate a licensed patent unless it materially breaches or terminates the license agreement.⁶ In this case, after entering into a patent license, the licensee subsequently challenged the patent's validity but continued to pay royalties, although under protest. Because the licensee paid royalties during the challenge, the court held that the license was never breached and the patent's validity could not be challenged since no justiciable case or controversy existed.

Combining these two decisions, while a licensee is not prevented from filing suit to challenge a licensed patent's validity, it has to materially breach or terminate the license agreement in order to create the necessary justiciable case or controversy. As such, a licensee has to put itself in peril of an infringement suit before it can challenge a licensed patent. This was the general rule until the Supreme Court's MedImmune decision.

The MedImmune Opinion

In 1997, MedImmune Inc. (Gaithersburg, MD) entered into a patent license agreement with Genentech Inc. (South San Francisco, CA). The license agreement covered Genentech's existing patent relating to chimeric antibodies and a pending patent application relating to the coexpression of immunoglobulin chains in recombinant host cells. According to the agreement, MedImmune agreed to pay royalties on the sales of licensed products, and Genentech granted MedImmune the right to make, use, and sell the licensed products.

In 2001, the coexpression patent application, or the Cabilly II patent, was issued.⁷ Soon after, Genentech sent MedImmune a letter stating that its Synagis product was covered by the Cabilly II patent, and that it should pay royalties for that product. MedImmune did not believe that royalties were due because the Cabilly II patent was invalid and unenforceable, and Synagis did not infringe the patent claims. MedImmune also believed the letter “to be a clear threat to enforce the Cabilly II patent, terminate the 1997 license agreement, and sue for patent infringement if it did not make royalty payments as demanded.”⁴ But if Genentech were to win such a suit, MedImmune would have to pay treble damages and attorneys' fees, and would be enjoined from selling Synagis, “a product that accounted for more than 80% of its revenue from sales since 1999.”4 With such risks in mind, MedImmune decided to pay the royalties, but “under protest and with reservation of all rights.”⁴

In order to clarify the matter, MedImmune filed a declaratory judgment action seeking to find the Cabilly II patent claims invalid and unenforceable, and to rule that Synagis did not infringe the patent's claims. Nonetheless, the District Court granted Genentech's motion to dismiss the case for lack of a case or controversy, citing Gen-Probe Inc. v. Vysis Inc. The Federal Circuit court affirmed the dismissal, also relying on the Gen-Probe decision.

However, on appeal, the Supreme Court held that a case or controversy did exist and that MedImmune could sue for declaratory judgment under the Declaratory Judgment Act.⁸ This act allows companies threatened with an infringement suit to preemptively sue for invalidity, unenforceability, and noninfringement of a patent, provided there is an “actual controversy.”⁸ Because an actual controversy is required for a court to have jurisdiction under the act, the Court had to determine whether such an actual controversy existed, even though MedImmune did not breach the license agreement and paid royalties.

Citing a number of cases involving government action, the Supreme Court noted that “where threatened action by government is concerned, we do not require a plaintiff to expose itself to liability before bringing suit to challenge the basis for the threat…The plaintiff's own action (or inaction) in failing to violate the law eliminates the imminent threat of prosecution, but nonetheless does not eliminate Article III [Declaratory Judgment Act] jurisdiction.”⁴ Similarly, the Court asserted that a licensee should not be required to expose itself to liability (i.e., treble damages and attorneys' fees for patent infringement, possible injunction) in order to create the necessary controversy to challenge a licensed patent. This decision effectively overturned the Federal Circuit's decision in Gen-Probe.

The Supreme Court also found that licensee estoppel or specific provisions in a license agreement prohibiting a challenge by a nonrepudiating licensee have a no bearing on whether an actual controversy existed for filing an action under the Declaratory Judgment Act. Instead, the Court noted that if licensee estoppel or particular provisions of the license agreement “preclude this suit, the consequence would be that [Genentech would] win this case on the merits . . . [and not] that Article III [Declaratory Judgment Act] jurisdiction is somehow defeated.”⁴ Even though the Court ruled that a nonrepudiating licensee can demonstrate an actual controversy, the licensee's suit can still be dismissed for reasons such as licensee estoppel or specific provisions in the license agreement prohibiting such challenges.

Effects of MedImmune

The MedImmune decision could affect existing and future patent license agreements. IVD manufacturers should prepare future patent agreements by keeping this decision in mind, and review existing agreements with the thought of possibly renegotiating them.

Existing Agreements. MedImmune offers current licensees opportunities to renegotiate existing patent license agreements, especially those that are overly favorable to licensors. Specifically, since a licensed patent's validity or enforceability can be challenged without having to breach the license agreement, a licensee can renegotiate an agreement to obtain more-favorable terms. Such terms could be decreased royalty rates in exchange for giving up the right to challenge the patent.

The ability to challenge a licensed patent may also provide a licensee an opportunity to litigate an unfavorable license agreement without subjecting itself to the liability of treble damages and attorneys' fees. While previously a licensee would have to breach or terminate the agreement and suffer the consequences, a licensee may now challenge a patent without having to breach, which reduces the risk of potential liability to litigation costs. To a licensee paying significant royalties, this would provide a strong financial incentive to challenge a licensed patent, since the only downside, besides litigation costs, would be continuing to pay royalties during litigation.

Likewise, current licensors should consider the possible effects of MedImmune. Such effects include increased risk of litigation and costs associated with litigation. This is especially troubling for small companies and universities that may not have the capital to litigate a challenge to one of its licensed patents. Such entities should be wary of being strong-armed into renegotiating existing license agreements.

Future License Agreements. From the MedImmune decision, licensees have gained significant benefits in negotiating and executing future license agreements. Perhaps the single greatest benefit is that a licensee can enter into a license agreement knowing that it still can challenge the licensed patent without having to breach the agreement. This permits a licensee to avoid the many risks associated with losing a patent infringement suit brought by the licensor and to postpone deciding whether to file a declaratory judgment action by simply entering into a patent license agreement. A licensee may insulate itself from liability to treble damages and attorneys' fees by entering into a license agreement, and then determine, from the safe harbor provided by the license, whether to challenge a licensed patent's validity, enforceability, and infringement.

MedImmune may also allow a licensee to negotiate more-favorable license terms. Specifically, in exchange for surrendering the right to challenge the licensed patent, a licensee can negotiate more-favorable royalty rates, payment terms, and other clauses. However, the enforceability of a provision forbearing a licensee's right to sue in the absence of a breach is questionable, particularly for public policy reasons. Nevertheless, a licensee may include such a provision in an agreement.

While licensees obtained many advantages, MedImmune left licensors with substantial challenges. Because a licensee can challenge a licensed patent without breaching the license agreement, a licensor must now consider provisions that might not have previously appeared in license agreements.

For example, such provisions would allow a licensor to collect increased royalties, collect a substantial portion of the royalties earlier during the license period, or prohibit or dissuade a licensee from filing suit in the absence of breaching or terminating the agreement. Examples of such provisions include simply requiring higher royalty payments.

A licensor should also consider placing provisions in an agreement that require a large up-front, lump sum payment or accelerated payments. Alternatively, license provisions could require graduated payments that result in higher royalty rates during the first quarter or third of the license agreement period, and lower royalty rates during the remainder of the period. Since a challenge to a licensed patent does not allow a licensee to recover royalties already paid and adds to a licensee's costs from the litigation, such provisions would create an economic disincentive for a licensee to challenge the patent.

In addition, a licensor could place provisions in an agreement that provide for decreased royalty rates if a licensee surrenders its right to challenge a patent in the absence of breaching the license agreement (although such provisions raise public policy concerns). In a similar provision, a licensor has the right to increase the royalty rate if a licensee unsuccessfully challenges a licensed patent.

Likewise, licensors can seek to control possible challenges to a licensed patent by including provisions in an agreement that provide them the right to terminate a license if the licensee challenges the licensed patent (again, the enforceability of such provisions is questionable).

A licensor can also attempt to limit such challenges by using arbitration provisions. Such provisions would require validity, enforceability, or infringement disputes
to be arbitrated, instead of being litigated.

Conclusion

In the aftermath of MedImmune, lower courts will have to deal with not only the Supreme Court's holding but also the many questions left unanswered. As Genentech's Cabilly II patent was found to be invalid by the U.S. Patent and Trademark Office two months after the MedImmune decision, resolving any questions through further litigation of this particular suit looks to be unlikely, at least in the near future. Regardless, both licensees and licensors should consider the MedImmune decision when renegotiating and preparing patent license agreements, as its full effect remains to be seen.

Photo by CombiMatrix Corp.

To respond effectively to recurring influenza A epidemics and a possible flu pandemic, virulent influenza A isolates must be identified quickly. Unfortunately, RNA viruses such as influenza represent a moving target for vaccines, diagnostics, and therapeutic approaches. Influenza, a single-stranded RNA virus, is subject to a high level of mutation, recombination, and genetic diversity. As a family, RNA viruses are prone to this same high level of mutation due to a lower fidelity during replication of the viral genome. Changing the genetic makeup of the virus helps it adapt to its environment and evade the host immune system.

The influenza genome is somewhat unusual in that its genetic material is divided into eight separate segments. The primary antigenic determinants for the virus reside on two of the eight segments: the hemagglutinin (H) and the neuraminidase (N) genes that code for glycoproteins that mediate the virus's attachment and entry into the host cell. There are multiple varieties of these proteins: 16 different H subtypes and 9 different N subtypes, which have been recorded in 50 combinations (144 combinations are theoretically possible). If a host cell is infected with multiple virions, it is possible for these segments to recombine to form new hybrid strains. Because an infectious event generates a large number of viral progeny (around 1000 viral progeny per single infectious event), the rate at which mutants can be created, due to stochastic mutation and recombination, can become very high. In fact, roughly 70% of viral progeny are mutant strains of the infecting virus.

Given the large number of viruses generated per infectious event and the high level of mutation and recombination for influenza, it is no surprise that there are many strains of influenza in circulation, and new strains appearing rapidly. The large genetic diversity manifests itself as antigenic variation. This means that the ability of the host organism to successfully employ the same immunological defenses against the virus over a sustained period is limited. Because the virus remains in a state of genetic flux and can afford to test new mutant strains due to the high number of viral offspring, it can also evade immune surveillance, become vaccine resistant, and, most disturbingly, develop a resistance to drug therapy.

(click to enlarge) Table I. Comparison of selected commercial immunoassay systems for blood testing.

Type A viruses are believed to have been responsible for most known human influenza pandemics, and avian influenza viruses are therefore key contributors to the emergence of new human flu pandemics.1 The natural reservoir for most Type A influenza strains is wild and domesticated birds, especially species that live in aquatic habitats such as ducks, geese, swans, gulls, and sandpipers. The spectrum of influenza subtypes found in avian populations is remarkably broad compared with the number that have been isolated from humans. However, there are also some influenzas found in humans that do not infect birds (see Table I).

Infections of wild-bird populations present a particularly difficult challenge. The response to infection varies widely between bird species, and the geographical locations can vary significantly. To compound this problem, although avian influenza has caused large-scale death in birds, it certainly is not the only cause of mass mortality in the avian population.

Transmission of Avian Influenza

How the virus is spread among birds is not entirely understood. Transmission through avian species likely occurs through one of two routes: the migration of wild birds and the movement of farmed poultry. In the former case, the incidence of dead birds, including prominent species such as swans, has been at the forefront of public attention. These situations have sometimes been associated with outbreaks of avian influenza. However, since migratory birds are also susceptible to other lethal infections such as avian cholera and botulism, not all dead birds are the victims of H5N1 infections. In fact, between June and mid-August of 2006, more than 18 significant epidemics involving migratory birds were identified in the United States, none attributable to avian influenza.

The second mode of transmission, through farmed stocks of poultry, is more complex. Avian influenza infections may move between poultry stocks and migratory birds. Although outbreaks of H5N1 are certainly lethal in chickens, there has been evidence suggesting that the symptoms of the disease can be masked in chickens coinfected with another strain of influenza. Furthermore, immunization programs designed to vaccinate broiler stocks against other pathogenic strains of avian influenza (e.g., H9) may confer adaptive immunity onto flocks exposed to the H5 virus. This latter, disturbing possibility provides for a Typhoid Mary scenario in broiler stocks that are moved over large distances or even across international borders. Therefore, the diagnoses of mixed infections—whether real or caused by vaccination—are critical pieces of information.

Diagnostic Challenges

One challenge of creating influenza tests is effectively gauging the pathogenicity of a strain. As the mortality of sentinel species like birds may be due to a variety of causes, infections may provide only limited information. In addition, the effects of a virus in birds may be distinct from those in humans (e.g., avian influenza has been associated with encephalopathic pathology in certain bird species). Whether an influenza epidemic is associated with high mortality is critical. Although this connection can be inferred through DNA sequencing, it is difficult to determine using immunoassay methods. Indeed, lower-pathogenicity forms of H5N1 have been found in wild birds in North America on multiple occasions, in 1975, 1986, 2005, and 2006.

Using conventional diagnostics to differentiate the higher-pathogenicity form of influenza, which is not antigenically distinct, from the lower-pathogenicity form is time-consuming. During an outbreak, slow or incomplete diagnosis could lead to serious economic and public health issues.

Should the H5N1 bird flu virus become able to spread more easily through person-to-person transmission, it could develop into a pandemic human influenza virus that could undermine the public health security and economic vitality of countries around the globe.^2-4

There is already evidence that the virulence of the Asian H5N1 virus has increased in poultry, animals, and humans since it first infected humans during a 1997 outbreak in Hong Kong.^2,5,6 Significantly, mutations in the virus have caused severe disease in domestic ducks and wild waterfowl, which formerly carried the virus without developing symptoms. This has been accompanied by a collateral increase in the frequency and severity of H5N1-caused disease in nonhuman mammals.^2,6,7

Morbidity and mortality from the H5N1 virus have also increased among children and adolescents. For example, there were significantly higher rates of clinical disease and death among children in Thailand in 2004 than during the initial 1997 outbreak in Hong Kong.⁸ More than half of the people with confirmed bird flu infections have died, and at least half of all confirmed and suspected fatal cases have occurred in previously healthy children and young adults under 20 years of age.⁹ Death rates from bird flu are now higher among infants and children than among adults. In Thailand, there is a fatality rate of 89% among children younger than 15 years.¹⁰

For public health workers, the rapid change in the genetic makeup of influenza provides a challenge. First, the variation in the influenza viral strain requires a complex annual vaccination strategy that infers the most likely strain for the upcoming year. This process is necessary because it takes a considerable amount of time for the vaccine to be produced, tested, and distributed, and is typically repeated every one to two years as new vaccine-evading strains appear. Second, there is evidence that the mutation and recombination of the virus—sometimes referred to as genetic drift and genetic shift, respectively—can result in drug-resistant variants. As the virus adapts, drug therapies such as Tamiflu from Hoffman-La Roche Inc. may become ineffective. Finally, and perhaps most disturbing, current diagnostic tools for identifying influenza from the natural reservoirs of the disease (e.g., wild birds) are either slow or insensitive, or provide an inadequate level of certainty about the pathogen present in the sample.

Given the threats that H5, H7, and H9 influenza strains represent to human health and agriculture, the standard toolbox of diagnostic assays should be expanded beyond simple immunological or basic polymerase chain reaction (PCR) assays. Rapid knowledge of the exact strain, the origin of the strain, and the probable characteristics of the virus are critical for monitoring a disease outbreak and preventing its spread.

To develop timely diagnostics, especially with the level of genetic variability observed in influenza, it is important to know the genetic makeup of emerging strains. Restricting this information for geopolitical or economic reasons (e.g., technology-licensing strategies) seems at odds both with medical needs as well as with the desire of the IVD industry to develop effective tools for analyzing flu. The recent decision by the Centers for Disease Control and Prevention (Atlanta) and the Association of Public Health Laboratories (Silver Spring, MD) to make genomic sequences for new influenza viruses available to the research community through publicly accessible databases is an important step toward ensuring that outbreaks are effectively monitored and that IVD tests remain up-to-date.

Technical Approaches for Detection and Identification

A variety of technical approaches exist for identifying influenza. Cost, certainty of outcome, assay sensitivity, time to answer, test complexity, and, sometimes, instrument portability are important factors. Because conventional tests, whether close to the bedside or in the field, have significant limitations, a combination of methods is generally used in critical scenarios.

Measuring influenza reliably can be challenging for three reasons:

• Flu infections in humans often exhibit transient viremia. This means that the virus is only shed at high titers for a brief period (around three to five days after infection). Thus, limitations in assay sensitivity are a common problem.
• The most effective sampling procedures tend to be the most unpleasant (e.g., nasopharyngeal washes). As a result, sample quality can vary widely.
• Improper clinical sample shipment and storage, as well as delays in testing, often result in degradation of the sample and false-negative results.

Unless effective tests can be run promptly at the sample-collection site, significant discipline and sampling training is required. To overcome some of these limitations, samples are often stored in viral transport media, buffers designed to preserve the virus. Another option for amplifying critical sample titers is to culture the virus in the laboratory, then to test it using serological or molecular methods. This process is extremely time-consuming.

Thus, ascertaining whether a flu is of the lethal A (H5N1) strain requires that a sample be frozen at the collection site and shipped to a secure laboratory. Once there, the virus is grown in eggs, isolated, and genetically sequenced—a process that takes four or five days, not including shipping time, and runs the risk of having the samples defrost and ruined in transit. By the time a result is available, the patient may have died.

Immunological Assays. Current methods for assaying influenza can be divided into two categories: immunological assays and molecular, or nucleic acid–based, assays. Immunological assays include agglutination assays, and immunofluorescence and enzyme-linked immunoassays, as well as rapid strip-type tests.

Immunological methods are the most established of these technologies for influenza diagnostics, although molecular tests are becoming commonplace. Immunological assays are relatively quick and provide basic information (e.g., the H or N type of the virus), but little else. In addition, a number of these tests have limited sensitivity and a significant false-negative rate.¹¹ Tracking emerging diseases with serological assays is particularly challenging. Generating and testing antibodies is the most time-consuming of these procedures.

Molecular Diagnostics. Molecular methods such as real-time PCR are extremely sensitive, reasonably fast, and can be adapted to new diseases. They often alleviate the need for tedious culturing of the virus. As such, these techniques can complement serological assays. An advantage for any nucleic acid method is that the sample can be stored in denaturing buffers that help reduce false negatives.

However, since real-time PCR methods rely on one or two data points to make a measurement and are frequently subject to spurious signals, the technique often requires verification by a secondary method such as DNA sequencing. In general, the problem with additional amplification products generated by real-time PCR has been addressed by adding one to four dye-quencher probes per reaction. These provide an additional level of confidence, but also significant cost. In addition, because real-time PCR is so specific, false-negative results can appear due to pathogen mutations at the priming sites. Real-time PCR is more flexible than serological assays, but it still provides a limited amount of information (e.g., it cannot easily distinguish between closely related sequences or mutations).

There are two ways to determine pathogenicity of avian influenza in birds such as chickens. One approach is to analyze the gene sequence and to determine whether it looks like a virus with pathogenic potential. The alternative is to inoculate young chicks and observe mortality over a 10-day period. Given that in vivo testing is slow and often impractical, it is reasonable to extract as much information as possible from a sample. In fact, sequence information for influenza can yield strain information that may infer pathogenicity, susceptibility to current vaccination strategy, and drug-resistance characteristics.

Microarrays and Other Methods

A number of approaches can be used to gain extra information about the genetic composition of a virus after the nucleic acids have been isolated and amplified. These methods include microarrays, mass spectrometry, and sequencing. Each of these techniques adds more time and cost to the assay, but they increase certainty and provide significantly more information. Mass spectrometry is effective and highly sensitive, but the equipment is large and complex. Sequencing also has advantages, but the process requires a great deal of complex equipment to be dedicated for a single clinical purpose.

(click to enlarge) Figure 1. Microarray assay data from avian H9N2 influenza A sample interrogated with thousands of probes (a). Amplitude (y-axis) corresponds to electrical signal (picoamperes per electrode) associated with each influenza-strain- specific probe. Multiple loci are probed per strain type (x-axis). Strain-specific probes are grouped so that individual signals from loci can be observed individually. Summary data are also shown (b). Assay time is 4 hours.

Microarrays can provide single-base resolution, if desired, on nucleic acids and can concurrently interrogate a nucleic acid sequence with thousands of probes (see Figure 1).^12-14 This is in stark contrast to the one to four probes that can be employed in a real-time PCR run. Currently, microarrays can identify several thousand different genotypes (e.g., all known influenza strains) and provide sequence information for critical loci in the genome (e.g., known areas that confer drug resistance).

Microarrays can also analyze specific regions at disparate loci without needing to sequence in a linear fashion through whole genomes like conventional sequence analysis. This is useful as only limited genome regions tend to be informative when differentiating between related strains.

The advent of customizable microarray technology (i.e., programmable, application-specific probes) means that interrogating new or varying strains of a virus is straightforward. Because the array can be updated and manufactured in a shorter period than it takes for a new pathogen strain to appear, validation of the array content remains a rate-limiting step. The validation and testing of these systems will become more efficient as the bioinformatics and rules defining microarray probe design improve.

Technical Hurdles. Until recently, microarrays suffered from a number of technical limitations that slowed their dissemination. For example, early microarray techniques for multipathogen detection required assay times as long as 48 hours. This was due to slow amplification techniques and long hybridization times. Improvements in the speed of whole-genome amplification techniques—a reduction from 16 hours to 1 hour, the adoption of pan-specific PCR schemes—has reduced the total assay time of multipathogen detection to 4–5 hours. In addition, the combination of orthogonal PCR amplification and microarray technology permits PCR to function more broadly while improving the sensitivity and hybridization kinetics of the microarray.

Figure 2. An electrochemical microarray reader, laptop, and semiconductor microarrays by CombiMatrix Corp. (Mukilteo, WA), for rapid influenza typing.

Another challenge for multiplex technologies is that microarray cost and the complexity of instrumentation and handling have been high. However, technologies adapted from the semiconductor industry are helping to change this situation. True chip technologies, in contrast to microarrays patterned and spotted on glass, can be read directly using well-established electrochemical detection techniques already used in devices such as blood glucose monitors. These technologies are scalable, meaning that the cost per test can be adapted to the appropriate market and that improved chemical characteristics of the device can make them robust enough for multiple uses. These developments translate to order-of-magnitude drops in price and more-robust, less-expensive, and more-compact instrumentation for multiplex molecular analysis.

The integration of electrochemical detection on semiconductor microarrays enables a new generation of simple microarray instruments that ensure the seamless transit of samples through nucleic acid extraction, amplification, and hybridization onto the array. These technologies help simplify the microarray process and increase assay quality and data interpretation while decreasing the risks of sample cross-contamination. Using embedded semiconductor technologies in the field to deploy multiplex pan-pathogen technologies may aid sample handling and preservation, limiting the transport of infectious agents and assuring chain of custody.

Conclusion

In an era of rapid multinational travel, increasing populations, and global trade, emerging diseases can spread quickly and cause significant socioeconomic damage. The IVD industry plays a critical role in monitoring the emergence of new infectious agents by taking a leading role in surveillance, as well as in guiding therapeutic development. Though challenges remain, fundamental advances in how microarrays are manufactured and used demonstrate that the technology has tremendous potential as a tool for simultaneously screening for multiple pathogens.

(left to right) Andy McShea, PhD, is vice president at CombiMatrix Corp. (Mukilteo, WA). Amit Kumar, PhD, is chief executive officer at CombiMatrix. Joe Dudley, PhD, is chief scientist, biosecurity and bioinformatics, at EAI Corp. The authors can be reached at amcshea@combimatrix.com, akumar@combimatrix.com, and jdudley@eaicorp.com.

The benchtop low-pressure plasma system developed by PVA TePla America Inc. (Corona, CA) (left) is designed for cellular manufacturing and laboratory use. The company’s atmospheric Plasma Pen (right), engineered to keep voltages and current safely inside its body, is used for in-line applications and for spot treatment.

The technology of energizing a gas to produce a glow discharge, or plasma, has become a powerful tool in solving surface preparation problems in the medical device industry. Plasma is used to ultraclean and sterilize surfaces and also to promote the adhesion of biocompatible coatings to in vivo devices and biological materials to in vitro diagnostic platforms. Indeed, plasma can either activate surfaces for purposes of cell or biomolecule immobilization or, conversely, produce nonstick surfaces for antibiofouling or metered drug-dispensing applications. The functioning of microfluidic devices can be greatly enhanced by plasma, too.

Plasma treatment makes microchannels on clinical diagnostic devices wettable to biofluids without having an effect on the properties of the analyte itself. Plasma is also used for low-technology applications such as promoting marking with ink on catheters and bonding syringe needles to hubs with adhesive. And, since plasma is a dry surface treatment technique, there are no waste chemicals to dispose; this makes it an environmentally friendly process involving very few consumables.

This article discusses how gas plasma can be an enabling technology in the manufacture of IVD platforms. It focuses on the way gas plasma controls surface energies and tailors surface chemistry to promote the attachment of biological materials.

The Nature of Plasma

Understanding the science behind gas-plasma surface modification requires first knowing exactly what plasma is. Plasma is a state of matter, equivalent to a solid, liquid, or gas (see Figure 1). When enough energy is added to a gas, the gas becomes ionized and enters a plasma state. Electrons break free from the pull of their atoms or molecules and are then available to transfer energy to other moieties present through electronic collisions. The active components of plasma include ions, electrons, radicals, excited species (also called metastables), and photons, among others. The collective properties of these active species can be controlled and harnessed so as to perform a variety of surface treatments, including nanoscale cleaning, activation for surface wettability, chemical grafting, and coating deposition.

(click to enlarge) Figure 1. Basic states of matter. The principal difference between the plasma and gaseous states is that the plasma state is electrically conductive.

Chemically, plasma is a highly reactive environment that can be employed to change the properties of surfaces without affecting the bulk material. The energy carried by this partially ionized gas in fact can be controlled so that it contains low heat energy. This is achieved by coupling the energy into the free electrons rather than the heavier ions, and it allows heat-sensitive polymers such as polyethylene and polypropylene to be treated.

The energy is coupled into the gas most commonly by the creation of an electrical field between two electrodes at low pressure in a vacuum chamber. This is the operating principle behind fluorescent lighting.

Plasma can also be generated at atmospheric pressure. At one time, atmospheric plasmas were too hot to be used as a surface treatment tool, but the technology has advanced recently. Low-temperature plasmas produced at atmosphere now can be suitable for treating even the most heat-sensitive polymers. More and more applications using atmospheric plasma are appearing as this technology gains ground on low-pressure plasma generation.

Effects on Surface Properties

A solid contaminated by hydrocarbon molecules adsorbed on its surface can be cleaned by plasma-excited oxygen species that readily attack the organic contaminants. The oxygen removes the adsorbed hydrocarbon material by converting it to carbon dioxide (CO₂) and water through a basically simple mechanism (see Figure 2). Many initialization mechanisms involving different excitation states of oxygen as both a free radical and diatomic molecule are possible. The surface-adsorbed hydrocarbon can itself be excited by electronic collision with the plasma, thus providing an additional set of possible reaction pathways.

(click to enlarge) Figure 2. Plasma-generated oxygen radicals attack surface-adsorbed hydrocarbons.

For surfaces sensitive to oxidation, plasma-activated hydrogen is an alternative for surface cleaning. Not only can hydrogen reduce organic surface moieties to volatile hydrocarbons, it can reduce oxides of copper, nickel, silver, and other metals, as well.

The chemical characteristics of a plasma are mostly determined by the feed gas. Oxidizing atmospheres are created by molecular oxygen and nitrogen, nitrous oxide (N2O), and CO₂, for example. These gases are used to render surfaces wettable, or hydrophilic, with respect to polar solutions. This is achieved through plasma-induced covalent bonding of oxygen, which produces functional groups such as carbonyls, carboxylics, and hydroxyls on the surface. These polar groups increase the surface energy so that, for example, tissue cells adhere better, or analytes dispensed onto diagnostic platforms will flow more easily through microfluidic vias.

Reductive plasma atmospheres are created by molecular hydrogen, a mixture of hydrogen, argon, ammonia (NH₃), etc. Reducing plasmas have proved useful in the activation of fluorocarbon substrates such as polytetrafluoroethylene (PTFE). PTFE is a material well suited for implantable medical devices because of its inertness and biocompatibility.

Its properties become disadvantageous, however, when PTFE needs to be processed—for example, for the attachment of synthetic scaffolds to encourage tissue growth on in vivo devices. But reductive plasmas resolve issues such as these by reducing the overall fluorine concentration at the device surface through the replacement of fluorine atoms with functional groups such as hydroxyls. In the example cited, surface hydroxyl groups provide anchor points for support of the synthetic scaffolds.¹

Some applications require the subject material to be etched. Fluorine-containing gases such as nitrogen trifluoride (NF₃), sulfur hexafluoride (SF₆), and carbon tetrafluoride (CF₄) are very good for etching hydrocarbon polymers, silicon and its oxides, and nitrides, among other materials.

In addition to the strong chemical effects of a plasma, the technology produces directional effects that also play an important role. Particles carrying momentum to the surface can physically remove more-inert surface contaminants such as metal oxides and other inorganics and can cross-link polymers to lock the results of surface treatment in place.

Figure 3. A scanning electron microscope image of a cross section of polycarbonate PECVD-coated with a 1-µm layer of silicon oxide (SiOx), showing how well the PECVD process contours the varied topography of the polymer surface. The pit toward the right side of the image is approximately 4 µm deep and 4 µm wide.

Polymer coatings can be grown on surfaces through a process called plasma-enhanced chemical vapor deposition (PECVD). PECVD works by activating species, such as monomers, in the plasma and inducing their polymerization on the workpiece substrate (see Figure 3). Barrier, nonstick, and scratch-resistance properties are among those that some PECVD coatings can introduce. Other coatings may be designed to contain chemical functional groups, such as NH₂ (amine), OH (hydroxyl), or COOH (carboxyl), that provide specific binding sites for further grafting (for example, to immobilize proteins or sensor agents for biological materials) or increasing the affinity of antithrombogenic, lubricious, type IV collagen, or other functional coatings. Surface chemical properties of the deposited coating are determined within the first few tens of nanometers of the surface.

Processing IVD Platforms

The full scope of gas plasma applications in the medical device industry is very large indeed. This article focuses primarily on applications that relate to the manufacture of diagnostic device platforms. In that arena, plasma technology is employed to clean surfaces in preparation for downstream processing and to activate surfaces to promote biomolecule and cell adhesion. The latter end is achieved by changing surface polarity, by grafting specific functional groups to the substrate, or by polymerizing a coating onto the surface. To better understand how plasma tunes surfaces to make them suitable for the application requirement, some important cases might be considered.

Microfluidic Devices and Hydrophilicity. Surface energy is a property of materials that determines such factors as their wettability and their susceptibility to biofouling. In general, materials with high surface energies are hydrophilic and wettable to such fluids as blood, bacterial-cell suspensions, buffers, inks, adhesives, and various other adsorbates and coatings. Low-energy surfaces, on the other hand, are termed hydrophobic and characteristically exhibit nonstick behavior.

Typically, microfluidic devices have to have hydrophilic surfaces so that the analyte will flow smoothly and consistently through the microchannels to the detection and processing elements. Such flow is accomplished by means of a variety of pumping methods, including electroosmotic, thermal, and mechanical techniques. However, microfluidic platforms usually are made from polymeric materials—for example, acrylic, polystyrene, or polydimethylsiloxane (PDMS)—that are inherently hydrophobic.

One of the major problems caused by the hydrophobic nature of these materials is bubble trapping in the microchannels, which inhibits fluid flow. Even when the channels are primed with an alcohol-and-buffer solution, air bubbles can pose a problem. Gas plasma treatment, by oxidizing the surfaces of the microchannels to make them hydrophilic, can prevent the formation of air bubbles.

Flow rates are also affected during electrokinetic pumping by surface-charge densities. Electrokinetic pumping, used to drive fluids through microchannels, wors on the principle of electrokinesis, the conversion of electrical energy into kinetic energy. A charged surface will attract particles of opposite charge in the electrolyte. This allows the particles remaining in the fluid to be pumped electrophoretically through the vias with greater ease. Charging surfaces with plasma has been shown to be effective in supporting electrophoretic or electroosmotic flow.²

Immunoassays, Microarrays, and Tissue Culture Media. Platform substrates for such clinical diagnostics as immunoassays, microarrays, and cell culture media are predominantly fabricated from synthetic polymers. While such materials are ideal for this industry by virtue of being inert, mechanically stable, and inexpensive, their surface properties introduce inherent limitations. Specifically, binding sites for bioactive molecules or cells are inadequate. Bioactive entities cannot anchor themselves effectively to these surfaces. Strong and uniformly dispersed binding sites are important prerequisites for the immobilization of biological analytes and for in vitro cell cultivation. Synthetic-polymer platforms must be surface-modified so as to improve their properties to support cell proliferation and biomolecular adsorption. Application of gas plasma to the surface of these analytical devices effects the necessary changes.

Increased Cell Growth Yields

Animal- or plant-derived tissue cultures grown in vitro have to be supplied with nutrients, hormones, and other growth factors that are provided naturally when the cells grow in vivo. Tissue cells attach to solid surfaces, where they then proliferate in a liquid nutrient medium. In the case of animal cells, this would be serum. The surface properties of the growth medium must be conducive to uniform cell attachment and growth. Before these properties are tuned, however, the surface must be free of contamination. Mold release agents, volatile hydrocarbons, and other contaminating species can be removed from cell culture platforms by exposure to the energetic, yet cool, environment of plasma to ensure the proper pattern of cell attachment and proliferation (see Figure 4).

(click to enlarge) Figure 4. Not treating polystyrene wells may lead to nonuniform cell attachment and cell clumping (a) or areas with no cell attachment (b). A plasma-treated substrate promotes uniform cell attachment and proliferation (c).

The inherently hydrophobic nature of the polymeric materials from which culture media are manufactured does not favor tissue-cell attachment. Hydrophilic surfaces are needed instead. Oxidizing plasmas can be used to add oxygen groups to surfaces, thus increasing their polarity and rendering them hydrophilic.

Hydrophilic surfaces are attractive to tissue cells, inducing them to ad-sorb. Where a significant concentration of specific chemical functionality is required on a surface (as discussed in the next section), the application of gas plasma to the substrate can achieve the chemical graft or polymerization of monomer necessary to produce the desired functionality.

Roughened surfaces present a higher overall surface area than a smooth surface of equivalent extent. In theory, this equates to a higher number of potential cell-binding sites. Since cells typically are on the order of 10 µm in size, surface microroughening provides the greatest enhancement of cell adhesion.³ Surface roughening on a nano scale could not be expected to increase cell adhesion though, because the comparatively larger cells cannot take advantage of increased surface area on that scale.

However, nanoscale roughening can increase drug-induced differentiation and apoptosis. The reasons for this are unclear. An increased number of cell receptors might be the explanation. Or, perhaps enhanced signaling pathways to the nucleus account for this performance benefit. Whatever the mechanism behind it, this effect has important implications for improving tissue scaffold development on implantable devices.

Surface topography can be selectively altered in a plasma environment by either accelerating ions toward the surface or chemically etching it.

Capacitively coupled radio-frequency plasmas generally exhibit a net directional flow of ions toward the substrate. This is an effect of the relative response times of ions and electrons to the polarity change in the electric field producing the plasma. Since electrons are much lighter than the ions, they respond more quickly. Therefore, substrates lying in the path of the electrons will be charged negatively. The positive ions subsequently are accelerated toward the negatively charged surface through the agency of electrostatic attraction. The impact of these ions on the surface to which they are drawn removes surface material.

Argon gas is well suited for microroughening surfaces in this manner. The energy of the accelerated ions can be controlled by adjusting the power and pressure settings of the plasma-generating equipment. For example, increasing the pressure parameter above 1 mTorr can greatly reduce the ion impact energy, if not eliminate it completely. This enables the surface-roughening effect of plasma to be switched off.

(click to enlarge) Figure 5. Atomic-force-microscopy images showing the nanoroughening effect of an oxygen plasma on polyethylene terephthalate, or PET. The untreated surface is on the left.

The application of oxygen plasma is a much milder process than the one just described. It can thus be used to nanoroughen polymeric materials by the action of mild chemical etching (see Figure 5).

The cumulative effect of the combination of surface cleaning, surface activation, and nanoroughening by means of plasma treatment is an increase in cell attachment of as much as 30% in comparison with untreated substrates, and more-uniform cell coverage.

Improved Biomolecule Adhesion

Gas plasma technology is able to solve problems involving the adhesion of biomolecules to such diagnostic substrates as immunoassay and microarray platforms. It does this by providing particular chemical functionality at the surface that allows covalent coupling of biochemical species to occur.

Carboxylic, hydroxylic, and amino functionalities are important and common types of chemical functionality that are readily obtainable through gas plasma processing. For example, in the manufacture of microarrays, amino functionality provides binding sites for the direct attachment of nucleotides and oligonucleotides to the working surface.

If steric hindrance interferes with direct binding of these large biomolecules, then primer molecules, sometimes called linkers, are used. Linkers provide space for the biomolecule to adsorb to the surface in the proper configuration. As it happens, linker molecules themselves require that surfaces be activated to help them anchor to the substrate.

Most often, straightforward treatment with oxygen plasma is enough to promote the binding of these molecules. However, in some cases, specific functionality is required. Some capture agents work more efficiently in either an acidic or a basic environment, for example. If the capture agent is linked via a carboxylic group, an acid environment is provided. Amino functionality will produce a more basic environment.

(click to enlarge) Figure 6. Gas plasma surface treatment adds chemical functionality either by exposing the surface to plasma containing the specific functionality (a) or by growing a coating on the surface via PECVD (b) using a monomer that already has the desired functionality.

There are two basic ways to functionalize a surface with specific chemical groups. One is to deposit a PECVD coating that introduces the desired functionality, and the other is to generate plasma in the presence of the functional group and allow the group to bind to the surface (see Figure 6). While the latter is the simpler method, the former results in a higher surface concentration of the functionality (10–20%). Using ammonia in the feed gas will result in NH₂ groups binding to the surface. Methanol is used to functionalize with hydroxyl groups, and a combination of methanol and carbon dioxide provides carboxylic functionality.

Unfortunately, during the deposition of these groups, some fragmentation reactions occur that can transform the primary functionality. For example, ammonia plasma will deposit primary amino groups along with secondary and tertiary amines, nitriles, imines, and other derivative compounds. The relative amounts of these groups that are deposited vary depending on the type of plasma system used and the parameter settings. However, this method can be expected to produce 2–8% of the desired functionality.

(click to enlarge) Figure 7. The morphology of a gel droplet deposited onto a polymer slide via ink-jet is the desirable one when the surface energy is controlled through use of the right plasma feed gases (a) and exhibits deformation when placed on a too-hydrophilic surface such as a PECVD coating containing amino groups (b).

Sometimes, providing the right chemical functionality alone is not enough. Amine groups, for example, increase surface energies, rendering surfaces hydrophilic. Overly hydrophilic surfaces may not be desirable when, say, depositing gel drop arrays onto a microarray platform, because the microdroplets may wet the surface inappropriately (see Figure 7). This type of wetting results in malformed droplets.

Again, gas plasma can solve this problem and preserve proper droplet morphology by controlling the surface energy, even in the presence of the amino groups. During plasma amination of the microarray platforms, fluorine chemistry can be added to the process in a controlled manner. The fluorine binds to the floor of the platform and increases its hydrophobicity such that the droplets retain their spherical shape. Fortunately, this process does not affect the surface concentration of deposited primary amines, nor does it interfere with the covalent binding of the gel to the platform.

Immunoassay platforms are evolving constantly, undergoing transformation into a variety of shapes, sizes, and configurations. The most common forms of these substrates are 96- and 384-well microplates. Gas plasma treatment is an established method of hydrophilizing such plates in order to promote the immobilization of antigens, antibodies, and other biologically active small molecules.

(click to enlarge) Figure 8. Bubbles of trapped air are common when fluid is dispensed into untreated wells whose surfaces are hydrophobic (a), but essentially nonexistent in plasma-treated wells whose walls can be completely wetted by the dispensed fluid (b).

The formation of bubbles in wells during fluid dispensing is a potential problem that plasma is used to control (see Figure 8). A bubble trapped in a well with a hydrophobic surface will produce erroneous spectrophotometer readings and may even cause spillage into neighboring wells owing to the well volume it occupies. Plasma treatment ensures complete wetting of the analyte in the wells and can virtually eliminate the possibility of bubble formation.

While the hydrophobicity of microplate reservoirs can result in trapped bubbles in the analyte, there are circumstances where reservoirs that are too hydrophilic can create problems as well. Excessive hydrophilicity results sometimes in the analyte wicking up the sides of the well onto the platform floor and potentially contaminating neighboring reservoirs.

One such case known to the author involved an immunoassay platform fabricated from a proprietary hydrophobic polymer. This polymeric platform accommodated a number of reservoir wells that had gold detector plates at their base. The gold had to be cleaned prior to deposition of the biosensor material, so the platforms were exposed to oxygen plasma. However, while this cleaned the gold nicely, it affected the reservoir sides such that the adverse result just mentioned occurred: the dispensed biosensor solution wicked up. The challenge for the plasma process engineer was to clean the gold plates while preserving the optimal level of hydrophilicity on the reservoir walls. This was achieved by supplying a combination of feed gases to the plasma generator, one to remove the hydrocarbon contaminants from the gold and a second to render the reservoir walls hydrophobic through the addition of fluorine groups. This success demonstrated the versatility and adaptability of the plasma surface modification process.

Resistance to Adsorption

Applications for nonstick fluoropolymer technology extend well beyond cookware. In vivo and in vitro medical devices may need to have surfaces that resist the adhesion of proteins or cells in order to maximize their hemocompatibility. Antithrombic activity can be controlled, for example, by coating surfaces with materials similar to PTFE.

A surface's suitability for adsorbtion is reduced by lowering its surface free energy—that is, the energy the surface has available to it for the formation of chemical bonds. One way of doing this for a medical device is to apply to it a thin coating that has an inherently low surface energy. Polymeric fluorocarbon coatings with nonstick properties readily adhere to a wide range of materials when deposited on their surface by PECVD. Gas plasma processing provides a reliable, biocompatible, and environmentally friendly method of reducing the surface energy of materials by polymerizing fluorocarbons onto a surface in a highly controlled environment. Any fluorocarbons present in the exhaust are captured by a scrubber at the pump outlet.

It has been reported that the interaction of DNA with polypropylene polymerase chain reaction plates can result in denaturation over time.⁴ This has implications for the storage of DNA in polypropylene vessels, possibly resulting in a reduction in both the quality and the quantity of DNA as it is stored over time. Studies have shown that oxygen plasma–treated polypropylene plates have a lowered adsorption affinity for DNA.⁵ Oxygen plasma generates a negatively charged surface. It is believed that this negative charge repels the phosphate backbone of the synthetic DNA and thus prevents the DNA from adsorbing to the surface.

Process Validation

Contact-angle measurements are used extensively as a measure of surface bondability. Untreated polymeric surfaces are low in energy, so water droplets applied to these surfaces bead up; they have high contact angles. This is because the cohesive forces of the water are stronger than the adhesive forces of the surface. On plasma-treated surfaces, water contact angles are very low because of the energy that has been added to the surface in the form of polar chemical groups (see Figure 9). This energy is used to bind to the water molecules, which causes the droplets to spread out across the surface. These are hydrophilic surfaces. Therefore, low surface contact angles can be taken as good indicators that a surface is wettable.

(click to enlarge) Figure 9. A bead of water on an untreated hydrophobic surface has a high contact angle (a), while the same surface following plasma treatment becomes hydrophilic, resulting in a low water contact angle (b).

X-ray photoelectron spectroscopy (XPS) and surface derivatization techniques are used to quantify the percentage of the surface that has been functionalized with the desired chemical groups. For example, the surface polymerization of allylamine will result in amino functionalization. To quantify the primary amine present, reagents selectively tag the primary amine groups with fluorine. Fluorine is used because it is easily detected by XPS and its chemical environment does not have to be distinguished. This is in contrast with the case of nitrogen, where many nitrogen-containing functionalities may coexist. Once the surface fluorine concentration has been determined by means of XPS, the original primary amine concentration can be derived.

Conclusion

The semiconductor industry has been using gas plasma technology in the manufacture of microchips for years. Its plasma processes were known to require a high level of sophistication, and plasma systems were tailored to that industry. More recently, however, plasma technology has expanded into the arena of surface-treating polymeric materials. But despite the merits and enabling capabilities of plasma technology in this new industrial application, the expansion of its use has been slow. One reason for this is that plasma solutions often have been associated with high cost and limited flexibility in terms of integration into the manufacturing process.

Demetrius Chrysostomou, PhD, is director of technology at PVA TePla America Inc. (Corona, CA). He can be reached at demetri@ pvateplaamerica.com.

Today, though, plasma technology companies are value-engineering their equipment in an effort to keep costs down. Not only that, they are also designing products to be highly flexible and versatile. Systems these days are available in batch and in-line configurations, and may operate at low pressures or at atmospheric pressure. They can easily be integrated into existing manufacturing lines, are quite easy to use, and can be operated by relatively low-skilled, and thus less costly, personnel.

Plasma technology is gaining greater recognition within the medical device industry because of its success as a surface cleaning and modification tool and its attractiveness as a dry, environmentally friendly process. It is no longer considered an excessively costly technology for filling surface preparation needs. Rather, it is seen as an effective process that facilitates manufacturing and provides a stepping stone to future technologies.

The Auto Laminator by BioDot Inc. (Irvine, CA)

Historically, IVD companies have produced lateral-flow immunoassays (LFIAs) by using batch methods in research and development and manufacturing processes. However, with the drive toward achieving quantitative LFIAs and the demands for higher-volume manufacturing, LFIA production is transitioning to in-line methods for research and development and manufacturing. Implementing in-line methods into research and development minimizes development costs and risks during the scale-up transition to manufacturing.

This article reviews current trends in manufacturing processes that IVD companies are implementing to achieve higher performance reprodu-cibility from test to test, as measured by coefficient of variability (CV), and better efficiency for highvolume manufacturing. This article also focuses on processes such as application of various chemistries onto supporting materials, drying of such porous materials, and laminating and cutting all processed materials into finished test strips that are adequate for LFIAs.

Introduction to Lateral-Flow Immunoassays

The lateral-flow immunoassay format was initially developed in the 1970s. In 10 years, it became a standard platform for various point-of-care (POC) immunological tests. The benefits of this format were the following factors:

• Ease of use.
• Small amount of sample required.
• Adequate level of sensitivity.
• Manufacturability on a large scale.
• Stable final-product shelf life at room temperature.
• Able to implement with a reader technology.
• Ease of regulatory approval (since the format was well known in the market).
• Inexpensive to manufacture.

For many years, the lateral-flow immunoassay format was very popular and was implemented in many tests. However, recent market needs have created higher demands, requiring LFIA tests to become more than qualitative tests. The evolution of lateral-flow tests toward true quantitative formats is an area of active research and development, especially for tests for diseases with high societal costs such as cholesterol, cardiac arrest, diabetes, and HIV testing.

(click to enlarge) Figure 1. A typical lateral-flow immunoassay test.

A lateral-flow immunoassay device is composed of various materials and reagents (see Figure 1). The standard components include a backing card, membrane, sample pad, absorbent pad, conjugate pad, and sometimes a wick. Modern lateral-flow tests have several improvements over the traditional model. For example, the sample pads have become efficient blood separators, allowing assays to be run with whole-blood samples without any sample centrifugation or clotting.^1,2

Due to some constraining patent issues, many lateral-flow assay manufacturers have recently invested in research and development to develop multiplexing assays on complex platforms for specific applications. There is also an acute effort to improve the design of current lateral-flow substrates to increase reproducibility and simplicity of use, such that a venipuncture sample would suffice.

Existing Manufacturing Processes. There are many ways to process LFIA tests, depending on how they will eventually be used. Most IVD manufacturers use either an in-line process or a batch process, which has been far more common until recently. The batch process starts with a roll or sheet of each material (i.e., membrane, sample pad, conjugate pad, wick material) and individual backing cards. Each material is processed independently, entailing considerable operator interference. Once all of the materials are processed, they are laminated onto the backing cards. The cards are then cut into 4–7-mm strips that are assembled into the final test cassette.

The in-line process starts with a roll of all of the materials, and usually involves three types of modules. The first is the reagent dispensing and drying module, the second is the slitting module, in which processed webs are cut down to narrower widths, and the third is the lamination module. A slitting module is needed only when the materials are provided as wide webs instead of narrow rolls.

Batch Process

In a batch process, a roll of membrane is cut into 300-mm strips. LFIAs use nitrocellulose membranes, which represent the analytical part of the final product. To be sufficiently sensitive and stable, the nitrocellulose membrane must exhibit high protein-binding selectivity and protein-stabilization potential. Lines of proteins are deposited onto the membrane by either a contact or noncontact method, and serve as test and control lines.

After dispensing, the membrane is dried and then blocked to prevent nonspecific binding. The blocking step may be done by impregnation in a dip tank or by spraying, and the membrane may be dried in an oven at 37°C for one hour. While the blocking step is commonly done, it is optional and may be omitted depending on a test's design.

Available in a roll format, the conjugate pad is made of glass fiber or polyester material and should be cut into individual sheets. It is very hydrophobic, and should be pretreated to render it hydrophilic and capable of absorbing a colloidal gold or latex solution. This solution contains the conjugate antibody that forms the second part of the assay. As the sample being tested moves through the conjugate pad, the conjugate antibody is released and binds to the sample.

Pretreatment can be done by impregnation in a dip tank or by spraying, in which the conjugate pad is dipped and agitated into a solution designed to make it hydrophilic without affecting the antibodies. In some cases, manufacturers of conjugate pads may treat them in advance to render them hydrophilic (e.g., by adding PVA), eliminating the need for additional pretreatment. The pad is then blotted to remove excess fluid and dried in an oven at 37°C for one hour.

Following these steps, the gold or latex particle solution containing the conjugate antibody is dispensed onto the pad by using an aerosol-type spray.^3-6 An alternative method involves adding the antibody solution to the pretreatment solution, and dipping the pad into the resulting bath. While this method saves one processing step, it does not result in the same penetration of conjugate antibody into the pad.

Also available in a roll format, the sample pad is made of cellulose material and should be cut into individual sheets. The sample pad absorbs any fluid applied to it and makes it compatible with the rest of the assay (e.g., by changing its pH, filtering it, separating out some of its constituents, etc.). For some applications, the sample pad should be pretreated by dipping it into a specific buffer, blotting it, and drying it for one hour at 37°C. The wick material is also available in a roll or sheet format, and should be cut into individual sheets.

Limiting Factors. The batch method is the same for both research and development and manufacturing processes, and can be scaled to higher-volume manufacturing by using duplicate systems. However, the batch process requires multiple operator interventions and includes several steps that cannot be well controlled and cause variability in the final product, which affects the quantification of the LFIA. Such variability emerges during the dispensing, blocking, drying, and assembling steps for all LFIA test components.

In particular, the blocking and pretreatment steps for the membranes, conjugate pads, and sample pads are areas of concern. The dipping process involved in these steps is poorly controlled, which leads to variability in the results. Another significant area of concern is the drying process. Batch ovens do not produce uniform temperatures, resulting in a poorly controlled drying process especially when wet components are placed in them. Reproducible drying is important since it eliminates variability in the resolubilization of the constituents.

Moreover, drying membranes in an oven is cumbersome and slow, and affects the overall manufacturing process and cost.

Converting a batch process into an in-line process involves multiple transformations of several steps in the batch process. By doing so, the impregnation process and drying times are considerably shortened. But despite such limitations, the batch process is still readily used and is adequate for producing qualitative LFIA formats.

Throughput Consideration. With the batch process, manufacturing volumes can be as high as 2 million to 3 million parts per year. However, manufacturing volumes can be more than 10 million parts per year by using a set of in-line equipment. In addition, the in-line approach results in lower CVs compared with the batch process.

In-Line Process

When IVD manufacturers encounter limitations in producing their LFIA tests due to test-to-test variability, they can turn to an in-line process, an alternative to the batch process. The in-line method is designed to meet the specific needs of demanding applications and provides lower CVs because of tighter-tolerance processes.^3,4 It allows multitasking between dispensing (contact or noncontact) and impregnation (in a dip tank) applications. It also has the capacity for in-line drying and quality control monitoring.

The current drive toward developing novel test formats and quantitative tests requires higher demands on the dispensing, blocking, drying, cutting, and laminating processes, the manufacturing of the tests, and the equipment. In order for lateral-flowbased assays to meet such demands, IVD manufacturers should select reagents, substrates, and materials that are compatible with those processes. For developing LFIA with a high level of complexity and reproducibility, adopting an in-line approach provides a robust and controlled manufacturing process.

Compared with batch processes that require multiple interventions from qualified operators, the in-line approach can be fully automated, modular, and configured based on individual application needs. It minimizes the risk of operator failure, and provides more control and symmetry over the process compared with batch processing. The following are various modules that can be configured into an in-line reel-to-reel system (starting with a roll of raw material and ending with a roll of processed material), or an autolamination system (starting with several rolls of processed material and ending with laminated cards).

Web Control and Dispensing. This module consists of a payout reel with tension and web speed control. It also includes web tracking systems that locate the web edge relative to the dispenser positions and align the web edge to a common position on the take-up reel. The purpose of the tracking systems is based on the camber inherent in the rollstock of materials that are knife slit from the master rollstock. The camber is the edge deviation from a straight line that is inside a roll-formed piece and becomes apparent as the piece is unrolled.

The camber results in a drift of the web edge relative to a fixed reference point of 2–5 mm over the length of a 50-m roll. Taking the example of test and control lines dispensing on nitrocellulose, the result of this drift is that the line position can be offset from the membrane edge by as much as 2–5 mm. This displacement is a source of not only increased product variability but also offsets in the positions of the test and control lines once the final product has been inserted into a plastic cassette.

Different types of dispensers driven by tandem pumps can be mounted to the control system.^4,5 The tandem pump is a configuration of two syringe pumps that are connected to one dispenser and are working in an offset mode to alternately fill and dispense each pump. This provides a constant dispense output over long web lengths. As mentioned above, the test and control lines could be striped on the nitrocellulose membranes using either contact or noncontact method dispensing. While the contact dispenser represents a cheaper alternative that necessitates little maintenance, it is not designed for quantitative tests.

For example, FrontLine dispensers by BioDot Inc. (Irvine, CA) drag a meniscus of fluid on top of a nitrocellulose membrane at a speed slow enough to allow the meniscus to be absorbed by the porous and hydrophilic membrane, and draw a line of sample into the membrane.

(click to enlarge) Table I. Coefficient of variability (CV) of the FrontLine contact dispensing method versus the BioJet noncontact dispensing method.

By contrast, the BioJet dispenser by BioDot ejects drops of defined volume with high reproducibility and improved coefficient of variability on the drop volume compared with FrontLine's contact approach (see Table I).^6-8 The antibody or proteinaceous reagent inside each drop binds instantly to the nitrocellulose membrane due to its high protein-binding property. With the appropriate center-to-center drop pitch and drop volume, the drops overlap to form a continuous line.

The dispensing module also accommodates a camera system for inspecting dispensed lines (see Figure 2). The test and control lines can be assessed for continuity, position relative to an edge, and position relative to each other. Those parts of the lines that fail to meet inspection criteria are marked with a visible ink and rejected when the corresponding laminated card is cut for final assembly.

Dip Tank. This module consists of a reagent reservoir with a roller system to impregnate a moving web, and includes a refill system controlled by a reagent-level sensor. The refill system maintains a constant, slow flow of fluid into the tank, ensuring a consistent level of fluid and a more consistent solute concentration than a batch tank, in which solute drag is uncontrolled. This module can also be provided with an enclosure and, when appropriate, a dedicated fume-exhaust system.

Dry Tower. The dry towers have a vertical web path to minimize the footprint or length of the composite ma-chine. The dry tower can also be configured horizontally for drying formats that cannot be accommodated with the vertical design. Drying module features include the following details:

• Variable path length.
• Small footprint with a vertical six-foot dry path.
• Independent temperature- control zones along the web path with a temperature range of ambient up to 80°C.
• Forced-air convection from normal to web surface.
• Noncontact surface temperature sensors along the web path.
• Independent convection flow paths from blower to web surface with exterior venting.

Figure 2. A reel-to-reel system by BioDot Inc. (Irvine, CA) with camera control and dry towers.

The dry path for each tower is six foot long and is composed of six heater zones, three in each tower (see Figure 2). The air input for the convection drying comes from three fans with variable speed control. The air flow from each fan has an independent flow path that directs the air to both sides of the web and exits through a vent on top of the dry tower. In this manner, dry air is continuously fed to the moving web surfaces along the web path. The dry path and drying time are increased by adding extra dry towers.

The temperature can be controlled and can range from room temperature to 80°C. Noncontact IR temperature sensors are positioned along the dry path to measure the web surface temperatures. Due to the endothermic nature of evaporation, the web temperature does not reach the convection air temperature inside the drying tower until the moisture has been completely removed. This allows for high temperatures during the initial drying process and lower temperatures (around 40°C) when the web temperature approaches the convection air temperature. The kinetics of the drying process in an in-line method are enhanced compared with a batch oven.

Lamination and Cutting. The in-line system's modular design can accommodate adding other types of modules for value-added processes. For example, when materials are provided as wide webs, a module can cut the webs into strips rather than re-roll them. A laminating module can laminate the reagent-treated webs to other materials such as adhesives with release liners, plastic backing, etc. Such laminating modules will laminate membranes to a plastic backing after the test and control lines have been dispensed.

In-Line Slitting. This module is used to slit wide webs into narrower web formats. The machine consists of a reel feed system, a rotary blade system, and a number of take-up reels. The take-up reels are designed to be transferred directly to the in-line lamination systems. The rotary blade system is composed of a series of cutting blades with spacers such that the cut width can be customized by using spacers of different widths. For example, a 100-mm-wide web of conjugate material could be sprayed with 10 conjugate lines, then dried, rerolled, and taken to the slitter where it is slit into 10 narrower webs, each 10 mm wide. Impregnated webs may also be processed as 100-mm-wide webs and then slit to the appropriate lengths for automatic lamination.

Figure 3. An auto-laminator system by BioDot Inc. with backing card feeding module, dispensing module, and laminating module.

In-Line Lamination. These modules assemble the various component layers that have been pretreated, slit to the appropriate widths, and re-rolled into a laminated card or roll. Such rolls are laminated onto a plastic backing once the precut liners have been released. Similar to the reel-to-reel systems, the in-line lamination systems have a modular design to accommodate a wide range of LFIAs (see Figure 3). The different types of lamination modules include the following characteristics:

• Membrane lamination. This module consists of a feed reel for the membrane with tension and speed control, a take-up reel for the membrane protective cover when required, and a lamination roller. An important feature of this module is its web tracking system that senses the membrane edge and aligns it to a defined position on the plastic backing. This station can laminate both backed and unbacked membranes.
• Multiple material feeds. This module includes three-roll feeds for the conjugate, sample, and absorbent pads with lamination rollers. Take-up reels for various release liners may also be included. Mechanical guide systems can reference the different materials to the plastic backing.
• Vision inspection. A vision system inspects the different laminate placements. Those out of position are ink marked for removal during the final cutting process.^8,9
• Reroll/cut. This module cuts the laminate into strips using a guillotine cutter or rerolls it for feeding into the strip cutting operation. If the reroll option is selected, the system is configured with a large-diameter core to prevent any stress damage from the rolling to the laminated card.
• Additional options. Other modules include adding plastic overlays to the top of the laminate and performing dispensing operations at any point during the lamination process.

Conclusion

While there are currently several methods for processing LFIA tests, IVD manufacturers are primarily using batch and in-line processing. However, even though batch processing remains a valid method for lateral-flow assay manufacturing, its inherent deficiencies that are due to multiple operator interventions prevent it from obtaining CVs as low as in-line processing. With the current market's demanding specifications for LFIA tests, IVD companies are investing into new processes to produce rapid diagnostic tests with better CVs.

Controlling the complete process is crucial, from quantitative dispensing of reagents, blocking, and drying to aligning materials onto backing cards. In order to meet such manufacturing requirements, research and development laboratories are also transitioning to in-line systems to produce adequate lateral-flow assays. Moreover, the volume of rapid diagnostic tests is exploding, pushing IVD manufacturers to take into account higher throughput considerations. In-line processing can offer advantages for the development and manufacturing processes of rapid diagnostic tests.

(left to right) Thomas C. Tisone is president of BioDot Inc. (Irvine, CA). Helene Citeau is applications scientist at BioDot. Barbara McIntosh is vice president of sales and marketing at BioDot. The authors can be reached at tom.tisone@biodot.com, helene.citeau@biodot.com, and mcintosh@biodot.com.

Senator Edward M. Kennedy (D-MA) introduced the Laboratory Test Improvement Act (S. 736), which is intended to grant FDA the authority to regulate laboratory-developed tests. This bill has been referred to the Senate Committee on Health, Education, Labor, and Pensions, which is chaired by Kennedy.

The legislation will require labs providing home-brew tests to submit evidence to FDA that supports their analytical and clinical validity. All the information submitted to FDA will be compiled into a database, which will subsequently be made available to the public on the Internet. Under specific circumstances, the bill allows FDA to require home brews to go through the 510(k) or PMA process, after giving labs the opportunity to correct the information. Such circumstances include if the information on the test in the database is inadequate, if it shows the test is not comparable to an FDA-approved test kit, or if the test is a direct-to-consumer test.

Labeling for home-brew tests would also have to indicate whether they have not been approved by FDA, and healthcare providers and patients will receive results noting such information.

Even though the bill appears to level the playing field by making labs as accountable as IVD manufacturers, some industry analysts believe the proposed review framework for home-brew tests would place enormous burdens on FDA.

“The bill would impose significant new responsibilities for the Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD), but without giving it new resources,” says Jeffrey N. Gibbs, JD, director at Hyman, Phelps & McNamara (Washington, DC). “The extra workload from home brews will detract from OIVD's ability to review submissions. The bill will also impose new costs on laboratories, hurting the IVD industry's customers. In addition, the bill will create significant confusion due to the ambiguous language and provisions, which cannot benefit the industry.” 

Other analysts believe that if labs developing home brews decide to discontinue doing so because of the increased regulation, cutting-edge tests for certain rare diseases will no longer be available.

The Kennedy bill drastically upsets the playing field such that the effect could be the end of innovative home-brew tests, which will be detrimental to public health,” says Thomas M. Tsakeris, president of Devices and Diagnostics Consulting Group Inc. (Rockville, MD).
 

The Cobas 6000 by Roche Diagnostics (Indianapolis).

Roche Diagnostics (Indianapolis) is making two acquisitions that will further increase its stronghold in life sciences. In early April, Roche announced it would acquire BioVeris Corp. (Gaithersburg, MD), a maker of healthcare diagnostics, for about $600 million. A few days before, Roche had announced it would acquire CuraGen Corp.'s subsidiary, 454 Life Sciences (Branford, CT), a developer of sequencing technology, for up to $154.9 million.

While still subject to regulatory and shareholder approvals and other closing conditions, the BioVeris transaction is expected to close by the third quarter of this year. The 454 Life Sciences transaction does not require shareholder approval and was to have closed by the second quarter.

Roche has a history with both companies. Since 1996, Roche has had a limited license to utilize BioVeris's detection technology in its Elecsys immunochemistry product line. Roche's Elecsys product line is already the fastest growing portfolio of its lab diagnostics business, according to company reports. By acquiring BioVeris, which has about 200 employees, Roche will be able to expand its ECL business into new market segments.

“We licensed the technology for use in human diagnostics testing,” says Mary Beth Myers, director of marketing and communications for Roche's centralized and molecular diagnostics divisions. “Once we acquire BioVeris, we will own all the ECL patents, which means we will be able to expand our immunoassay business. Segments including life science research, life science development, patient self-testing, veterinary testing, drug discovery, drug development, and clinical trials all become open once we own the patents.”

The clinical trials segment alone is about $333 million and growing at about 10% a year, thanks to an increasing number of clinical trials and larger numbers of patients enrolled in those trials. An increasing number of Roche's IVD customers are also expanding their business into the area of clinical trials, Myers notes.

Roche has also been acting as the exclusive worldwide distributor of the 454 Life Sciences' Genome Sequencer systems and associated reagents to all markets with the exception of regulated diagnostics. Through this acquisition, Roche will obtain access to 454 Life Sciences' future generations of sequencing products and the use of 454 Sequencing in regulated diagnostic applications, says Tim Harkins, a spokesman in marketing applied sciences at Roche.

Harkins says the acquisition is a natural evolution for the two companies. “Many different segments within Roche were talking to people within 454 … so it's just formalizing and strengthening a very strong working relationship we already had,” he says.

Harkins adds that the acquisition is significant because it will allow Roche to go after existing and emerging markets in genetic sequencing technologies. The traditional sequencing market is valued at around $700 million; including forensics, it is valued at about $1 billion, he says.

454 Life Sciences' technology enhances genomic data and sequencing applications, enabling the discovery, development, and clinical testing of new vaccines and drugs, and allowing personalized medicine to become a reality, Harkins says. Roche plans to maintain the 454 Life Sciences facility in Branford with its 167 employees as a fully integrated part of the Roche organization.

The acquisitions by Roche are not surprising to industry analysts. “Roche has generated a tremendous cash position and has been actively seeking to deploy capital for strategic acquisitions,” says Jeffrey Ellis, head of diagnostics M&A for CrossTree Capital Partners (Tampa, FL.)

Ellis adds that the BioVeris and 454 acquisitions are “logical steps to bring in-house proven products and technologies with which Roche already has a significant vested interest.” While the transactions are not transformative acquisitions as were the recent acquisitions of IVD companies by General Electric (Milwaukee) and Siemens Medical Solutions (Malvern, PA), they point to a trend of continued consolidation in the IVD industry, Ellis notes.

Doyia Turner, a spokeswoman for Roche Diagnostics, confirms Roche's acquisitions were not in response to industry competition from those well known for their diagnostic imaging systems. “While we do look at them as important competitors, we have been in this space for a long time,” she says. “They may have the experience with imaging, but we have the experience with the clinician and the lab, and that is going to be new for them.”

Ellis expects more acquisitions for Roche and its competitors. The combination of “large, cash-rich IVD companies and a surge in innovative new IVD companies will lead to more mergers and acquisitions for those smaller players that can prove commercial success,” he says.
 

The Triage Meter by Biosite Inc. (San Diego).

Inverness Medical Innovations Inc. (Waltham, MA) and Beckman Coulter Inc. (Fullerton, CA) were vying for the heart of Biosite Inc. (San Diego) in a love triangle that heated up during the last few months. It is a bit ironic, too, as Inverness and Beckman both wanted Biosite's expertise in cardiology testing technologies that help clinicians determine quickly whether a patient has congestive heart failure or is having a more emergent cardiac event.

Biosite, a biomedical company that has also made discoveries in the proteomics arena aimed at advancing medical diagnostics, had been talking to prospective buyers for about a year. According to Biosite, over 70% of U.S. hospitals currently use the company's Triage rapid diagnostic tests. Its total revenues in 2006 were $308.6 million. Valued at $55.38 per share the day before the first offer, Biosite's stock is now worth more than $90 per share.

Beckman Coulter, a large instrumentation company specializing in biomedical testing, enjoyed $2.5 billion in sales in 2006. On March 25, 2007, the company announced it would enter into a definitive merger agreement to acquire Biosite's out-standing common stock for a cash tender of $85 per share, or $1.55 billion.

Scott Garret, president and chief executive officer of Beckman Coulter, called the merger “an exciting transaction that grew out of our successful relationship with Biosite over the past four years.”

However, on April 5, Inverness, a maker of home pregnancy and fertility tests and rapid point-of-care diagnostics, confirmed it made an unsolicited cash tender bid for Biosite for $90 per share, beating Beckman Coulter's offer by $5 per share. Inverness, a company only slightly larger than Biosite and about half the size of Beckman, had less than 5% of previously acquired Biosite common stock and planned to delve into cardiology with this acquisition.

Ron Zwanziger, chairman, president, and chief executive officer of Inverness, said the acquisition “would make for a powerful long-term strategic fit by enabling Inverness to leverage Biosite's strength in proprietary protein markers and robust cardiovascular platform.”

But Zwanziger's earlier comments made Inverness sound like a jealous lover, citing “serious concerns regarding the integrity of a supposedly competitive bidding process that would lead Biosite's management to enter into a preemptive merger agreement with another party rather than fully explore a combination with us.” Nonetheless, Zwanziger remained “hopeful that Biosite's board would respond favorably.”

Garret called the offer from Inverness “unsolicited, highly speculative, and conditional” in light of Inverness's statement that it would require additional due diligence to confirm financing. However, when Inverness submitted signed commitment letters from its proposed financing sources, Biosite's board officially stated that the offer was a “superior proposal” as defined in the initial agreement with Beckman Coulter. Zwanziger was “extremely pleased.”

On May 2, Beckman Coulter countered with its own offer of $90 per share. Inverness quickly responded with a new binding offer of $92.50 per share about a week later. Zwanziger said that Inverness expected an announcement by Biosite that it was a “superior proposal” by May 9, or it might exercise its right to withdraw. Despite the threat and Biosite's refusal to deem the offer superior at that time, Inverness stayed in the running with its offer set to expire May 16.

Jeff Ellis, head of diagnostics M&A at CrossTree Capital Partners (Tampa, FL), said he thought Inverness would get the deal and could price it up even higher than $92.50 per share if necessary. “The only question is whether Beckman will respond now and if Inverness has to up its price again or whether they'll let it go,” Ellis said. “I won't be surprised if they just let it go.”

On May 14, Biosite announced that it deemed Inverness' latest offer of $92.50 per share a “superior proposal” to the revised merger agreement with Beckman Coulter. On May 15, Beckman announced that it would not go above its offer of $90 per share, claiming that doing so would not serve its stockholders. Garret said that while it still felt “the combination of Biosite with Beckman Coulter is strategically sound,” it also believed that $90 per share was “a full and fair price for Biosite.”

Garret added that, “Although we do not agree with this conclusion, we expect that Biosite will terminate its existing merger agreement with Beckman Coulter and, concurrently, pay Beckman Coulter a termination fee of $54 million.” Biosite said in a separate statement that Inverness had agreed to pay its $54 million termination fee.
 

Photo by iStockphoto

Secretary of Health and Human Services (HHS) Mike Leavitt outlined the department's Personalized Healthcare Initiative, which intends to achieve gene-based medical care combined with health information technology. The initiative includes some measures that could affect the IVD industry, particularly those manufacturers developing molecular diagnostics.

Leavitt said HHS will review existing structures for ensuring that genetic tests are accurate, valid, and useful. The objective is to ensure that responsibilities are appropriately assigned among HHS agencies to address the analytical and clinical validity and utility of tests.

According to HHS officials, the department has seen a large number of new molecular and genetic tools being developed. However, there is a lot of confusion regarding the framework in which such tests can get market approval, and whether they should be reviewed by FDA or subject to CLIA oversight. A number of issues have also been raised during the past few years relative to enhanced understanding of the performance of tests, their accuracy, and how the information can be used.

“There is a robust interest in the development of new technologies that look at genetics, gene expression profiles, and many new types of genetic tests,” says Greg Downing, project director for the Personalized Healthcare Initiative. “We are trying to develop a better understanding of the evidence and the support for their integration into healthcare, and examining whether there are needs for new capabilities and new ways to look at the information about them.”

Leavitt also said the American Health Information Community (AHIC) will develop recommendations to identify health IT standards for including genetic test information on electronic health records.

HHS officials added that the goal is the exchange of such information in a more efficient manner, such that it can be retrieved, archived, and utilized in different applications. The department is working with AHIC to evaluate the opportunity to develop standard reporting systems into electronic health records of genetic laboratory test results and family history information. HHS is also working with standards organizations such as Health Level Seven and others, and it has prioritized the inclusion of genetic tests into the electronic health record as an important tool.

“I think everyone would agree that one of the major challenges in our healthcare system today is the lack of adequate information and the ability to exchange information about what has happened in a patient's healthcare experiences,” says Downing. “The Secretary's efforts through AHIC have been centered on the electronic health record as one of the tools to help develop information so that we have common or interoperable ways of collecting information about laboratory tests or electronic prescriptions.”

In addition, Leavitt noted that the FDA critical path initiative is currently organizing work across various science and regulatory areas to improve product development, especially for gene-oriented drugs and diagnostic tests. Furthermore, FDA will publish a regulatory guidance on the codevelopment of drugs and diagnostic products this fall.

Secretary Leavitt

HHS officials confirmed that in the critical path compendium, there are a number of opportunities related to clinical diagnostic tools; among those, several genetics tests have been discussed as having potential. Overall, the department is looking at how to modernize the medical device development process.

“We anticipate this year the release of a guidance on the codevelopment of diagnostic tests and therapeutics in clinical trials,” says Downing. “This guidance should provide some advice to industry in helping guide the use of various tools and clinical trials in evaluating new therapeutic agents. In another area, the voluntary submission of pharmacogenomic data has gone well in terms of being able to develop a broader information base about the interface of tests with therapeutic agents.”

However, HHS officials realize that IVD manufacturers are concerned about whether proper reimbursement for these tests will be available. Officials noted that the primary focus has been developing evidence to demonstrate the utility of such tests in clinical populations. For example, the department is looking at options through its health IT agenda to determine whether capturing information through these tests' electronic records is a feasible way to encourage their utility and potential reimbursement strategies. HHS will continue to develop better longitudinal information by determining not only how well the test performs but also how it informs choices about appropriate therapies and the outcomes of patients.

“One of the major barriers hampering reimbursement is developing evidence of the opportunity that these technologies offer to improve the quality of care,” says Downing. “Getting that information with regard to market approval and the longitudinal aspects of this are difficult. What we would like to do is to try to emphasize using current resources to improve the ability of gathering evidence about the outcomes of these tools. That is not meant to be in the context of premarket aspects, but rather better understanding what is the information that these tools are providing and how are they enabling us to improve the quality of healthcare either through improved response or diminishing adverse events.”

Additional information about HHS' Personalized Healthcare Initiative can be accessed via the department's Web site at www.hhs.gov/myhealthcare.