Analysis of Degradation Properties of Biopharmaceutical Active Ingredients as Caused by Various Process Cleaning Agents and Temperature

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

The biotechnology industry assumption in the cleaning of product contact equipment is that both acidic and caustic solutions and high temperatures effectively degrade residual active pharmaceutical ingredients. The necessity of conducting research to qualify such assumptions is relevant when considering the cleaning of equipment between batches of the same product or cleaning for product changeover within a manufacturing suite. The focus of this study was to examine the claim that “typical” cleaning regimens found in the biopharmaceutical industry effectively degrade residual proteins from an equipment surface. Degradation studies seek to understand the breakdown effect a cleaning agent and/or heat has on particular protein therapeutics. By utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis technology to quantify the degradation effects on protein therapeutics, it was discovered that all caustic cleaning agents did not completely degrade the protein. While most basic and neutral pH cleaning solutions did have a degradation effect on protein therapeutics, most acidic cleaners did not. The small percentage of acidic cleaners that successfully degraded protein therapeutics required the presence of elevated temperature. Through analysis of degradation data, it was found that the combination of heat and a caustic cleaning agent together yielded the most degradation of a protein and was most effective. INTRODUCTION The effects of cleaning agents used during cleaning operations on equipment utilized in the manufacture of protein therapeutic products, including active pharmaceutical ingredients (APIs), are not completely understood. A common perception in the biopharmaceutical industry is that caustic cleaning agents degrade (or break down) protein therapeutics, and that acidic, neutral, or enzymatic cleaners may or may not have similar effects. Moreover, a similar degradation effect may be observed with a high temperature and may be time dependent. While this mechanism is not completely characterized, the assumptions and potential implications may affect a manufacturer’s cleaning program philosophy. For example, should a manufacturer test for API residuals using a product-specific assay when the cleaning agent breaks it down? Furthermore, a strong science and risk-based approach to cleaning should define cleaning conditions, such as concentration, time, and temperature based upon a fully characterized cleaning process. The purpose of this report is to answer these questions by presenting the results of a study that examined and ultimately defined the degradation effects of cleaning solutions, temperature, and exposure duration on biopharmaceutical active ingredients. In addition to degradation, the mechanism investigated in this report, a cleaning agent may employ sequestering, Journal of Validation technology [Summer 2009] 69 P E E R R E V I E W E D emulsification, dispersion, wetting, chelating, or saponification for removal of soils from surfaces. Some of these mechanisms (i.e., sequestering, emulsification, chelating, and saponification) are not immediately relevant to cleaning API. Moreover, mechanisms such as dispersion and wetting are expected to be a precursor to the degradation effect studied in this report. Degradation is, therefore, an appropriate tool to study and characterize common cleaning regimes in an API manufacturing environment. In order to characterize the degradation effects, a novel application of existing technologies was developed. The technique employed the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether the API degraded into smaller fragments of the original protein therapeutic. SDS-PAGE separates proteins solely by their size. Sodium dodecyl sulfate (SDS) is an anionic detergent, which micellizes proteins by wrapping around or binding the polypeptide backbone. In so doing, SDS confers a net negative charge to the polypeptide in proportion to its length; the denatured polypeptides become rods of negative charge with equal charge or charge densities per unit length. In addition, SDS-PAGE also separates proteins independently of pH and subsequent amphoteric changes. These characteristics render SDSPAGE an appropriate analytical technique for quantifying the degree of degradation of the studied proteins. Cleaning process efficacy is achieved through a combination of time, action, chemical/concentration, and temperature (TACT). This study did not evaluate the effect of action, because it is directly related to how process equipment is cleaned (i.e., static soak versus spray device). Because this is a laboratory study not specific to any particular piece of equipment, the “action” contribution was not examined. However, the remaining factors were studied at length, applying experimental values typically found in a biopharmaceutical manufacturing setting. A number of cleaning agents and APIs were examined in order to determine if the effects and results observed were applicable categorically or simply specific to a certain entity. Cleaning chemicals were divided into caustics, acids, and neutrals including enzymatic cleaners in order to determine if there were common effects related to a particular pH range. The materials of construction chosen represent the predominant equipment surface materials typically found in bulk biopharmaceutical manufacturing processes. Refer to the “Experimental Materials and Design” section for more detail on experimental variables and study design. This study investigated the effects of time, chemical, and temperature on therapeutic protein degradation rate in order to gain additional information on the independent contribution of the variables to the efficacy of a cleaning cycle. Analyzing the degradation characteristics of each cleaning agent would be expected to aid in answering questions such as: Which cleaning agent works best for APIs? Do all basic and neutral cleaning agents degrade APIs? Do all acidic cleaning agents degrade APIs? How long does it take for the protein to degrade? Answering questions such as these will result in more effective and efficient cleaning procedures and could result in cost savings for a company (1). EXPERIMENTAL DESIGN, MATERIALS/ EQUIPMENT, AND PROCEDURES Design The following components were utilized in the design of the studies. Cleaning Agents. An array of cleaning agents was selected for these studies. The cleaning agents selected were divided up into three categories based on their working pH: acidic, neutral, and basic. With a large number of cleaning agents, it was probable that certain generalizations could be established representative of each of these three categories. Tables I, II, and III list each cleaning agent within its specific category as well as the name of the manufacturer, pH of the solution (neat and actual tested), and the concentration(s) tested. The cleaning agent concentrations were determined based on the manufacturer’s recommended concentrations for cleaning proteinaceous soils. For commodity chemicals, such as NaOH, KOH, Cocktail, and phosphoric acid, the concentrations used for this experimentation are those typically used in cleaning cycles. The pH values of the neat and diluted cleaning solutions were measured using pH strips. API Soils. Five representative API soils were selected to use during these experiments (see Table IV). The soils selected were chosen based on availability and as representatives of typical soils manufactured at Wyeth Biotech, Andover, MA. Degradation experiments were carried out for each API in combination with each cleaning agent. The gels were set up in a manner to test multiple variables at once. Each gel tested for the effect of cleaning agent alone, heat (temperature) alone, and the combination of heat (temperature) and cleaning agent. Within each of these testing criteria contact time was varied to determine if there was time dependency. Time intervals of 6, 30, and 60 minutes were tested to determine its influence. The maximum of 60 minutes was chosen, as this was anticipated to be an effective duration for the clean70 Journal of Validation technology [Summer 2009] i v t home.com gxpand jv t .com K A T H L E E N K E N DR I C K , A L F R E D O C A N HO T O, A N D M I C H A E L K R E U Z E ing agent exposure portion for a cleaning cycle. To aid in the comparison of the results were two lanes of controls. The controls were the same dilution as the experimental lanes and added to the gel just prior to execution. The final lane was filled with a molecular marker. This marker aided in determining the amount of effect (degradation or aggregation) that was occurring due to the varied conditions to which the protein was introduced. Materials and Equipment The following materials and equipment were utilized in the studies: • Gels—ClearPAGE 4-20% gradient Cat# FK42012 • Gel box—C.B.S. Scientific Co model DCX-700 • Power supply—VWR model EPS 4000 • Orbital shaker—VWR model DS-500 • Coomassie Brilliant Blue • Molecular weight markers—Lonza ProSieve Color Protein Markers Cat #50550 (173,117,76,51,38,26, 8,12,and 9 kDa). SDS-PAGE Solutions. The following SDS-Page solutions were used: • Loading Buffer (4X Solution) • Sodium Dodecyl Sulfate (SDS) • Ethylenediamineteracetic Acid Disodium Salt (EDTA) • Tris pH 6.8 • Glycerol • Purified Water • Running Buffer (10X Solution) • Tris Base • Glycine • SDS • Purified Water Table I: Basic (test solution pH greater than 9) cleaning chemicals and specifications. Chemical Name Manufacturer pH of Neat Solution Concentration Tested pH of Solution Tested CIP100 Steris ~14 2.0% >13 NaOH Mallinckrodt 14 0.1N >12