Total Organic Carbon (VCSN and VWP) and HPLC Analysis for ...TECHNOLOGY/APPLICATION Total Organic...

42-5763, 2009 PDA J Pharm Sci and Tech M. Queralt, E. García-Montoya, P. Pérez-Lozano, et al. Cleaning Validation in a Pharmaceutical Pilot PlantTotal Organic Carbon (VCSN and VWP) and HPLC Analysis for

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TECHNOLOGY/APPLICATION

Total Organic Carbon (VCSN and VWP) and HPLC Analysisfor Cleaning Validation in a Pharmaceutical Pilot Plant

M. QUERALT,1 E. GARCIA-MONTOYA,2 P. PEREZ-LOZANO,2 J. M. SUNE-NEGRE,2 M. MINARRO,2

and J. R. TICO2

1Post-graduate student, Pharmacy Department, University of Barcelona; 2Professor, Pharmacy Department, Faculty ofPharmacy, University of Barcelona © PDA, Inc. 2009

ABSTRACT: This paper presents a useful method using total organic carbon analyzers employing both combustionand wet oxidation for validating equipment cleaning procedures and verifying cleaning in a pharmaceutical pilotplant. The results are compared with those obtained using high-performance liquid chromatography. The studysummarizes the initial steps that should be taken into account and focuses particularly on the solutions to some of themost critical considerations (e.g., glass material, detection and quantification limits, recovery). Also described are thecalculation of control limits and the good results obtained.

KEYWORDS: Cleaning validation, Total organic carbon, Pilot plant, GMP.

1. Introduction

Cleaning is one of the critical processes in pharma-ceutical manufacturing. Equipment contaminationmay come from any of the materials that have been incontact with the equipment surfaces. It is critical toavoid carryover of trace amounts of either active orother materials from one batch to another in order toavoid cross-contamination of the subsequent product.For that reason, equipment used in pharmaceuticalmanufacturing must be cleaned meticulously (1, 2),and the cleaning procedure used must be validated(3–5). In the specific case of a pharmaceutical pilotplant (6 – 8), the fulfilment of current good manufac-turing practices (cGMPs) can be difficult, as the pro-cesses carried out in the plant will differ considerablyfrom those used in industrial production. In a pilotplant active product ingredient (API) differs for eachproject, a combination of different pieces of pilotequipment is used for the same dosage form, theequipment uses variable loads for the equipment, theformulation of a single product still in the develop-ment phase may change, complex samples may exist

for which no specific analytical method is yet avail-able, and speed and efficiency are necessary in thedevelopment of new formulations for new products.So, it seems useful to do a cleaning verification priorto work as it ensures a degree of safety even if thecleaning method has been previously validated.

Besides using officially recognized analytical methodsin the cleaning, validation studies (9) are of particularimportance in pilot plants due to the specific workingconditions and risks presented.

Total organic carbon (TOC) is suitable for use in thevalidation of cleaning procedures applied in pilotplants but also can be used to demonstrate the efficacyof the cleaning procedure just before equipment oper-ation. TOC analysis is a non-specific method for an-alytical assessment in the validation of cleaning pro-cedures in the pharmaceutical industry. Table I showsa summary (10) of standard analytical methods andoffers a comparison of the different methods based onthree parameters: specificity, sensitivity, and cost.

One advantage of TOC over these traditional analyti-cal methods is its larger spectrum, since it can detectany residue containing organic carbon (this is ideal fordetecting residues following pharmaceutical cleaningprocesses). In addition, TOC analysis is a very sensi-tive method that can detect analytes in parts per billion

Corresponding author: Dra. Encarna Garcıa-Montoya,Faculty of Pharmacy, University of Barcelona, Avda.Joan XXIII s/n. 08028 Barcelona. Tel. 0034 93 403 4712. [email protected]

42 PDA Journal of Pharmaceutical Science and Technology



(11–13). One of the first studies to apply TOC analysisto cleaning validation was published in 1990 by Baffiet al. (14). This study is notable for its validation ofthe TOC method (the authors verified linearity, accu-racy, recovery, and the precision of measurements ofthe possible contaminants) and for determining a de-tection limit of 0.1 ppm and a quantification limit of0.5 ppm, which were required to demonstrate theabsence of trace levels of biopharmaceutical residues.Many subsequent studies have demonstrated the broadapplication of this analytical technique to pharmaceu-tical validation (10, 15–18).

On the other hand, TOC is a rapid technique that canbe used to analyze and control the equipment prior tooperation, thus ensuring no cross-contamination be-tween one product and the next (19). Due to thecomplexity of the formulations used in a pilot plant, itis preferable to analyze the residues present aftercleaning as a whole, rather than attempting to identifysingle components (12). TOC analysis can help todetect residues that are not identified by high-perfor-mance liquid chromatography (HPLC) (detergents,

cross-contamination, etc.). This paper describes thepreparatory phases of the study and presents the val-idation results for a piece of equipment in a pilot plant,establishing the required specification of TOC loadprior to operation.

1.1. Characteristics of the Equipment Used in theStudy

The present study used Shimadzu TOC analyzers,models TOC-VCSN and TOC-VWP, both of whichwere employed in the analysis of liquid samples (Ta-ble II lists the manufacturer’s specifications).

1) TOC-VCSN: combustion catalytic oxidation/NDIRmethod

This analyzer uses high-temperature combustion (cat-alytically aided platinum at 680 °C) as the oxidationmethod (20). Figure 1 illustrates how the device de-termines the presence of three possible types of typesof carbon (C).

TABLE IGeneral Reference: Analytical Methods in Cleaning Validation (10)

Method Specificity

Able to Detect Cost

API ExcipientsCleaning

agents

Waste/Biopharmaceuticals

API*: LOW

*****: HIGH

HPLC yes yes yes yes ****

TLC yes yes yes **

TOC yes yes yes yes yes ***

Spectrophotometry yes yes yes yes **

pH No yes *

Conductivity No yes *

Gravimetric No yes yes *

ELISA yes (BIO) yes *****

Electrophoresis yes (BIO) yes

TABLE IIEquipment Specifications Given by the Supplier

TOC-VCSN TOC-VWP

Detection limit (provided by supplier) 5 ppb 0.5 ppb

Injection type multiple simple

Injection volume 10–2000 �L 350–20400 �L

Sample Aqueous (there is a solid and gas option) Aqueous

43Vol. 63, No. 1, January–February 2009



2) TOC-VWP: wet oxidation/NDIR method

This analyzer uses wet chemical oxidation combiningsodium persulphate and UV radiation as the oxidationmethod (21). Figure 2 illustrates the three possibletypes of carbon that the device can determine.

1.2. Significance of TOC Measurements

TOC analysers can, in a single sample, independentlymeasure the amounts of three types of carbon: totalcarbon (TC), total organic carbon (TOC), and inor-ganic carbon (IC). Also, TOC can be distinguishedbetween purgable carbon (POC) and non-purgable car-bon (NPOC). So, the relation is TC � TOC � IC, andTOC � NPOC � POC.

Of the three measurements taken by the TOC analyz-ers, it is assumed in cleaning validation in the phar-maceutical industry that the TOC load is equivalent tothe NPOC, since the POC represents volatile com-pounds such as organic solvents that are almost neg-

ligible if the cleaning is carried out in accordance withthe standard operating procedures (SOPs). This greatlysimplifies the analysis of routine sample readings suchas those taken in this study.

2. Previous Considerations

When considering the use of TOC analysis for clean-ing validation, first it is important to resolve certainproblems that could affect later validation results. Thefollowing steps should therefore be considered beforeproceeding to the validation itself:

1. Qualification of the TOC equipment, according tothe pharmacopeia

2. Selection of the prior treatment (cleaning) of theglass material

3. Calculation of the precision of the TOC analyzer

4. Linearity of the method: calibration lines

5. Selection of the contaminant to monitor

6. Calculation of acceptance limit for residues in theprototype

7. Validation of the TOC method for determiningdetergent and API residues

8. Calculation of the recovery and the accuracy ofAPI A� at the acceptance limit

Finally, in the established validation method it isparticularly important to use the appropriate type ofswab with low carbon content (10, 22). The three typesof analyzer (TOC-VCSN, TOC-VWP, and HPLC) takereadings from the same residue samples obtained dur-ing the equipment validation procedure in order todetermine the functionality and equivalence of thethree analytical methods.

2.1. Qualification of the TOC Equipment

Qualification is based on the pre-use test given in thepharmacopeia in order to verify that the equipmentfunctions correctly. The suitability of the system orTOC equipment is determined according to the meth-ods described in the Real Farmacopea Espanola(RFE), 3rd ed. (23) and the United States Pharmaco-poeia USP30 NF25 (24), through the analysis of a

SAMPLE

Valve

Syringe

IC reactorIC CO2

O2 (transportator gas)

Gas O2Bubble

Phosphoric acid

NDIRdetector IC

Valve

Syringe


TC reactor

Combustion tube

Gas O2bubble

TC CO2

NDIRdetector TC

Valve

Syringe

TC reactor

NPOC CO2

Gas O2bubble

Clorhidric acid

POC

NDIRdetector NPOC


IC

Combustion tube

Figure 1

TOC VCSN utilities scheme.




solution prepared with a substance that oxidizes withdifficulty (1,4-benzoquinone) and through comparisonwith another substance that oxidizes easily (sucrose).If the analyzer functions correctly it should oxidizeboth substances equally and the differences betweenthe two analyses should be comparatively small.

The system is suitable if the efficiency of the responseis no less than 85% and no more than 115% of thetheoretical response.

2.2. Treatment of the Glass Material Prior to Use inthe TOC Analysis

According to USP30 NF25 (24), there must be anefficient cleaning system to remove organic material

from the glass material used in determining the TOC.The three standard systems are (23) the following:

1) Chromic mixture (must be handled with care)

2) Dilute nitric acid

3) Alkaline detergent

As a general rule it is advisable to work with laboratoryglass material that is dedicated exclusively to TOC anal-ysis. It is also important to rinse the glass material withhighly purified water several times prior to use. In thiscase, the effects of treatment with a chromic mixture andwashing with an alkaline detergent were compared todetermine the most suitable option.

Valve

Syringe

IC Reactor

IC CO2

N2 (transportator gas)

Gas N2bubble

Phosphoric acid

NDIRdetector

IC

Valve

Syringe

N2 (transportator gas)

TC reactor

UV lamp

gas N2bubble

Sodium persulfateTC CO2

NDIRdetector

TC

Valve

Syringe

IC reactor

IC CO2

Gas N2bubble

Phosphoric acid

POC IC

UV lamp

TC reactor

Sodium persulfate

NDIRdetector

NPOCNPOC CO2

SAMPLE

Figure 2

TOC-VWP utilities scheme.




2.3. Precision of the TOC Analyzer

This parameter expresses the degree of concordance(degree of dispersion) between a series of measure-ments obtained from a single, homogeneous sampleunder predefined conditions. The parameter is deter-mined using the repeatability method (25) by calcu-lating the relative standard deviation (RSD) of tenaliquots of a standard sample containing 1 ppm C.

According to the AOAC (American Association ofOfficial Analytical Chemists) (26), the limits for theRSD are established according to the concentration ofthe substance in the sample to be validated. In thepresent study a RSD of up to 8% is accepted forconcentrations of 1 ppm.

2.4. Calibration Curves

For cleaning validation using TOC analysis, cali-bration curves must be made utilizing specific stan-dards with exactly known carbon content (16, 17).Two calibration curves were prepared: (a) one forthe calculation of TC and NPOC, with a standardsolution of potassium hydrogen phthalate, and (b)another for the calculation of IC, with a standardsolution of sodium hydrogen carbonate and anhy-drous sodium carbonate.

The curves were prepared using the working concen-tration from the cleaning validation, so it was advis-able to use seven points that range between 0.05 and 5ppm. Curves from 5 to10 ppm were also prepared incase the samples contained higher levels of C (theseare not shown in the study as they were not used).

The detection limit (DL) and the quantification limit(QL) were obtained from the calibration lines. Theselimits were calculated using the ICH method (27),which is based on the standard deviation of the re-sponse and the slope of the calibration line. The av-erage SD was calculated for the areas corresponding toconcentrations of between 0.05 and 5 ppm of carbon.To obtain the method’s noise level, the average SDwas then divided by the slope of the calibration line.This was multiplied by 3 to obtain the DL and by 10to obtain the QL. After obtaining the theoretical valuesof DL and QL, five single samples were prepared forthe two levels to experimentally demonstrate that thetwo limits correspond to the DL and QL proposedbefore.

2.5. Monitored Contaminant (28)

The worst-case criterion is applied to select the con-taminant with the lowest solubility in water (thereforethe most difficult to clean) and/or the lowest dailytherapeutic dose (TD) (therefore the most active ortoxic) as the prototype.

A cleaning validation check sheet (listing each APIused in the pilot plant) was prepared for each piece ofequipment analyzed. When new APIs pass through theequipment they must be analyzed if they represent anew worst-case scenario with respect to those alreadyincluded in the checklist. If this is the case, the accep-tance limit must be recalculated; if the new API doesnot constitute a worst case, the established limit re-mains valid. The checklist can be designed as an Excelspreadsheet to facilitate the calculations.

2.6. Acceptable Residue Limit of the Prototype

If the equipment is cleaned after production of productA and then used to produce product B, the contami-nant limit (active ingredient or excipients of A, resid-ual detergent, or degradation product) subsequentlyapplied to B must meet the basic organoleptic criterionand the calculated general acceptance limit (2, 29, 30).

● Organoleptic criterion: absence of visible contam-ination (6, 31, 32) (4 –20 �g/cm2) when viewedwith visible or UV light. Absence of unusualodours.

● General criterion: the limit will be the lowest valueobtained from the two methods described below.

Method using the fraction F (safety) of the therapeuticor toxic dose of contaminant A�: the volume of con-taminant present in the daily therapeutic dose of prod-uct B that will subsequently be produced by the equip-ment must be lower than a predetermined fraction ofthe therapeutic dose (TD) or toxic dose of the contam-inant. Product B refers to the final product containingAPI B� and its corresponding excipients. The patienttakes a given quantity of product B (e.g., 900 mg intablet form, which contains 500 mg of the API aspi-rin). However, the contamination due to API A� isdiluted in the 900 mg of final product. In this case, theworst possible case should be chosen as product B,which will be the smallest batch size, since the residueof A� would be concentrated in B.




In practice, F takes values of between 0.001 and0.025—see Table III (8, 33). F � 0.001 indicates thatone-thousandth of the TD of the API of contaminantA� is the maximum amount that can pass into the TDof product B. The three security factors of ten in thefraction 0.001 represent three considerations: the firstis due to the fact that drugs are usually inactive at 0.1of their TD; the second is a prevention factor (toensure inactivity); and the third is due to the robust-ness required in any cleaning validation.

An example of the required equation would be

ppmA� �F�DT A� min

DT max product B.,

where DT A� min is the therapeutic dose or toxic doseminimum of the API A�, and DT max product B is thedaily maximum dose of the product B.

Criterion based on the presence of trace amounts(ppm): Traditionally, an upper limit (34) between 1 to10 ppm of the contaminant A in product B was con-sidered appropriate. However, in ordinary cleaningand batch production, accepted limits can be 100 –1000 times higher, and when using very active prod-ucts the limits can be 10 –100 times lower. If TOCanalysis is used for cleaning validation, the chemicallimits have to be converted into their equivalent TOCvalues (35), as these will obviously be lower. Thisgeneral limit can then be transformed into the specificlimit according to the sampling method used in thecleaning validation.

2.6.1. Rinse Water Validation Method: This is themethod chosen for equipment that is difficult to dis-assemble or with areas that are difficult to reach(pipes, dispensers, etc.) because it encompasses all ofthe surfaces of a piece of machinery. It is usually adirect analysis (36) performed through a small number

of steps when the volume of solvent can be easilymeasured. The major disadvantages of this method areas follows: it does not remove strongly adhered resi-dues, the residue is diluted to a certain extent, some ofthe rinse liquid may be lost, the type of solvent (sol-ubility, safety, etc.) and the quantity used can affectthe outcome, the distribution of the contamination inthe equipment is not known, the use of non-aqueoussolvents can cause problems, and, finally, not all typesof equipment can be validated using this method be-cause some cannot retain the rinse water (in thepresent study, which uses a Glatt coating pan, thisvalidation method cannot be applied).

Finally, when using this method the equipment shouldbe cleaned according to the approved SOP and thensubmerged in or rinsed with fresh rinse water, fromwhich the samples for analysis should be extracted. Itis recommended that the recovery of the method becalculated, even though this step was previously omit-ted for soluble APIs. The formula used to calculate theacceptance limit is as follows:

LimitppmA��RINSE� �N��gA�/ml� � V�l �

W min�kg�,

where ppm A� (rinse liquid) � maximum ppm A� in B,N � mg A�/mL (limit of active ingredient/mL), V �volume of rinse liquid (l), and Wmin � minimumweight of a batch (kg) or weight of the smallest batchthat can be produced by the equipment used.

2.6.2. Surface Sampling Validation Method: This isa direct, reproducible method. It is useful when thecontamination is assumed to be homogeneous and canbe extrapolated to the total surface area of the equip-ment. However, if only the most inaccessible points ofthe equipment are sampled (hot points or criticalpoints) and the results are extrapolated to the totalsurface area of the piece of equipment analyzed

TABLE IIISecurity Factors of Different Pharmaceutical Forms (9, 33)

Pharmaceutical Form Security Factor (F)

Investigation, allergenic or toxic products 100.000 to 10.000

Parenteral products 10.000 to 5.000

Opthalmic products 5.000 to 1.000

Oral products (tablets, capsules, etc.) 1.000 to 5.000

Topical products (creams, emulsions, etc.) 100 to 1.000




(worst-case scenario of uniform contamination), itmay be necessary to modify the cleaning method,which complicates the cleaning method and its vali-dation. It is important to record what will be sampled,the points in the piece of equipment from which sam-ples will be taken (accessible and inaccessible pointssuch joints, cracks, waste pipes, etc.), and the materialof the surfaces at these points. This should ensure amore accurate extrapolation of the total contaminationand will avoid unnecessary “extra cleaning efforts”. Itis also necessary to take into account the auxiliaryequipment that comes into contact with the product,such as filters, utensils, etc.

The amount of contaminant found in the dispensers(critical points that can potentially cause selectivecontamination) will be released entirely into the firstunits produced (worst-case selective contaminationscenario). A special sampling material is required(brush or swab) which must be as inert as possible andshould determine the percentage recovery of themethod. Possible contamination and/or interferencewith the contaminant should also be considered. Thesolvent is selected taking into account the solubility ofthe contaminant, the nature of the contamination, thetype of surface, and the sampling material. The ana-lytical method always includes pre-treatment of sam-ples, which includes dissolution, filtration, extraction,etc.

It is essential to calculate the recovery factor in orderto correct the values obtained when analyzing thesamples. In biotechnology, recoveries of around 15–20% are acceptable. The U.S. Food and Drug Admin-istration (FDA) accepts �50%, although in practice itrequires the percentage to be in the range of from 70to 100% (37, 38). The RSD of the samples should bebelow 10% in all cases.

The surface area of the sample should be considered(normally 25 cm2); it should ensure that the percent-age recovery is sufficiently high that the analyticalmethods can evaluate the products. If the DL and QLof the method are low, the surface area will have to beincreased.

The following formula is used to determine the accep-tance limits applied to the surface sampling:

LimitppmA��sup� �M � S �cm2�

R � P min �kg� � 10,

where ppmA� (sup) � maximum ppmA� in B, M �microg A�/cm2 (limit of the active ingredient/cm2),S � surface area of the equipment in cm2, R �average analyte recovery (%), and Pmin � minimumweight of a batch (kg) or weight of the smallest batchthat can be produced by the equipment used.

2.7. Validation of the TOC Method for DeterminingDetergent and API Residues

TOC curves are used to demonstrate the linearity ofthe possible contaminants (detergents and APIs) be-tween the concentration of the contaminant and thequantity of carbon in ppm C to establish the limita-tions of the analyzer and of the method (13, 39). It istherefore assumed that any carbon residue is indicativeof existing contamination, irrespective of its source.

2.8. Recovery of API A� and Accuracy of the Method

This test verifies the percentage recovery of themethod as recorded in the analysis, which is thenapplied to the results of the cleaning validation of theGlatt coating pan. First this is done by loading astainless steel plate (40 cm2) that has been washedwith low-TOC water (five rinses) and then spiked withthe standard API A� solutions. The plate is then left todry for 2 h in normal conditions but “protected” in athermostatized oven at 25 � 2 °C exclusively for theTOC test.

Three concentrations of API A� (5.70 ppm, 2.87 ppm,and 1.43 ppm) were prepared, which were then pipet-ted onto separate stainless steel plates. In addition, afourth plate without the standard solution was used asa blank. A sample was taken from the pipetted platesusing a premoistened swab which is placed in a100-mL flask. The resulting solution was read by theTOC and HPLC analyzers, and recovery of three stan-dards were calculated using the following equation:

AverageTOC�“swab”� � blank�“swab”�

TOC max theoreticalAPI� 100

� % recovery.

Finally, the test was repeated for the acceptance limitof A� in the final solution (based on the knowledgethat the surface area of the equipment to be sampledwill be approximately 400 cm2 and the flask has avolume of 100 mL), and the maximum permissibleconcentration of the solution was calculated. Theplates were pipetted in triplicate (samples 1, 2, and 3).




In addition, three other samples at the same concen-trations of API A� (5.70 ppm, 2.87 ppm, and 1.43ppm) were prepared, which were then quantified di-rectly in the three analysers. The method’s accuracywas calculated by comparing the results with the realconcentrations of the solutions.

3. Comparative Analysis of Residues inTOC-VCSN, TOC-VWP, and HPLC Equipment

The two TOC methods were compared with the HPLCmethod for validating the cleaning of the Glatt coatingpan in the pharmaceutical pilot plant of the Universityof Barcelona to determine the comparative function-ality of the three methods. The validation consists ofdetermining the results of three cleaning cycles. Dueto the characteristics of the equipment (Figure 3), onlythe surface sampling validation method can be used.

Consequently, once the equipment had been cleanedfollowing use with the monitored prototype product or

worst case, samples were taken from areas of approx-imately 400 cm2 with the preselected swab. The swabwas ultrasonicated for five minutes in a calibratedflask containing 100 mL of highly purified water. Theresulting solution was filtered through a 0.45-�m Mil-lipore Millex-HV Hydrophilic PVDF filter that willretain any possible insoluble residues, and readingswere then taken with the TOC and HPLC analyzers.

The TOC analyzer detects all contaminants containingcarbon, so that any residue containing carbon will beassumed to be the prototype contaminant; this gives anacceptance limit similar to that produced by the HPLCtechnique.

4. Results

The results for each of the pre-operation tests carriedout in the study (explained in Section 2) and the resultsof the comparative analytical study described in Sec-tion 3 are reported.

4.1. Qualification of TOC Equipment

As can be seen in Table IV, the two units meet theoxidation efficiency requirements, where the wet oxi-dation equipment (TOC_VWP) is more efficient(103.6%) than the combustion unit (TOC_VCSN,111.8%).

4.2. Previous Treatment of Glass Material

The NPOC of the high-purity water was determined inthe eight flasks treated with chromic mixture and inthe eight untreated flasks washed with basic detergent(Figures 4 and 5). As can be seen in Figure 4, there areno statistically significant differences between threegroups, while a statistically significant difference canbe seen for the sample coming from flasks treated andanalysed using wet oxidation.

cover

Coatingpan

base

Air-inlet

Figure 3

Glatt coating machine, with its parts marked.

TABLE IVTOC Equipment Qualification Results

TOC-VCSN Combustion TOC-VWP Wet Chemistry

Benzoquinone (rss) (ppm C) 0.7156 0.6495

Sacarose (rs) (ppm C) 0.6542 0.6301

High purity water (rw) (ppm C) 0.1316 0.0944

rss rw (ppm C) 0.5840 0.5551

rs � rw (ppm C) 0.5226 0.5357

Answer (%) 111.75% 103.62%




From Figure 5, it can be also observed that the treat-ment reduced the amount of C in the flask and thedifference among flasks in the group. In spite of thisadvantage, due to the delicate handling required bychromic mixture, it is advisable to use material de-signed exclusively for TOC analysis and prior to useto wash the material with an alkaline detergent andrinse it four times with water with low TOC content.

4.3. Precision of the TOC Analyzer

To calculate this value, a standard solution was pre-pared which contained 1 ppm C (with standard phtha-late solution for TOC) and ten readings were taken.The results can be seen in Table V. The RSD is 6.5%for the combustion TOC and 4.6% for the wet com-bustion TOC, both of which are below the RSD indi-cated by the AOAC for concentrations of 1 ppm (8%),which indicates that the two analyzers are sufficientlyprecise.

4.4. Phthalate TOC/API A� HPLC Lines

The three curves were read in the three differentsystems. Results are shown in Table VI, along with thedetection and quantification limits. It can be seen thatthe background noise is greater for TOC than forHPLC, as TOC detects all types of carbon compoundwhereas HPLC detects only one compound.

After obtaining the theoretical values of DL and QL,five single samples were prepared for the two levels.For the first sample, the DL values describing preci-

sion were above the established limit (10% Eurachemmethod), and for the QL the RSD was below this limit,since we obtained 8.15% for combustion TOC, 7.55%for wet TOC, and 4.13% for HPLC.

4.5. Monitored Contaminant

The aim of the study is to validate the cleaning of aproduction unit with a total surface area of 3433.9 cm2

and a theoretical capacity of 0.8 kg. The list of tech-nical specifications will help to determine the indicatorAPI of the validation (Table VII), which takes intoaccount the API that had passed through the unit forthe previous product and the detergent used in thecleaning process.

According to the reference consulted (28), the moni-tored contaminant will be the substance with thesmallest therapeutic dose (TD) or LD50 (lethal dose inthe case of the detergent components), and which hasthe lowest degree of solubility in water. The indicatorprototype selected for the validation was API A�.

4.6. Acceptance Limit of Prototype

As described above, first the general limit is calculatedand then the limit for validation is determined usingrinses water or the surface sampling method.

1) Acceptance limit calculation

This is calculated using the therapeutic fraction crite-rion (see the following formula); if it is lower than themaximum dose limit of 10 ppm (organoleptic method),the lower of them is chosen:

ppm A� �0.001 � 25000 �g/day

1 g/day� 25 ppm.

Sample's dispersion

ppm

C (

TO

C)

Vcsnnt Vcsnt Vwpnt Vwpt0

0.05

0.1

0.15

0.2

0.25

0.3

Figure 5

TOC-VCSN and TOC-WP dispersion graphic.

Vcsnnt

Vcsnt

Vwpnt

Vwpt

0 0.05 0.1 0.15 0.2 0.25 0.3

ppm C (TOC)Figure 4

TOC-VCSN and TOC-WP distribution using boxand moustaches graph.Vcsnnt: No-treated flask analysed by combustionVcsnt: Treated flask analysed by combustionVwpnt: No-treated flask analysed by wet oxidationVwpt: Treated flask analysed by wet oxidation




Since the 10 ppm limit is lower than the calculatedtherapeutic fraction limit (25 ppm), it is consideredthat there can be a maximum of 10�g of API A� in 1 gof the quantitative formula of B.

Using TOC, API A� contains 57.34% carbon, whichmeans that the limit will be

ppm TOC A� �57.34 � 10 ppm

100� 5.73 ppmC A�,

2) Calculation of the acceptance limit for surfacesampling method

The formula used to calculate the surface limit is givenin Subsection 2.6.2. So, if the maximum total amountis 5.73 ppm C for the piece of equipment, the limit canbe calculated for cm2. In this case, M � 2.33 mg/cm2

of API A� or 1.34 mg/cm2 of C. The latter limit, takenfrom the TOC analysis results, means that all organicresidues are assimilated as API A�, thus the strictestpossible limit is applied.

The limit is not calculated for the rinse water methodbecause this is not suitable for the type of equipmentvalidated in this study (see Figure 3).

4.7. Validation of the TOC Method for DeterminingDetergent and API Residues

In all cases a satisfactory curve with at least 5–7 pointsis obtained. The determination coefficients r2 are

shown in Table VI. These results confirm the lineardetection of the possible contaminants using both theTOC and HPLC techniques.

4.8. Recovery of API A�

It is observed that recovery at high concentrations (5.7ppm C) is greater in TOC than in HPLC, whereasHPLC provides greater recovery at lower concentra-tions (1.43 ppm C; see Table VIII). It was found thatat low TOC concentrations the TOC amount of thesampling devices can influence measurement errorbecause at low levels the percent influence is moreimportant than in higher levels of TOC.

The API A� solution, which will be pipetted onto theplate, is prepared as a solution with the concentrationof the acceptance limit of A� in the final solution,which is calculated as follows:

��2.33 microg/cm2� � 400 cm2�/100 mL

� 9.32ppm API A� � 5.34 ppm C API A�

The solution is prepared and the procedure is repeatedthree times. The results are shown in the last lines ofTable VIII. For swab recovery of A� at the surfaceacceptance limit, a general value of 70% is acceptablefor each of the three methods.

In addition, accuracy was calculated with the threestandard solutions prepared in the three levels assayed.

TABLE VObtained Results To Prove Precision

Instrumental System RepeatabilityTOC-VCSN (area, mV/min)

Instrumental System RepeatabilityTOC-VWP (area, mV/min)

1 10.11 191.20

2 10.35 187.80

3 11.40 191.90

4 10.72 180.70

5 9.88 182.30

6 10.28 174.30

7 9.50 178.40

8 9.40 178.90

9 9.45 177.00

10 * 201.20

SD 0.66 8.41

Average 10.12 184.37

RSD (Precision) 6.52 4.56

* Value eliminated by Dixon criteria.




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TABLE VIIIRecovery in Three Levels of the API A�

Surface Area of Plates Sampled: 40 cm2

Amount of standard 5.70 ppm C TOC-VCSN (ppm C) TOC-VWP (ppm C) HPLC (ppm API A�)

Sample 1 6.336 5.884 7.680

Sample 2 6.436 5.823 6.410

Sample 3 6.916 6.455 7.440

Blanc filtered swab 1.315 1.245 *

High-purity water blank 0.237 0.287 *

5.7 ppmC real 6.306 5.694 9.890

Average 6.563 6.054 7.180

RSD (%) 4.724 5.758 9.359

% recovery 86% 89% 73%

Amount of standard 2.87 ppm C TOC-VCSN (ppm C) TOC-VWP (ppm C) HPLC (ppm API A�)

Sample 1 2.484 2.441 1.680

Sample 2 3.562 2.074 3.073

Sample 3 4.061 3.969 3.332

Blank filtered swab 1.315 1.245 *

High-purity water blanc 0.237 0.287 *

2.87 ppmC real 3.353 2.404 5.059

Average 3.369 2.828 2.694

RSD (%) 23.925 35.538 32.612

%recovery 55% 75% 54%

Amount of standard 1, 43 ppm C TOC-VCSN (ppm C) TOC-VWP (ppm C) HPLC (ppm API A�)

Sample 1 1.655 1.480 1.495

Sample 2 1.596 1.493 1.606

Sample 3 1.425 1.371 1.105


High purity water blanc 0.199 0.239 *

1.43 ppmC real 1.698 1.527 2.472

Average 1.559 1.448 1.402

RSD (%) 7.664 4.627 18.320

% recovery 16% 16% 57%

Surface Area Sampled in the Equipment: 400 cm2

Amount of standard 5, 34 ppm C TOC-VCSN (ppm C) TOC-VWP (ppm C) HPLC (ppm API A�)

Sample 1 5.69 4.96 18.16

Sample 2 4.74 4.99 14.97

Sample 3 5.90 5.31 20.34


High purity water blanc 0.23 0.28 *

Average 5.44 5.09 17.83

RSD (%) 11.41 10.70 15.12

% recovery 75% 71% 70%

* Does not apply.




The results are displayed in Table VIII, showing anacceptable accuracy in all the cases.

4.9. Cleaning Validation Results for the Glatt Pan

The cleaning of the coating pan is validated by sam-pling the internal surface that comes into contact withthe prototype product. The parts of the equipment thatdid not come into direct contact are also considered,such as the base air inlet and the cover of the Glattcoating pan (Figure 3), because these areas also pro-vide a risk of cross-contamination. The acceptancelimits are calculated independently according to therespective surface areas.

Table IX shows the TOC results and the equivalentHPLC results for the three cleaning validation cycles.The results are adjusted (by 70% recovery) to obtainreal values of the contamination recorded.

The cleaning of the coating pan is therefore validated,as the results are within the established limits.

5. Discussion and Conclusion

Table X summarizes the findings of the study bycomparing the three techniques used to validate theprocedures for cleaning equipment in the pilot plant. Itwas proved that the analytical method is very practical

TABLE IXTOC and HPLC Cleaning Validation Results

Area(cm2)

SampledArea(cm2)

TOC (ppm C) Results (3 cycles)CoatingMachineLimits

(ppm C)

HPLC1050(ppm API A�)

Results(3 cycles)

CoatingMachine

Limits APIA� (ppmAPI A�)

CSN(combustion)

WP (wetchemistry)

1° 2° 3° 1° 2° 3° 1° 2° 3°

Coating pan 3433.90 400 0 0 0 0 0 0 5.34 0 0 0.10 9.320

Base 2700 400 2.01 0.79 2.01 1.38 0.97 0.78 5.34 0.621 0.22 0.21 9.320

Entrance 395.84 395.84 0 0 0 0.04 0 0 5.29 0 0 0.15 9.220

Air entrance 360 360 0.36 0 0 0 0 0 4.81 0 0 0.15 8.390

Cover 2474 400 0 0 0.61 0.06 0 0.78 5.34 0.031 0.10 0.23 9.320

TABLE XSummary of Difficulties and Advantages of the Three Methods

Concept TOC-VCSN TOC-VWP HPLC

Maintenance cost ��

Validation/method qualification time consumed/year � � �

Qualification cost/year � � ��

Analysis standard time (calibration curve � samples) � ��

Reactants cost ��

Staff ��

Standard cost/analysis ��

% obtained recovery (API A�) 75% 71% 69.8%

DL (ppm C) 0.0068 0.0002 0.0664

QL (ppm C) 0.0227 0.0006 0.2215

Method precision (CV %, 1 ppm) 6.52 4.56 2.23

Accuracy (% recovery, 1.43 ppm) 105 90 100



Standard required Universal (potassiumphthalate)

Universal Specific (API)




and suited for use in a pilot plant, as TOC analysisprior to operation provides further security by addingan extra component to the verification of the cleannessof the production line.

From the comparative study it can be concluded thatthe TOC-VWP shows lesser variability of results anda lower DL than does the TOC-VCSN, making it moresuitable as an analytical method for cleaning valida-tion due to its higher sensitivity (because it is expectedthat a low load of TOC is present in the samples, thusit can be used to analyze samples from devices con-taining certified low amounts of TOC). The two TOCanalyzers are suitable for interpreting the results in thelimit tests and provide accurate measurements.

Comparison between the TOC and HPLC analyticalmethods reveals TOC analysis to be more practicalthan HPLC, as it does not require the preparatorystages prior to validation according to the API underanalysis and is also faster. As well for a pilot plant,TOC analysis can serve as a general equipment clean-ing indicator for products that have no specific vali-dated cleaning procedures. Because the technique isspecific to the active ingredient concerned, less vari-ability is observed with HPLC than with the two TOCmethods (2.23%). In addition, TOC analysis cannot beused for organic solvents and is only applicable towater-soluble samples (so that it must sometimes besupplemented by specific methods such as HPLC anal-ysis or performed for solid samples, requiring thor-ough cleaning of all material in order to minimizeinterference), which is not the case of HPLC analysis.

HPLC is a specific and accurate method; however,TOC is generally much faster. The major savings intime and associated cost gained by using TOC are dueto the speed of method development and analysis.Method development is an iterative process involvingrunning a sample and checking results. Because sam-ples are run quickly and analysis results are obtainedwithin seconds when using TOC, method optimizationis reached faster than when using the slower HPLCmethod. After method development and optimization,time savings are obtained in the analysis a companyruns regarding the ongoing monitoring of its cleaningmethods.

Finally, the following working guidelines are sug-gested in the implementation of a validation cleaningplan followed by cleaning verification for day-to-dayroutine analysis, using TOC analysis:

1. The cleaning procedure for each piece of equip-ment in the pilot plant should be validated, indi-vidual records detailing the APIs that have beenpassed through the equipment should be compiled,and residue acceptance limits calculated, as pre-sented in Table VII.

2. A TOC limit should be established for each pieceof equipment validated.

3. A technical schema of the equipment should beutilized to enable rapid evaluation and calculationof new acceptance limits according to the charac-teristics of the new APIs used.

4. The pilot plant equipment should be evaluated priorto use by taking a swab from the critical point ofthe unit (as marked in the working sheet, TableVII) and performing an analysis in the TOC anal-yser (taking only 10 –15 min).

This analysis of cleaning verification prior to theequipment’s use is advised because equipment in apilot plant is often inactive for long periods, whichmeans that the effect of previous cleaning will havediminished and the equipment must usually be cleanedagain. Furthermore, in clinical batch production thatfollows this procedure, there is no need to follow thestandard practice of producing a placebo batch (inorder to remove possible contaminants) immediatelyprior to production of the pilot batch.

6. Acknowedgements

The equipment used for the study was ceded byIZASA S.A. (Barcelona, Spain).

7. References

1. European Commission. Working Party on Control ofMedicines and Inspections. Annex 15 to the EUGuide to Good Manufacturing Practice. Qualificationand Validation. European Commission: Brussels,2001. http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-4/pdfs-en/v4an15.pdf. Accessed De-cember 5, 2007.

2. Office of Regulatory Affairs, FDA. Guide to In-spections Validation of Cleaning Processes.www.fda.gov/ora/inspect_ref/igs/valid.html. Ac-cessed December 5, 2005.




3. Salazar, R. Validacion Industrial. In Su Aplica-cion a la Industria Farmaceutica y Afines, 1st ed.,Romagraf: Barcelona, Spain, 1999; p 386 – 414.

4. Center for Drug Evaluation and Research, FDA.Can Total Organic Carbon (TOC) be an accept-able method for detecting residues of con-taminants in evaluating cleaning effectiveness?Questions and Answers on Current Good Manu-facturing Practices, Good Guidance Practices,Level 2 Guidance; Equipment. http://www.fda.gov/cder/guidance/cGMPs/equipment.htm#TOC.Accessed 11/06/05.

5. LeBlanc, D. A. Equipment Cleaning. Encyclopae-dia of Pharmaceutical Technology; 3rd ed.; In-forma Healthcare USA, Inc.: New York, 2006; pp1580 –1592.

6. Forsyth, R. J.; Leblanc, A.; Voaden, M. A singleadulteration limit for cleaning validation in apharmaceutical pilot plant environment. Pharm.Technol. 2007, Jan 2, http://www.pharmtech.com/pharmtech/Validation�and�compliance/A-Single-Adulteration-Limit-for-Cleaning-Validatio/ArticleStandard/Article/detail/397397. Accessed12/02/2007.

7. Forsyth, R.; Van Nostrand, V. Using visible res-idue limits for introducing new compounds into apharmaceutical research facility. Pharm. Technol.2005, 29 (4), 134 –140.

8. Martınez, J. E. Cleaning validation for develop-mental, stability and clinical lots. Pharm. Tech-nol. 2002, 26 (11), 62–74.

9. Asociacion Espanola de Farmaceuticos de la In-dustria (AEFI). Validacion de Metodos Analıti-cos, 1st ed.; AEFI: Madrid, 2001; p 121.

10. Jenkins, K. M. Vanderwielen, A. J.; Armstrong,J. A.; Leonard, L. M.; Murphy, G. P.; Piros, N. A.Application of total organic carbon analysis tocleaning validation. PDA J. Pharm. Sci. Technol.1996, 50 (1), 6 –15.

11. Biwald, C. E.; Gavlick, W. K. Use of total organiccarbon analysis and Fourier-transform infraredspectroscopy to determine residues of cleaningagents on surfaces. J. AOAC Int. 1997, 80 (5),1078 –1083.

12. Gavlick, W.; Ohlemeier, L. A.; Kaiser, H. J. An-alytical strategies for cleaning agent residue de-termination. Pharm. Technol. 1995, 19 (3), 136 –144.

13. Anatel Corporation. The Determination of CIP-100 Residues on Stainless Steel Surfaces by TotalOrganic Carbon Analysis. Application Note12.99.3, 1999.

14. Baffi, R. A.; Dolch, G.; Garnick, R.; Huang, Y. F.;Mar, B.; Matsuhiro, D.; Niepelt, B.; Parra, C.;Stephan, M. Total organic carbon analysis methodfor validating cleaning between products in bio-pharmaceutical manufacturing. J. Parent. Sci.Technol. 1991, 45 (1), 13–19.

15. Smith, J. Selecting analytical methods from clean-ing compounds in validated process systems.Pharm. Technol. 1993, 17, 88 –98.

16. LeBlanc, D. Cleaning technology for pharmaceu-tical manufacturing. Pharm. Technol. 1993, 17,84 –92.

17. Holmes, A. J., Vanderwielen, A. J. Total organiccarbon method for aspirin cleaning validation.PDA J. Pharm. Sci. Technol. 1997, 51 (4), 149–152.

18. Westman, L., Karlsson, G. Methods for detectingresidues of cleaning agents during cleaning vali-dation. PDA J. Pharm. Sci. Technol. 2000, 54 (5),365–372.

19. Glover, C. Validation of the total organic carbon(TOC) swab sampling and test method. PDAJ. Pharm. Sci. Technol. 2006, 60 (5), 284 –290.

20. Shimadzu. TOC-VCSN User’s Manual. ShimadzuCorporation: Kyoto, 2001.

21. Shimadzu. TOC-VWP User’s Manual. ShimadzuCorporation: Kyoto, 2001.

22. Cleaning Validation Swabs. www.texwipe.com.Accessed January 17, 2006.

23. Real Farmacopea Espanola (RFE), 3rd ed.; Ma-drid, Spain, 2005; p 71.

24. United States Pharmacopoeia USP30 NF25,641�, 2007.




25. Anatel Corporation. Total Organic Carbon Anal-ysis for Cleaning Validation in PharmaceuticalManufacturing. Application Note 12.99.2, 1999.

26. American Association of Official AnalyticalChemists (AOAC) Requirements for Single Lab-oratory Validation of Chemical Methods. http://www.aoac.org/Ag_Materials/additives/aoac_slv.pdf. Accessed May 29, 2007.

27. International Conference on Harmonisation ofTechnical Requirements for Registration of Pharma-ceuticals for Human Use (ICH). Validation of ana-lytical procedures: text and methodology Q2 (R1).http://www.ich.org/LOB/media/MEDIA417.pdf.Accessed May 29, 2007.

28. Fontane i Miret, M. Validacion de Procesos deLimpieza en la Industria Farmaceutica. Thesis,Universidad de Barcelona, 2000.

29. LeBlanc, A. Establishing scientifically justifiedacceptance criteria for cleaning validation of fin-ished drug products. Pharm. Technol. 1998, 22(10), 136 –148.

30. LeBlanc, A. Establishing scientifically justifiedacceptance criteria for the cleaning validation ofAPIs. Pharm. Technol. 2000, 24 (10), 160 –168.

31. Forsyth, R., Van Nostrand, V. Application of vis-ible-residue limit for cleaning validation in apharmaceutical manufacturing facility. PharmTechnol. 2005, 29 (10), 152–161.

32. Forsyth, R.; Hartman, J.; Van Nostrand, V. Risk-management assessment of visible-residue Limits

in Cleaning Validation. Pharm. Technol. 2006, 30(9), 104 –114.

33. Hall, W. E. Validation and Verification of Clean-ing Processes. In Pharmaceutical Process Valida-tion, 3rd ed.; Nash, R. A., Wachter, A. H., Eds.;Marcel Dekker, Inc.: New York, 2003; Vol. 129,p 493.

34. Fourman, G. L.; Mullen, M. V. Determiningcleaning validation acceptance limits for pharma-ceutical manufacturing operations. Pharm. Tech-nol. 1993, 17 (4), 54 – 60.

35. Wallace, B.; Stevens, R.; Purcell, M. Implement-ing total organic carbon analysis for cleaning val-idation. Pharm. Technol. Aseptic Processing2004, Supplement, 40 – 43.

36. Le Blanc, D. Rinse sampling for cleaning validationstudies. Pharm. Technol. 1998, 22 (5), 66–74.

37. Hall, W. E. Validation and Verification of CleaningProcesses. In Pharmaceutical Process Validation,3rd ed.; Nash, R. A., Wachter, A. H., Eds.; MarcelDekker, Inc.: New York, 2003; Vol. 129, p 484.

38. Yang, P.; Burson, K.; Feder, D.; Macdonald, F.Method development of swab sampling for clean-ing validation of a residue active pharmaceuticalingredient. Pharm. Technol. 2005, 29, 84 –94.

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