Combination products can cause twice as many problems for companies in the testing of these devices since they require expertise in two highly regulated specialties—medical devices and pharmaceuticals. This article offers a number of areas manufacturers of these devices will have to consider.

By Steven Richter, Ph.D.

Analytical chemistry laboratory featuring a Microtest HPLC
Manufacturers seeking to ensure that their combination product meets all cGMP guidelines and passes muster with the FDA, ISO, and USP must first conduct multiple tests and processes on both medical device and pharmaceutical products. Whether the tests are outsourced or completed in-house, rigorous and exact procedures and tests must be adhered to for the product to succeed at market release.
Dosage Calculations
To begin, preliminary testing of a medical device should be conducted prior to sterilization validation processes. A microbiologist or biologist should test the product for bacterial contamination which will enable the determination of accurate sterilization dosages. The sterilization of residuals to ethylene oxide must be determined—and meet FDA guidelines—prior to release.

In 1992, ISO requirements called for laboratories and manufacturers to perform dose calculations due to the possibility of bacteria present on medical devices that may be resistant to radiation sterilization. At this time, ISO, along with participating laboratories, developed processes to determine if the organisms were resistant to radiation sterilization.
Sterilization Validation
Biological indicator sterility testing and processing is required in terms of validation of this process. While few medical devices are processed by steam, the majority of medical devices are either sterilized by ethylene oxide gas or radiation. Generally, a company would use indicators for ethylene oxide (EO) processes only, as biological indicators are not required for radiation sterilization monitoring.
Bacterial Endotoxins
Bacterial endotoxins are toxic in direct blood contact applications. Any device that can be used as a conduit to introduce fluids into the human body requires by law to be tested for endotoxins or pyrogens. Endotoxins are bacterial gram negative cell walls that can elicit a fever or pyrogenic response in a human. Pyrogens as opposed to bacterial endotoxins can be either bacterial or chemical.

  Endotoxin plate reader
Testing for endotoxins with validated methodologies will satisfy not only the pharmaceutical side, but also the medical device side. One test is the gel clot method. This method is a visual observation of a clotted gel that is based on horseshoe crab blood. The blood of the horseshoe crab clots in minor amounts of endotoxins. A chromogenic test, where the instrument is tested using software and a plate reader, determines the intensity of the reaction to the horseshoe crab blood. The intensity is then measured by software which calculates toxic product levels.The kinetic/chromogenic method is highly accurate and specific for bacterial endotoxin. The method proves far superior for reducing false positives that are seen with the gel clot method due to potential beta glucons. Beta glucans molecules are contaminates from fungal or environmental origins. Some researchers think that Beta glucans may be eliminated by recombinate reagents.
USP/EP Release Requirements
The USP/EP, versions 29/5, are highly respected organizations that govern pharmaceutical manufacturers. To put the USP/EP logo on a pharmaceutical label, it must comply with their strict requirements. The FDA scientists use the USP to prosecute medical device and pharmaceutical companies in terms of adulteration and mislabeling laws. There are five analytical methods that meet the USP requirements in determining the effectiveness of a combination product throughout its shelf life.
  • The antimicrobial effectiveness test (AET) is tested by challenging the preservative system to see how effective the pharmaceutical is throughout its shelf life. This involves inoculating the pharmaceutical article with bacteria and fungi to determine if their levels change over a 14- to 28-day period.
  • Microbial limits testing is generally used for non-sterile pharmaceutical articles that can cause disease when ingested or applied topically.
  • Antibiotic assays are performed using a cylinder plate method that quantitates the propensity of the bacteria to grow near the antibiotic.
  • Particulate testing is completed using a light scattering instrument to determine particulates in liquid parenteral products.
  • D-Value analysis determines if the resistance value is homogeneous. Spores often change their values so it is necessary to confirm that the correct resistant values are maintained.
    Microbial Identification
    Microbial identification is identifying bacterial and fungal contaminants, which is difficult to do using conventional means. Genetic (16s rRNA) bacterial and fungal identifications are state-of-the-art for fast, accurate results. Again, this system must be validated against the USP methods.
    Pharmaceutical/Biologics Testing
    Pharmaceutical testing includes ensuring that container closure systems work properly as well as stability and contamination testing of the product. Leading industry tests include:
  • Bacteriophage screening—When certain biologics are manufactured with bacterial cell cultures. A Bacteriophage is a virus that effects bacteria. These viruses can effect the cell and the drug products produced by the cultures.
  • The container closure system—An integral microbial barrier with a procedure that uses pressure to drive a methylene blue dye into the vials. This method is very robust and validatable.
  • In mammalian cell based products, the lab assays test for product contamination with mycoplasma organisms and mammalian viruses (9CFR). These biosafety tests are required for release of the biologics.
    Water Analysis
    FDA guidelines state that pharmaceutical manufacturing must use very high quality water. There are three tests to determine the quality of the water: TOC, Conductivity HPC/Bioburden, and endotoxin testing. TOC is total organic carbon test; it determines levels of contaminates in purified or WFIi water systems. Organic Carbon (any organic contaminant such as pesticide, cleaning solution, or drug product) has to be less than 500 parts per billion.

    Drugs made with inorganic material require other USP water tests. Conductivity is a tests that determine inorganic contamination levels. HPC or heterotrophic plate count is used to identify oligotrophic (water loving) organisms that will cause problems with drug product as well as downstream processing.
    Package Validation
    There are four different tests that can be used for package validation to ensure that the combination product remains intact. They include:
    • ICH stability testing
    • Transportation Test: Vibration and drop testing
    • Subjecting the product to a harsh environment of temperature and humidity: cycling
    • Determining if the package is a microbial barrier: Methylene blue dye testing
    In-Vivo/In-Vitro Biocompatibility
    When encountering in-vivo biocompatibility, the biocompatibility test matrix indicates the testing regimen. If a medical device is used in blood contact services as opposed to skin contact services then the required testing is much more involved.

    In-vitro biocompatibility was developed in the early 1990s; these methods were placed into the USP and ASTM for testing medical plastics. Medical plastics have a propensity for toxicity due to their mold role release agents. These tests are extremely important to get a product approved by the FDA. For change control, additional biocompatibility tests and chemistry tests (FTIR) can be utilized for those qualifications.
    Contamination control is an issue on the forefront of all pharmaceutical and medical device manufacturers. Microbial and chemical contamination in contact with the product can be adulterous (or is in violation of the Food & Drug & Cosmetic Act of 1976 as amended). Under the act, anything that is mislabeled or packaged incorrectly is a violation and can be prosecuted by the FDA inby terms of a recall or even prosecution and incarceration.

    Boiled down, when it comes to combination products, it becomes an issue of not only a medical device but also a pharmaceutical item is not manufactured under GMPs. When pharmaceuticals and medical devices join together, each entity brings with it specific guidelines and practices that must be adhered to. These are very unique challenges that can be easily met with the right team of individuals, experience, and resources.
    For additional information on the technologies and products discussed in this article, visit Microtest at

    Steve Richter, Ph.D. is president, scientific director, and founder of Microtest, 104 Gold St., Agawam, MA 01001. At Microtest, he manages the laboratory, testing facility, and manufacturing facility, and is also the technical expert in the microbiology laboratories. Dr. Richter has more than 25 years experience at all levels of microbiology and medical devices, having worked previously at the FDA in Boston. He can be reached at 413-786-1680 or