USP <88> Class VI BIOLOGICAL REACTIVITY TESTING– IS IT ENOUGH TO STOP THERE?

A White Paper

Dan Klees

Industry Manager, Pharmaceutical/Biotechnology

Endress+Hauser, Inc.

Greenwood, Indiana

December 10, 2003

ABSTRACT: Test protocols for biological reactivity testing are well defined by the U.S. Pharmacopeia, General Chapters 87 and 88. These test protocols involve invitro reactivity testing with mammalian cells and in vivo reactivity testing in animals, respectively. However, many Biopharmaceutical companies requireonly a Certificate of Compliance (COC) from equipment manufacturers that states the plastics, elastomers (or any other material) potentially in contact with the biopharmaceutical process meet U.S. Pharmacopeia Class VI Testing. It is the opinion of the Author that U.S. Pharmacopeia Testing per Class VI (one part of USP <88> testing), is not alone sufficient in determining the biological reactivity to that material. Additionally, the materials should be tested in accordance with USP <87>, in vitro testing with mammalian cells, to better determine potential reactivitythat may decrease cell growth and, therefore, limit profit per batch.

Biologics, also known as biological therapeutic products, consist generally of chemical compounds such as viruses, proteins, blood products or fractions, vaccines or gene transfer products. The common thread is that all biologics are derived from living cells, microbes or organisms.

To producemost biologics, cells or microbes are first cultivated in bioreactors or fermentors, respectively, in up-stream processing. The desired biological therapeutic compound is then recovered, separated and purified in down-stream processes. The growth or cultivation of cells and microbes takes place under controlled conditions; this process must be repeatable.

Biotech companies invest much effort and money into controlling of process. It is essential to recognize that the product is the process and “…the fundamental belief that to produce a safe, consistent biological product, the product is inextricably linked to the process…”.1 Control of the process must incorporate all aspects and requirements of the cGMP’s (Current Good Manufacturing Practice) to ensure regulatory compliance. This includes the necessary controlsand validation to ensure that the biologics have the safety, purity, identity, and efficacythat they purport to have.

The cGMP states “Equipment shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of a drug product beyond the official or other established requirements”.2 It should be noted that cGMP’s do not address or require maximizations of yields–only the calculation of theoretical yields! The maximization of yields is a necessary financial requirement in the BiotechIndustry and not a regulatory requirement.

Biotechnology companies obviously invest much in maximizing the yield of the desired biologics. The cell bank must be viable; the environment must be void of unwanted cells, organisms or chemical residue; the nutrition must be appropriate; the environment must promote growth for the cells or microbes, etc. Any conditions or procedures that stifle the required growth rate of the cells or microbes willlessen the yield of the desired therapeutic compound and therefore limit the profitability of that batch. In the up-stream processing, the industry takes great care in managing the cell master stock, cleaning and sterilizing the equipment, controlling the nutrition feeds and gasses, and promoting an environment which will maximize the desired cell or microbial growth. In down-stream processing, the industry again optimizes recovery, separation and purification of the biological compounds to maximize yields and profitability.

In addition, biological reactivity testing has been performed to further increase yield. It is obvious that any material that reduces cell or microbial growth will affect yield.

1Baseline Guide for Biopharmaceutical Facilities, International Society for Pharmaceutical Engineering

2Code of Federal Regulations, 21CFR 211.65(a)

One such biological reactivity test applicable to and often requested by the Biotech Industry is the testing of elastomers, plastics, polymeric materials and their extracts as described in the U.S. Pharmacopeia General Chapter <88>. This in vivo testing consists of three (3) tests: Systemic, Intracutaneous, and Implantation. The materials and their extracts are then classified according to the test results as meeting Plastics Class I through Class VI. A summary of the tests are as follows:

The above Class Testing of elastomers, plastics, polymeric materials and their extracts is performedin contact with animals. However, it is not enough to stop after completing only this test – the growth of cells and microbes can be retarded or completely stopped by chemicals that may pass Class VI testing. A case in point is sulfur-cured elastomers.

Some elastomers, namely rubbers, have been cured, historically, by using sulfur and/or sulfur containing compounds. Sulfur is listed as a GRAS (Generally Recognized as Safe) material, classified as an indirect food additive, intended for repeated use, when used as a vulcanization material or agent.3 Sulfur-cured elastomers therefore meet FDA GRAS requirements and also can pass USP<88> Class VI Testingyet the elastomer can negatively affect mammalian cell growth!4

The biological reactivity test of mammalian cells in vitro is described in the U.S. Pharmacopeia General Chapter <87>. “This test is designed to determine the biological reactivity of mammalian cell cultures following contact with elastomeric plastics and other polymeric materials with direct or indirect patient contact or of specific extracts prepared from materials under test.”5 The test protocol includes Agar Diffusion Tests, Direct Contact Tests and Elution Tests of the materials or extracts in determining the reactivity of mammalian cells.

When tested in accordance with USP <87>,most sulfur-cured elastomers will have a detrimental affect on mammalian cells and therefore will have a slight to severe reactivity grade. If a sulfur-cured elastomer were to be used in a biotech mammalian cell cultivation process, the yield of cells could be drastically reduced or even completely stopped in smaller batches – even though the elastomer may pass USP <88> Class VI testing and be a FDA listed GRAS material!

Summary

The Biotech Industry has been requesting Class VI testing from instrument and component manufacturers. However, the Class VI designation applies only to USP <88> biological reactivity testing, in vivo. If the industry requests biological reactivity testing in accordance with USP <87, in vitro, as well as USP <88>, in vivowith results designated by Class I through VI, cell or microbial culture growth can be protected thereby maximizingbiologics batch yields and profitability.

About the Author

Dan Klees is the Pharmaceutical/Biotechnology Industries Manager at Endress+Hauser in Greenwood, Indiana. He received his Bachelor of Science and Master of Science Degrees in Physics from MiamiUniversity. Dan Klees has 30 years of experience in the Regulated Industries, having held management positions in sales and marketing with Rosemount and Foxboro as well as executive management positions with integration and engineering firms TriCore and RES/TAVA Technologies. He is a member of ISPE and is a member of the ASME/BPE Standards Group Sub-Committee. Dan has published several automation and advanced control algorithms articles in leading technical journals. Dan Klees can be contacted at Endress+Hauser (317) 535-2715 or via e-mail

3Code of Federal Regulations, 21CFR 177.2660 (ii) Vulcanization Materials – (a) Vulcanizing agents

4Rubber Fab Technology Group,

5The United States Pharmacopeia Convention, Inc, USP 26, copywrite 2003