Development of a Point-of-care C-reactive Protein Device

Chemical Engineering Senior Design Project 1 U.C. San Diego, Spring 2011 ___ C-reactive protein (CRP) is synthesized by the liver, and in the early 2000s received much public attention when the mass media reported that chronic, lower level elevations of CRP could be a good risk factor indicator of cardiovascular diseases. In news reports, latching onto some medical journal articles, it was even asserted that a CRP blood test could be a more important gauge of heart diseases than a blood lipid panel analysis. The positive association between CRP and cardiovascular diseases, to an extent, is demonstrated mainly by epidemiological studies; the scientific mechanism is not well understood. In the scientific literature, CRP is considered a non-specific biomarker for inflammation. It is among the so-called acute-phase proteins. High levels of CRP in blood plasma is an indication of inflammation triggered by, for example, infection, trauma, inflammatory diseases, and even some cancer. Moderate changes in CRP level can also occur after strenuous exercise, heatstroke, and childbirth. Even though CRP cannot be used as a prognostic indicator of a specific disease, many medical professionals consider it a good general indicator of inflammation, and analogous to measuring body temperature, a quick reading of CRP can help physicians in making diagnostics. To monitor CRP level as part of health care management, the physicians or their nursing assistants must be able to make the measurements using point-of-care devices at primary care facilities. Point-of-care (POC) testings are generally performed at or near the location of patient care. Unlike a centralized testing lab, these off-site locations do not have expensive analytical equipment. These locations also have neither specially trained technicians nor the time to handle elaborate sample preparations. Hence, one of the important requirements is that POC procedures must be able to apply directly a small blood sample from pricking a finger, an oral swap, or simply saliva. In other words, any preparative or developing steps that are necessary for the sample must be integrated into the POC device. The tools used in POC diagnostics tend to be portable instruments or bench-top units with a small footprint. The trade-off is to yield complexity for the ability to deliver results quickly to the primary physician. The simplest POC devices, especially handheld units, generally operate on the basis of membrane-based test strips or self-contained disposable cassettes. With small bench-top instruments, the key is that no user intervention is necessary between sample loading and results retrievalwith the so-called sample-in, answer-out capability. Periodic machine maintenance is acceptable if it involves simple replacement of a depleted cartridge with a new one with replenished chemicals. The marketing department of Blax Beech, Inc., has recently performed an analysis and recommended the management to explore the market opportunity of launching a point-of-care CRP bioanalytical device within a short timespan, around a year or so. This development period includes filing for FDA approval. As such, the management considers it a priority to identify and license proven technology as a platform. If the platform requires the customer to invest in a benchtop system, the technology must be flexible such that it can be used for other biochemical assays that Blax Beech may subsequently extend into their markets. All these prospective assay products
1 With contributions from Beckman Coulter and Caliper Life Sciences. 1

should be able to use the same type of sample (as in blood, saliva, etc.) from the patient. Among available technologies, the Blax Beech management considers microfluidics to be the most attractive. Microfluidics is a field that involves the transport phenomena and chemical reactions of minute quantities of fluid in channels with dimensions in the tens of micrometers or even smaller. In conjunction with microelectromechanical systems (MEMS), one can fabricate the so-called lab-on-a-chip or micro-total-analysis systems ( TAS) that can integrate multiple processing steps on one single device. In terms of proven microfluidic systems, the Blax Beech management considers the one developed by the Swedish company Gyros AB a leading candidate. The Gyros products are built on the disposable compact disc (CD) platform. Depending on how the disc is designed and fabricated, the CD-based platform can use minute sample size, handle multiple samples, and perform multiple steps without the need of pumps or electrical fields. The Gyros products have been tested on performing immunoassays and enzymatic reaction-based assays. The main goal of this project is to design a CD-based CRP assay that makes use of the Gyros platform for POC applications. The first step is, of course, to design a biochemical assay for CRP that can be adapted to the Gyros machines. It is understood that your focus is on the engineering aspects, while the chemistry department of Blax Beech will provide and support the biochemistry aspects. Nonetheless, engineers do provide feedback to scientists regarding the feasibility of implementing a scientific protocol in manufacturing and production. Afterward, you either use one of the Gyros pre-fabricated CDs if they are applicable, or custom order a CD from Gyros that you specifically design for your CRP assay. Regardless, based on the CRP assay that you choose, you need to model and perform design calculations to ensure that the CD can provide all the necessary reaction, binding, and washing steps in terms of sequence, duration, and fluid volume. As a commercial product, you also need to address additional issues such as storability (or shelf-live) of the biochemicals used in the assay and federal regulations for clinical devices. The two FDA regulations that you want to be familiar with are the premarket approval (PMA) application, and the premarket notification 510(k) clearance. The Blax Beech management understands that with Gyros, a customer must purchase an expensive machine. The managers are open to suggestions on competitive point-of-care alternatives that have a lower entry cost. A design constraint for the likely less sophisticated alternatives is that they must be able to detect low level serum CRP, in the so-called high-sensitivity CRP tests. Report Requirements The design report follows the outline suggested in SSL&W, with the addition of the executive summary. Further details on what should be included in the design report can be found in that text. Write the document from the point of view of an engineer making a report and recommendation to the management in the company. 1. 2. 3. 4. 5. 6. 7. 8. Letter of Transmittal (exempted for the electronic submission system; FYI only for nominal circumstances) Title Page Table of Contents Executive Summary Abstract Introduction and Project Charter Innovation Map Concept Design, with subsections on customer requirements, product requirements,

9. 10. 11. 12. 13.

competitive and market analyses, product concepts, and other considerations, like federal regulation compliance, where applicable (SSL&W stated that all these issues must be addressed for each viable design concept, but you should consolidate common items, for example the patent search, where you see fit.) Product Design, with enough technical details and analysis to build a prototype Conclusions and Recommendations Acknowledgements Bibliography Appendix (leave this part in the binders in the CENG 176 Lab)

Resources The fist two authors of each article with multiple authors, as they appeared exactly in print, are used because that's all you need to search for the article (does not mean that is how you should do that in your report). There are many references necessary for this project. This list below is not comprehensive but should help you launch the project. C-Reactive Protein and Bioassays First, you need some background on bioanalytical assays, especial with immunoassays. For a tutorial on antibodies for your own background information, try the Millipore Antibodies Tutorial. It covers the basis of the so-called and extremely common ELISA even though it never addresses it directly. For a tutorial on the avidin-biotin system, go to Fisher Thermo Scientific. Find the AvidinBiotin Technical Handbook. It may be posted on their main page. If not, click into Technical Resources and find their page on Technical Handbooks. Click on the Avidin-Biotin link and download the PDF. FYI: You only need to read the first two pages. The rest of the handbook is not needed unless you have to do some antibody modification after you have purchased it. On this note, Thermo Scientific also has a handbook on crosslinking reagents. Thermo Scientific bought up the former Pierce Chemicals Co., which was the leading expert in conjugation chemistry. You can still see the Pierce name on the Web pages and in the URL of the site. Alvydas Mikulskis, Dave Yeung, et al., Solution ELISA as a platform of choice for

development of robust, drug tolerant immunogenicity assays in support of drug development, J. Immunol. Meth., 365: 38-49 (2011). This article gives you some background on ELISA. It is not written as a review, so not everything is necessary or relevant for your project.
Next, some articles related to CRP and detection methods. The one by Pepys & Hirschfield (2003) explains the biochemistry of the protein, and the one by Young & Rifai (2009) is just a two-page Op Ed. Try c-reactive protein detection or something similar in Google Patent. Youll need to screen out the patents not relevant for this project. That includes the ones making hybridomas or antibodies; ignore them. Its given nowadays that polyclonal or monoclonal antibodies can be purchased easily from a vendor. Nicolaos Christodoulides, Sanghamitra Mohanty, et al., Application of microchip assay system for the measurement of C-reactive protein in human saliva, Lab Chip, 5: 261-269 (2005).

 Filiz Ibraimi, Dario Kriz, et al., Rapid one-step whole blood Creactive protein magnetic permeability immunoassay with monoclonal antibody conjugated nanoparticles as superparamagnetic labels and enhanced sedimentation, Anal. Bioanal. Chem., 384: 651657 (2006). Mark B. Pepys and Gideon M. Hirschfield, C-reactive protein: a critical update, J. Clin. Invest., 111: 1805-1812 (2003). Marc Wolf, David Juncker, et al., Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks, Biosen. Bioelectronics, 19: 1193-1202 (2004). Yi-Ning Yang, Hsin-I Lin, et al., An integrated microfluidic system for C-reactive protein measurement, Biosen. Bioelectronics, 24: 3091-3096 (2009). Ian Young, Nader Rifai, High-Sensitivity C-Reactive Protein and Cardiovascular Disease, Clinical Chem., 55: 201-202 (2009). If you ever want to read more on the medical science of CRP, the entire February 2009 issue of Clinical Chemistry is a special on this topic. This two-page article is just the brief introduction. Microfluidics Review papers to get you going: Chun-Che Lin, Jung-Hao Wang, et al., Microfluidic Immunoassays, J. Lab. Automation, 15: 253-274 (2010). A nice review that covers a lot of topics, including CD-based assays. Vincent Linder, Microfluidics at the crossroad with point-of-care diagnostics, Analyst, 132: 1186-1192 (2007). They have their own products under development. Niels Lion, Frederic Reymond, et al., Why the move to microfluidics for protein analysis, Cur. Opin. Biotechnol., 15: 31-37 (2004). Couple of specific ones: Todd Thorsen, Sebastian J. Maerkl, et al., Microfluidic Large-Scale Integration, Science, 298: 580-584 (2002). A reference cited in many of the design projects used by SSL&W; you be the judge on its worthiness. Christopher J. Easley, James M. Karlinsey, A fully integrated microfluidic genetic analysis system with sample-inanswer-out capability, Proc. Natl. Acad. Sci. USA, 103: 19272 19277 (2006). This work is on the much more complicated genetic analysis, but their objectives may help your thinking on the constraints of designing a point-of-care device. The following references are related to the Gyros technology: Robert Gorkin, Jiwoon Park, et al., Centrifugal microfluidics for biomedical applications, Lab Chip, 10: 1758-1773 (2010). One of the authors (not listed here), Marc Madou, is a professor at UCI and a pioneer in this technology. One can easily trace back to his earlier papers with engineering analysis.

 L. Riegger, M. Grumann, et al., Read-out concepts for multiplexed bead-based fluorescence immunoassays on centrifugal microfluidic platforms, Sensors & Actuators A, 126: 455462 (2006). Jeanette Roman, Julia Qiu, et al., Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis, J. Pharmacol. Toxicol. Meth., in press (2011). Google Scholar found it as an advanced pre-print copy in February. Regulation Regulations on food and drugs are covered by Title 21 of the Code of Federal Regulations (CFR). FYI, it is accessible on line (www.gpoaccess.gov/cfr/). Medical devices are covered by many parts in the CDR and for this project, it is a little bit easier to go directly to FDA (www.fda.gov). The Approvals and Clearances entry under Medical Devices is more or less just an access to the database. To learn more about PMA and 510(k), you want to do a search inside the FDA site. Some of the instructions are under Development & Approval Process posted by the Center for Biologics Evaluation and Research (CBER). In this project, you are not going to write an application for approval or clearance. You just need to be aware of the procedures that may be involved in the product development process and more importantly if there are any regulations that may affect your design. Vendors Do a search with c-reactive protein" in www.biocompare.com. You should find companies that mainly market ELISA kits for research labs. Gyros, www.gyros.com. They have a Publications page that is not easy to find anymore. From there, it led us to the following conference abstract. Mats Ingana, Helene Derand, et al., Integrated Microfluidic Compact Disc Device with Potential Use in Both Centralized and Point-of-Care Laboratory Settings, Clinical Chem., Conference abstract, 51: 1985-1986 (2005). Abaxis, www.abaxis.com. They make a CD-based point-of-care product Piccolo Xpress. Abbott Point of Care, i-Stat Analyzer, http://www.abbottpointofcare.com. They made one of the earlier entries into the point-of-care market with a handheld unit. Technoclone, now part of Pathway Diagnostics (the device photo is in here), makes a niche compact point-of-care analyzer. There are other such companies. Your call if you need to research more of them. Business/Marketing ICON Group International, www.icongrouponline.com. Not free; why we did not make it to be a live link. But the UC San Diego librarian found us a free report. See the class Wiki site for the PDF. ImarketInc, www.imarketinc.com. Not free, even though cited by SSL&W too. So a good site if one has the money to buy the reports.
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 Strategic Business Insights (SBI), www.strategicbusinessinsights.com. Formerly the SRI Consulting Business Intelligence, a spin-off of SRI. Now, they provide a handful of free (teaser) reports and thats about it. The Yahoo! Finance Industry Center, http://biz.yahoo.com/ic/index.html, is a good place to get market performance data by industrial sectors, and for us, to track down the public traded companies. Reminder: this is not relevant to our project, but more for you general business knowledge. Specifically, you can try: Medical Instruments & Supplies, http://biz.yahoo.com/ic/520.html where you find, e.g., Baxter and Becton Dickinson Medical Appliances & Equipment, http://biz.yahoo.com/ic/521.html where you find Medtronic Scientific & Technical Instruments, http://biz.yahoo.com/ic/837.html where you find, e.g., Agilent and Bio-rad FAQs Additional information, especially pertaining to costing and economics, will be added here if we manage to find them. Otherwise, just a handful of FAQs. Why are there no technical design specifications within the problem statement? With product design, you have to find out what is the voice of the customer first. Then you translate that into technical product requirements. To a good part, you extract the design specifications from your understanding of the physiological parameters and microfluidics design from CRP related papers. For point-of-care devices, certainly existing commercial products should help to frame your design targets. How much do I need to know about biochemistry? Not much. This project is not on developing new detection methods but designing a device that utilizes established protocols. So you just need to know the definitions and principles behind immunoassays and the avidin-biotin bindingthey are the cornerstones of biological assaysto the extent that you can read the technical papers and apply the methods appropriately when you design your product. That is why we simply suggested a couple of tutorials from some companies in our Resources. Absent of that, you can read the texts for biochemistry labs. Your (engineering design) report does not need to explain the biochemical principles. What are the expectations of the progress reports? We conceivably may consider the four phases: First progress: Preliminary assessment of the marketplace and technology field; initial project charter Second progress: Concept design Third progress: Product design with rigorous engineering analysis Final design report Nonetheless, what you want to accomplish at each milestone is entirely up to your design group. You need to remember that this is a competition and you are evaluated by your peers.

 Do we have to do a marketing and profitability analysis? No. Unlike engineering economic analysis in preliminary process designs, marketing research and analysis are typically the turf of the marketing department in a company. The marketing folks will provide the marketing data, sales projection, entry price points, and strategic marketing and business plans. Thats why SSL&W wrote that product development is a team effort. (Saying that, you will have to do all this work yourself if you are starting up a new and very small company with your buddies.) As engineers, you just need to show that you have some knowledge about the market segment that your product will be targeting at. Hence, in your report, all you need is a very small section on the general medical diagnostic market, and perhaps the probable market volume of the proposed product. How expensive are those point-of-care products? Well provide more information when we get them. See, for example, the pamphlet from Yole Development. There can be a wide range in the price for one test, depending on the particular assay.