analysis of degradation properties of biopharmaceutical

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ABOUT THE AUTHORSKathleen Kendrick is Validation Technology Manager at Wyeth Biotech in Andover, MA. Contact Ms. Kendrick regarding this paper at [email protected] or 978.247.1474. Alfredo Canhoto, Ph.D., is Validation Manager, Cell Culture and Utilities at Genzyme Corporation in Allston, MA. Michael Kreuze is Principal Engineer, Wyeth Biotech in Andover, MA.

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P E E R - R E V I E W E D

Analysis of Degradation Properties of Biopharmaceutical Active Ingredients as Caused by Various Process Cleaning Agents and TemperatureKathleen Kendrick, Alfredo Canhoto, and Michael Kreuze

ABSTRACTThe 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 conduct-ing 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 stud-ies 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 clean-ers 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.

INTRODUCTIONThe effects of cleaning agents used during cleaning opera-tions on equipment utilized in the manufacture of protein therapeutic products, including active pharmaceutical ingredients (APIs), are not completely understood. A com-mon 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 tempera-ture and may be time dependent. While this mechanism is not completely characterized, the assumptions and potential implications may affect a manufacturer’s clean-ing program philosophy. For example, should a manufac-turer 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 clean-ing 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 clean-ing 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


emulsification, dispersion, wetting, chelating, or saponi-fication for removal of soils from surfaces. Some of these mechanisms (i.e., sequestering, emulsification, chelating, and saponification) are not immediately relevant to clean-ing 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 clean-ing 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 deter-mine 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 wrap-ping 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 SDS-PAGE an appropriate analytical technique for quantifying the degree of degradation of the studied proteins.

Cleaning process efficacy is achieved through a combina-tion of time, action, chemical/concentration, and tempera-ture (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 bio-pharmaceutical 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 manufactur-ing 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 sav-ings for a company (1).

EXPERIMENTAL DESIGN, MATERIALS/EQUIPMENT, AND PROCEDURES

DesignThe 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 work-ing pH: acidic, neutral, and basic. With a large number of cleaning agents, it was probable that certain generaliza-tions 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 concentra-tions for cleaning proteinaceous soils. For commodity chemicals, such as NaOH, KOH, Cocktail, and phos-phoric acid, the concentrations used for this experimenta-tion 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 repre-sentatives 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 clean-

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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 occur-ring due to the varied conditions to which the protein was introduced.

Materials and EquipmentThe 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 solu-tions 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

0.5N

1.0N

KOH Mallinckrodt 14 0.1N >13

0.5N

CIP92 Ecolab 14 2.0% 10-11

CIP150 Steris 14 1.0% 11-12

CIP1000 Steris ~14 2.5% >13

SoluJet Alconox ~14 2.0% ~12

Cocktail Proprietary Wyeth 14 N/A >13

Foam 140 Steris 14 1.0% ~11

Chematic 82 Dober 14 2.0% >14

Tergazyme Alconox N/A* 1.0% 10

CIP 95NA Ecolab 14 2.0% >13

*The neat concentration of Tergazyme is a powder.

Table II: Neutral (test solution pH 7 ± 2) cleaning chemicals and specifications.Chemical Name Manufacturer pH of Neat Solution Concentration Tested pH of Solution Tested

Chematic99 Dober 10 2.5% ~6

CIP90 Steris 11 2.0% ~6

CosaPur80 Ecolab 9 0.5% ~6

CosaPur84 Ecolab ~9 0.5% 6-7

DA-7645 Steris 10 2.0% ~6.5



• Fixing Solution• Methanol• Acetic Acid• Purified Water

• Coomassie Brilliant Blue Staining Solution• Methanol• Acetic Acid• Purified Water• Coomassie Brilliant Blue R-250 powder

• Destaining solution• Methanol• Acetic Acid• Purified Water.

Experimental ProceduresThe following procedures were used in the studies.

Working Concentration Determination. Dilu-tions of bulk drug substance (BDS) using USP water for injection (WFI) to be studied were prepared in the following concentrations: 1:50, 1:100, 1:200, 1:400, and 1:800. Gels were loaded as shown in Tables V and VI. All tubes of solutions were vortexed directly before loading.

Running Gels. Gels were placed in the gel box and run at 100 mA. A gel was considered complete when the dye front progressed down to just above the bottom of the gel casing.

Staining and Destaining Gels. Once the gel was complete, it was rinsed with WFI. The gel was then immersed in fixing solution and agitated at 50 RPM. The gel was again rinsed with WFI and then immersed in staining solution and agitated at 50 RPM. Finally, the gel was rinsed once again with WFI and immersed in destaining solution and agitated at 50 RPM twice.

Gel Analysis Using a Densitometer. Gels were scanned using “Quantity One” software (version #4.2.2). Data was analyzed by comparing the average optical

Table III: Acidic (test solution pH less than or equal to 5) cleaning chemicals and specifications.Chemical Name Manufacturer pH of Neat Solution Concentration Tested pH of Solution Tested

CosaPur85 Ecolab 3 1.0% 4-5

Foam 240 Steris 1 1.0% <2

CIP 72 Steris 1 2.0% <2

CIP 200 Steris 1-2 2.0% ~1

CIP 220 Steris 1-2 1.5% <2

Phosphoric Acid EMD 1-2 0.33% <2

Chematic91 Dober 1 2.0% <2

Chematic9301 Dober 2 2.0% ~2

Table IV: API information.

Protein Name

Average Concentration of BDS (g/L)

Observed Size (kDa)

Working Concentration

Product A 4 55 1:20

Product B 17 150 1:50

Product C 15 20 1:100

Product D 55 150 1:200

Product E 45 200 1:100

Table V: Working concentration gel #1 loading scheme.Lane Contents

1 38 μL of 1:50 BDS dilution 12μL of loading buffer


3 Blank



6 Blank



9 Blank

10 38 μL of neat BDS dilution 12μL of loading buffer


12 12 μL molecular weight marker 4μL loading buffer


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densities (OD) of the bands in lanes 1-9 to OD for lanes 10 and 11 (control lanes). The difference indicated the percent degradation of protein in a given lane.

Degradation Experiments. The procedure for degradation experiments was executed as described previously except gels for these experiments were loaded as described in Table VII. All other steps for running, staining, and analyzing gels were the same as described previously.

RESULTSThe goal of the degradation experiment was to use SDS-PAGE technology to quantify and qualify the degradative effects of temperature and/or cleaning agents on various APIs. This was accomplished by treating proteins with cleaning agents at concentrations as recommended by the vendor. The treatments that were quantified on an SDS-PAGE gel included the effect heat (temperature) had on the protein, cleaning agent effects on the protein, and the combined effect of the cleaning agent and heat (temperature) on the protein. Each gel also contained samples of the untreated protein as a control as well as protein molecular weight markers. Analysis of the gels resulted in a total of seven different effect categories (see Table VIII).

Figures 1-3 are some examples of the different effects observed on the gels. Lanes 1, 2, and 3 were specifi-cally designed to address the cleaning agent and protein interaction vs. time. Lanes 4, 5, and 6 were specifically designed to explain the interaction between cleaning agent, elevated temperature, and the protein vs. time. Lanes 7, 8, and 9 were specific to the interaction between the protein and heat alone without the influence of a cleaning agent. Lanes 10 and 11 (protein only) are the control lanes. Lane 12 is a molecular weight marker used to determine the approximate molecular weight of the protein.

Degradation Effect Pattern 1—No EffectFigure 1 illustrates the “No Effect” observed by use of 2% Chematic 9301 with Product A BDS 1:20. The overall effect observed with this gel is Pattern 1-No Effect.

From the gel and the corresponding densitometer read-ings, it can be seen that there was no apparent aggrega-tive or degradative effect on the API. Lanes 1-9 remain consistent with the controls in lanes 10 and 11.

Degradation Effect Pattern 4—Heat Alone, Chemical EnhancedFigure 2 illustrates the “Heat Alone, Chemical Enhanced” observed by use of 1% Foam 140 with Product B BDS 1:50.

The overall effect observed with this gel is Pattern 4-Heat Alone, Chemical Enhanced.

The gel and the densitometry data show that lanes 1-3 didn’t affect the API in comparison to the controls in lanes 10 and 11. Heat only (lanes 7-9) aids in degradation of the protein; however, the degradation is enhanced with the addition of chemical (lanes 4-6). Increased temperature alone will work but adding chemical will enhance the effect.

Degradation Effect Pattern 5—Heat Effect, Chemical StabilizedFigure 3 illustrates the “Heat Effect, Chemical Stabi-lized” observed by use of 2% CIP 200 with Product B BDS 1:50. The overall effect observed with this gel is Pattern 5-Heat Effect, Chemical Stabilized.

In this gel and in the densitometry reading, it was observed that when the solution is at ambient tem-peratures (lanes 1-3) there is no apparent degradation. When the temperature is increased to 60°C (lanes 7-9) a significant amount of degradation is seen. Lanes 4-6 are also exhibiting an effect but at a slower rate than lanes 7-9 and the effect appears to have been stabilized.

Summary of EffectsTable IX summarizes effects for the five biopharma-ceutical proteins tested in this study.

Of the 139 total gel effects that were observed, Chemi-cal Alone, Heat Enhanced (24%) and Only Chemical and Heat (27%) were the two categories observed most often. When only focusing on the basic and neutral cleaning

Table VI: Working concentration gel #2 loading scheme.Lane Contents



3 Blank



6 Blank



9 Blank

10 12 μL molecular weight marker 4μL loading buffer

11 Blank

12 Blank



agents, the same two categories were observed most often. However, when acidic cleaning agents were analyzed the top two effects that were observed were Heat Effect, Chemi-cal Stabilizing (35%) and No Effect (30%).

DISCUSSIONThe preceding experiments explored the degradation characteristics of representative APIs in the presence of various cleaning agents and temperatures. The deg-radation effect in most of the cases increased with an increase in temperature and time of exposure. Some cleaning agents had no degradation effect on protein APIs alone. However, when combined with increased temperature, they had an effect on either protein stabil-ity or aggregation. These findings and the novel use of standard biochemical tools and techniques have several implications for the biopharmaceutical manufacturing industry specific to cleaning regimes and associated cleaning validation efforts.

The degradation experiments were initially expected to fall into one of four categories: no effect; clean-ing agent (chemical) alone induced effect; heat alone induced effect; or combination of cleaning agent (chemical) and heat induced effect. Examination of the empirical results revealed that there were more subtleties in the resulting effects. The original effects needed to be further subdivided to acknowledge subtle-ties such as: chemical alone heat enhanced; heat alone chemical enhanced; chemical and heat synergy; only

chemical and heat effect; heat effect chemical stabiliz-ing; and chemical effect heat stabilizing.

The results indicate that for basic and neutral chemicals, the majority of the effects were primarily chemical with a time dependent heat enhancement or synergy. Con-versely, for the acidic chemicals, the primary effect was heat induced with a secondary chemical effect of either causing some degradation or aggregation. This portion of the data matches with the common perception that basic chemicals such as NaOH and KOH are the primary cleaning agents for proteinaceous soils. This generaliza-tion cannot be extended to all formulated caustic or basic cleaning agents as not all combinations of cleaning chem-istry and protein displayed degradation of the proteins tested. There should be an attempt made by organiza-tions involved in cleaning development and validation to understand the effect of their chosen cleaning agent and regimen on their API or residual process soils. This knowledge is fundamental in justifying cleaning agent chemistries, concentrations, times, and temperatures chosen in validating a robust cleaning cycle.

Furthermore, by understanding the strength and effects of cleaning cycle parameters, the risks associated with the changeover of equipment from one manufacturing process or API to the next can be mitigated. Likewise, residual drug substance carryover concerns and testing should be viewed in light of the effect of the cleaning agent on the process soils and API. If a protein is clearly degraded with a chosen cleaning chemistry, concentration, time and temperature, then the use of product specific assays to demonstrate residual removal are not appropriate markers of cleanliness. A non-specific assay such as total organic carbon (TOC) is more applicable and appropriate to ensure the removal of organic APIs or proteins that, with the use of the above-described techniques, have been demonstrated to be degraded.

The benefits of this work can be illustrated in a multi-product production services area. By having these degrada-tion studies complete for multiple soils, an argument can be made that small equipment parts may be cleaned together in a single load of a washer, assuming that the washer has been preprogrammed to deliver cleaning parameters that meet the threshold necessary for all individual soils con-tained within the load. This understanding could justify the necessary wash and rinse times and temperatures of a cleaning cycle.

Fundamentally this set of experiments sought to address the axiom of “Know thy Process.” Because the cleaning pro-cess is so integral to the biopharmaceutical manufacturing industry, many resources and time are utilized to qualify a cleaning procedure. By performing studies to understand

Table VII: Loading scheme and experimental sample preparation for degradation studies.Lane Contents

Protein (working concen-tration)

Chemical (working concen-tration)

Time (minutes) Temperature

Loading Buffer

1 19ml 19ml 5 Ambient 12ml



4 19ml 19ml 5 60oC 12ml

5 19ml 19ml 30 60oC 12ml

6 19ml 19ml 60 60oC 12ml

7 38ml N/A 5 60oC 12ml

8 38ml N/A 30 60oC 12ml

9 38ml N/A 60 60oC 12ml

10 38ml N/A N/A Ambient 12ml

11 38ml N/A N/A Ambient 12ml

12 Molecular weight standard (12ml) and 4ml loading buffer


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the effects of cleaning chemicals on process soils, compa-nies can design a cleaning cycle that is more efficient and increase the level of compliance with regulatory expecta-tions. As required by risk assessment tools such as hazard analysis and critical control points (HACCP) and failure mode and effects analysis (FMEA), there is an increased emphasis on scientific fundamentals and reducing risk by characterizing process soils and their interactions with cleaning agents and parameters that may be necessary to address the same. In addition, acute process understand-ing, done in a proactive manner outside the manufacturing facility, may lead to cost savings.

CONCLUSIONSThe application of SDS-PAGE to investigate and charac-terize the effects of cleaning processes on APIs provides a powerful tool in support of cleaning process development and validation. Gel electrophoresis indicates degradation (or aggregation) products due to contact with cleaning solution and/or elevated temperature.

Regarding what these techniques demonstrated for actual effects of cleaning agents on API degradation, con-clusions are made categorically as follows:

• Most but not all caustic cleaning agents degrade protein therapeutics. This study showed that when cleaning

Table VIII: Gel effect categories.Classification # Name of Effect Description

None

1 No Effect No effect from any independent or combined tem-perature or chemical interaction

Primarily Chemical

2 Chemical Alone, Heat Enhanced An increased effect when chemical and temperature were combined, but no effect with temperature alone

3 Chemical Effect and Heat Stabilizing Chemical had an effect, but with the addition of heat the strength of the effect decreased (aggregation and/or polymerization fall into this category)

Primarily Heat

4 Heat Alone, Chemical Enhanced An increased effect when heat and chemical were combined, but no effect with chemical alone

5 Heat Effect and Chemical Stabilizing Heat had an effect, but with the addition of chemi-cal the strength of the effect decreased (aggregation and/or polymerization fall into this category)

Combined Chemical and Heat

6 Only Chemical and Heat Effect No effects for heat alone or chemical alone, only an effect when chemical and heat were combined

7 Chemical and Heat Synergy Chemical had an effect and heat had an effect, but combined there was a stronger effect (aggregation and/or polymerization fall into this category)

Table IX: Overall effects observed for the five biopharmaceutical protein therapeutics tested.

Effect Total %Base/Neutrals % Acids %

No Effect 22 16 10 10 12 30

Chemical Effect

Chemical Alone, Heat Enhanced

34 24 34 33 0 0

Chemical Effect, Heat Stabilizing

4 3 4 5 0 0

Heat Effect

Heat Alone, Chem-ical Enhanced

8 6 6 6 2 5

Heat Effect, Chemical Stabiliz-ing

18 13 4 5 14 35

Combined Effect

Only Chemical and Heat

37 27 28 28 9 23

Chemical and Heat Synergy

16 12 13 13 3 7

Totals 139 99 40



agents were effective at degrading API, they were both time and concentration dependent.

• The assumption that all high temperature-cleaning cycles cause a break down of protein therapeutics has also been refuted. Furthermore, elevated temperatures combined with caustic cleaning agents were shown to be the most effective combination; however, this generalization was not true for all situations.

• With respect to the acidic cleaning agents, in general they did not exhibit a high degree of degradation. When degradation was observed, higher temperature enhanced the cleaning effect.

• The industry-wide assumption that heat and caustic

cleaning together are the most effective combination was confirmed.

It is also important to note that aggregation was observed in many of the SDS-PAGE results. This aggregation was observed with both acidic and basic cleaning agents. It has been demonstrated that aggregation is affected by external factors including temperature, pH, and protein concentration, all of which were factors in this study (2). For this reason, more testing should be done to determine the cause of aggregation in the select gels where it was observed.

1 2 3 4 5 6 7 8 9 10 11 12

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Figure 1: 2% Chematic 9301 with PRODUCT A BDS (1:20).

Lane Number Lane Contents Densitometer Reading

1 Protein and Solution, 5 min, Ambient 90.32



4 Protein and Solution, 5 min, 60°C 90.32



7 Protein, 5 min, 60°C 83.87

8 Protein, 30 min, 60°C 77.42

9 Protein, 60 min, 60°C 87.10

10 Protein Control 93.55


12 Protein Markers N/A

1 2 3 4 5 6 7 8 9 10 11 12

kDa174

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Figure 2: 1% Foam 140 vs. PRODUCT B BDS (1:50).








7 Protein, 5 min, 60°C 64.71

8 Protein, 30 min, 60°C 35.29

9 Protein, 60 min, 60°C 41.18




The overall effect observed with this gel is Pattern 1- No Effect.

The overall effect observed with this gel is Pattern 4- Heat Alone, Chemical Enhanced.


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ACKNOWLEDGMENTSThe authors thank Richard Wright and Priscilla Jennings for their technical assistance and numerous discussions throughout the project. In addition, thanks to all involved with long hours of degradation runs, data entry, review, and technical assistance: Meghan Pearson, David Hindson, Rod Azadan, Kristen Nobles, James Snow, Jennifer Strand, Stanley Garib, Kate Crotty, Kelli Barrett, Linda Marshall, Debra Pierzynski, and Brandon Sullivan

REFERENCES1. FDA, Guide to Inspections, Validation of Cleaning Processes,

1993.2. W. Wang, “Protein Aggregation and its Inhibition in Biophar-

maceutics,” International Journal of Pharmaceutics, 289, 1-30, 2005. JVT

GENERAL REFERENCESBailey, Rochelle, “Effect of 0.1N NaOH on rAHF Activity and

Structural Integrity,” Genetics Institute, 1991. Bowser, Tim, “CIP Systems Critical to Validatable, Cost-Effective

Cleaning,” Cleanroom, Volume 21, No.02, February 2007.Dawn Chemical, Inc. Website: www.dawnchemical.com/shop/

scripts/page.asp?p=_chemistryofcleaning&s=dawnchemical, viewed on August 3, 2006.

Tsai, A.M., van Zanten, J.H., Betenbaugh, M.J., “Study of Pro-tein Aggregation Due to Heat Denaturation: A Structural Approach Using Circular Dichroism Spectroscopy, Nuclear Magnetic Resonance, and Static Light Scattering,” Biotechnol-ogy and Bioengineering, 59 no.3, 273-280 (1998).

Lemmer, K., Mielke, M., Pauli, G., Beekes, M., “Decontamina-tion of surgical instruments from prion proteins: in vitro studies on the detachment, destabilization and degradation of PrPSc bound to steel surfaces,” Journal of General Virology, 85, 3805-3816 (2004).

Cabra, V., Arreguin, R., Vazquez-Duhalt, R., Farres, A., “Effect of temperature and pH on the secondary structure and pro-cesses of oligomerization of 19kDa alpha-zein,” Biochimica et Biophysica Acta, 1764, 1110-1118 (2006).

ARTICLE ACRONYM LISTINGAPIs Active Pharmaceutical IngredientsBDS Bulk Drug SubstanceEDTA Ethylenediamineteracetic Acid Disodium SaltFMEA Failure Mode and Effects AnalysisHACCP Hazard Analysis and Critical Control PointsOD Optical DensitiesSDS Sodium Dodecyl SulfateSDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel

ElectrophoresisTACT Time, Action, Chemical/Concentration, and

TemperatureTOC Total Organic CarbonWFI Water for Injection

Figure 3: 2% CIP 200 with PRODUCT B BDS (1:50).








7 Protein, 5 min, 60°C 78.57

8 Protein, 30 min, 60°C 35.71

9 Protein, 60 min, 60°C 21.43




The overall effect observed with this gel is Pattern 5- Heat Effect, Chemical Stabilized.

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