October 15, 2014 (Vol. 34, No. 18)

Performing Critical Cleaning and Validation Studies on a Prospective Basis

Contamination of a biologic intended for human use can be traced back to the turn of the last century, when untested diphtheria antitoxin isolated from the serum of horses contained tetanus and led to the deaths of more than a dozen children in 1901. This, and subsequent contaminations, heralded the arrival of good manufacturing practices (GMPs) in 1906, and later, cell culture technology began a new chapter in controlling contamination.

Today contamination is considered rare. Of more than 15,000 lot-release results from in vitro virus adventitious assays performed at BioReliance, only eight were positive (0.05%). Yet on the rare occasions that viral contamination occurs, it makes headlines and can be detrimental to finances and production timelines, causing facility shutdowns for cleaning and validation, and lost product due to contamination. In some cases, product shortages result, impacting patients.

Recent high-profile contamination issues have also put greater focus on cleaning and disinfection validation studies on a prospective basis—before a problem occurs, not just in response to an event. Regulators expect manufacturers to conduct cleaning validation studies in addition to risk assessments, and regardless of history, contamination control procedures should exist. Prevention is key.

Some of the most common prevention methods—cell and virus characterization, raw materials testing, clearance studies, and lot-release testing—are discussed in this article. 

Cell and Virus Characterization

Regulatory requirements for cell and virus characterization have three major components to establish identity, purity, and suitability for use:

  1. Source, history, and generation of material
  2. Cell-banking procedures
  3. Characterization and testing

Identity testing may be based upon either phenotypic or genotypic methods. Purity testing is performed at various points for bacterial, fungal, mycoplasma, and viral contaminants that might have been introduced during the cell-line generation, banking, and/or manufacturing processes. In addition, the cell substrate must be evaluated for the presence of latent and/or endogenous viruses (or viral nucleic acid sequence).

Concerns about the stability and tumorigenicity potential of continuous cell lines, especially new diploid and previously uncharacterized cell substrates, or for products not subjected to extensive purification (e.g., live virus vaccines), will necessitate extensive evaluation for licensure of the product. All of the characterization data provides a basis for risk-benefit decisions for biological product licensure.

Raw Materials Testing

Each lot of raw materials used in a product’s manufacture must be control tested to assure safety and traceability, and adherence to GMPs. Any cross-contamination potential, including sanitary measures used in producing and storing the raw material, must be evaluated.

Whenever animal-derived material is used in development or manufacturing, adventitious agent contamination must be considered (Figure 1). Many cell-culture systems require additives derived from bovine or porcine origin such as fetal bovine or newborn calf sera, insulin, transferrin, various growth factors, and trypsin. Testing by in vitro assays for the presence of bovine and porcine viruses is required and described in Title 9 of the Code of Federal Regulations (Figure 2).

Implementation of viral reduction steps for culture medium or other raw materials provides an additional assurance of safety. The level of potential viral reduction is verified by viral clearance studies. 

Figure 1. Biological therapeutics are derived from a number of biological sources, and each source brings a unique risk of viral contamination.

Clearance Studies

Because of sampling volume limitations and the inability to detect low levels of a contaminant by direct testing, the evaluation of a downstream purification process for virus reduction is essential to ensuring a product’s safety. Viral (or any agent) clearance evaluation is performed by scaling down the purification steps that may potentially contribute to the clearance and adding a virus to the starting material for each step (virus “spiking”).

The spiked starting material is then taken through the purification procedure. By quantifying the amount of virus in the product pre- and post-purification, the amount of virus that can be cleared by this step can be quantified.

A study must be designed using relevant model virus/agents such that meaningful data is obtained. The selection of the production steps, scaledown of the production steps, evaluation of column sanitization and reuse, cytotoxicity and interference testing, and validated and accurate endpoint assays are all critical in a clearance study.

When it comes to cleaning, disinfection-efficacy studies involve replication of the surface disinfection procedure at small scale to verify the clearance of spiked infectious agents. An infectious agent (e.g., bacteria, spore, mycoplasma, virus, etc.) is dried onto a small coupon of representative surface.

The disinfectant is applied to the coupon, mimicking the procedure used in the facility, and any remaining infectious agent is recovered and quantitated using an infectivity assay. The sequence of steps when designing a disinfection-efficacy study is detailed in Figure 3.

Figure 2. Schematic of in vitro adventitious virus assay used for screening biological raw materials. The test article is inoculated onto several cell lines, and various endpoint assays are used to detect any potential viral contaminant.

Lot-Release Testing

Pure source materials are important; however, contaminants may enter a biological manufacturing process through “portals of entry” that include cell substrates, raw materials, equipment, facilities, and/or operator. In addition, low levels of contaminants may not be detected during initial screening, but become evident during the bioproduction process.

Every lot of product, both for preclinical and clinical studies, requires a series of tests on both unprocessed and purified bulk material. Testing on unprocessed bulk includes sterility, mycoplasma, and virus testing. Purified bulk material may need testing for sterility, residual DNA, and host cell proteins.

History, both past and recent, shows that one cannot afford to be complacent. New technologies, new cell substrates, and new biologics will present new challenges. It is no longer a question of if but when a contamination event will occur, and manufacturers need to prepare accordingly.

The importance of, and approaches to, prevention are detailed and required by regulatory entities worldwide. Although replacing raw materials of animal origin with nonanimal-derived sources will remove some of the potential points of viral entry, it is naive to believe replacing serum will avoid all forms of contamination.

Rather, it is important to test for contamination at every point of the process, including the final product before lot release, and to validate cleaning processes to ensure product safety

Figure 3. Overview of disinfectant efficacy study steps: (A) obtaining coupons to represent disinfected surfaces; (B) drying challenge agent onto the coupon surface (with or without representative “soil”); (C) accurately mimicking the cleaning procedure using worst-case processing conditions; (D) recovery of any residual challenge agent.

Kathryn Martin Remington, Ph.D. ([email protected]), is principal scientist and Audrey Chang, Ph.D., serves as executive director, global development services, at BioReliance.

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