Manufacturing Considerations for IVD Reagents Used in Clinical Chemistry and Special Chemistry Laboratories

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In-vitro (IVD) reagents are critical components in the clinical chemistry and special chemistry testing laboratories. Without them, these laboratories are unable to generate the information required for patient diagnosis, monitoring, and treatment.

Reagent types

Chemical, biochemical, and immunochemical reagents are dependent on several different elements working in unison to generate accurate and precise test results. Regardless of the platform or format involved, most reagent systems follow these basic steps:

1. The sample and appropriate diagnostic reagent(s) are placed into a reaction vessel.
2. This sample/reagent test mixture is incubated, typically at a temperature of 37 °C, for a given period of time.
3. The test reaction is stopped once the preset time is reached and a quantifiable change is observed.
4. This change varies per procedure, but is usually an increase or decrease in spectrophotometric absorbance, a change in the intensity of light produced or not produced, or an increase/decrease in the optical density or turbidity of the test mixture, etc.
5. The change is measured and compared against a known change that has a definitive value assigned to it, most often coming from a calibration material or standard material, which is then calculated into a test result.

Chemical reagents are the most basic of the three and are typically designed as a single- or two-reagent system. They incorporate dyes, buffers, surfactants, and basic chemicals to form the working reagent(s). An example is the bromocresol green (BCG) reagent used to measure serum albumin levels. This common diagnostic reagent routinely measures the serum component based on the binding of BCG dye, specifically with albumin, to produce a colored complex. Absorbance of the resulting complex is measured spectrophotometrically and is directly proportional to the albumin concentration in the sample.

Biochemical reagents are more complex and typically require more working components. Also designed to be a single- or two-reagent system, these reagents are frequently enzyme-driven and incorporate buffers, stabilizers, coenzymes, indicators, surfactants, and preservatives. The reagent used for the determination of glucose, a commonly run diagnostic test in the clinical laboratory, is a biochemical reagent. For this test, glucose is acted on by the enzyme hexokinase in the presence of adenosine triphosphate and magnesium to produce glucose-6-phosphate. Glucose-6-phosphate dehydrogenase then specifically reacts with glucose-6-phosphate, with the concurrent reduction of NAD to NADH. The NADH produced absorbs light at a specific wavelength and can be spectrophotometrically detected as an increased absorbance and is proportional to the glucose in the sample.

 Figure 1 – Samples of raw materials used in immunochemical reagent manufacturing. Shown here are antibodies, antigens, buffers, conjugate markers, and disease-state sera.

Immunochemical-based reagents are composed of two or more reagents and incorporate numerous steps and working components, including buffers, conjugates, wash solutions, detection reagents, and serological markers—namely antibodies and antigens. These reagents are typically referred to as immunoassays and are frequently employed to measure analytes found at relatively low concentrations in the body (Figure 1). They include the reagents used to measure special proteins, cancer markers, infectious disease agents, hormones, vitamins, etc.

Manufacturing considerations

Important factors that should be considered when choosing a reagent manufacturer and supplier include:

• Industry experience: What is the supplier’s experience and knowledge of the IVD market? Do they have an established reputation within the industry? Are they financially sound?

 Figure 2 – Columns used in the manufacture of conjugated antibodies.

• Manufacturing capabilities: Can they handle bulk options and meet both immediate and projected needs? Does product coverage include other associated assay components? A key consideration for vendors of immunochemical-based diagnostic tests (i.e., immunoassays) is antibody supply. Antibody generation is a complicated and intricate production process that requires a great deal of time and expertise. Those who are not producing their own antibodies should consider sourcing antibodies from a reliable supplier. An antibody supplier should be able to offer a full range of custom services, including polyclonal production; monoclonal production; purification and conjugation; freeze-drying; and antibody fragmentation, as well as bulk custom formats, purified antigens, and sera (Figure 2). Since production of antibodies is complex and their use in diagnostic assays changes over time, it is critical that the supplier keep customers apprised of regulatory issues that can potentially affect key assay components.

• Regulatory and quality compliance: Vendors should be in compliance with all associated regulatory organizations, not just the FDA. A repeat pattern of noncompliance and ongoing issues with any regulatory body should be cause for concern. The same applies to any vendor operating without certified quality standards (such as ISO 9001 and ISO 13485) and without validated procedures and processes.

 Figure 3 – QC testing of raw material components used in reagent manufacture.

• Product performance: Performance refers to tests for overall test imprecision, method comparison/correlation to a device, test interferences, and assay linearity. Test sensitivity and specificity testing claims need to be addressed for immunochemical-based diagnostic reagents. In particular, relevant cross-reactivity data should be obtained to support specificity claims. The reagents need to be within limits acceptable to regulatory and end-user expectations. In addition, the product’s lot-to-lot consistency should be considered. The reagents, raw materials, and/or both should perform exactly the same way they did a year ago and exactly the same way a year from now (Figure 3). End-users expect this, as do the regulatory bodies.
• Technical support expertise: It is important to select an experienced vendor that can quickly and efficiently resolve any issues.
• Vendor audits: Frequent visits to the supplier are recommended, as are regular audits.
• Cost considerations: While each component in a diagnostic reagent contributes to the overall cost, significant cost savings can be realized by changing one element (such as a detection buffer) that can affect the sensitivity of the test, resulting in reduced volume requirements for key reagents and reduced assay costs. End-user value of the test versus component cost should also be taken into consideration, for example, a faster test, or one with improved performance may cost more, but may be able to save on time and labor.

Conclusion

To achieve high performance at the lowest possible cost, the individual test components and the chemical, biochemical, and immunochemical interactions of any reagent system must come together to produce a reliable and accurate patient test result. In order to produce diagnostic reagents that meet current and future demands, understanding of the various reagents used for clinical testing applications, as well as the breadth of sourcing options available for assay components, is needed. Use of strategic/tactical purchasing and manufacturing decisions could provide cost savings, while also addressing current and anticipated directions in the IVD industry.

The authors are with the Immunologicals Group of Binding Site Inc., 6730 Mesa Ridge Rd., San Diego, CA 92121, U.S.A.; tel.: 800-633-4484; e-mail: [email protected]; www.us.bindingsite.com. The authors wish to thank Ms. Elizabeth A. Janetschek for her assistance in the editing this article.