INTRODUCTION:

Cleaning validation is a documented process that proves the effectiveness and consistency in cleaning of the pharmaceutical production equipment¹. Validation detects and analyses optimization potential and supports implementation. Pharmaceutical manufacturer often make a large number of product types in one facility. Often there are several different strengths prepared of the same product. The cleaning problems include large number of processes and product types manufacture within one facility. The number of cleaning methods, assays and types of equipment to be tested are often staggering².

The current good manufacturing practices (cGMP) regulations recognize that cleaning is a critical issue to ensure product quality. Virtually every aspect of manufacturing involves cleaning, from the initial stage of bulk production to the final dosage form. The code of federal regulation (CFR) states that equipment and utensils shall be cleaned, maintained and sanitized at appropriate intervals to prevent malfunctions or contamination that would alter safety, identify, strength, quality or purity of the drug product beyond the official or other established requirements ³.

FDA considered the potential of cross contamination to be significant and to pose a serious health risk to the public. FDA expects firms to have written procedure called Standard Operating Procedure (SOPs), detailing the cleaning processes used for various pieces of equipments⁴.

Multiple product facilities (also referred to as multi product or multi use facilities) clearly represent a more difficult challenge. Procedurally steps must be taken in multiple product facility to ensure that cross contamination potential are eliminated. Changeover of the product from one product to another must be carefully controlled⁵.

As per USFDA and MCA guidelines separate manufacturing facilities are required to be created for the production of Penicillin and Cephalosporin group of antibiotics. But WHO guidelines allow the production of said dosage forms in same premises. Especially when common facilities are used for the production or various pharmaceutical products including that of Penicillin group of antibiotics, steps must be taken to ensure that cross-contamination potential is eliminated.

Looking at the risk of potential hazards of cross-contamination of Amoxycillin Trihydrate, the present study was undertaken with a view to assess the effectiveness of leaning procedure being followed for equipment used in manufacturing of selected dosage forms of Penicillin group of antibiotic in one of the modern pharmaceutical manufacturing plant, approved by WHO⁶.

TABLE I: PRODUCT DETAILS AMOXICILLIN TRIHYDRATE SYMOXYL CAPSULE

Brand Name	Symoxyl Capsule: 250 mg
Generic Name	Amoxycillin Trihydrate
Therapeutic Category	Antibacterial
Therapeutic Dose	250 mg
Largest Daily Dose	4.5 g
Solubility	-Slightly soluble in water, in ethanol (95%), and in methanol. -Practically insoluble in ether and in fixed oils. -Soluble in dilute solutions of acids and alkali hydroxides.

MATERIALS AND METHODS:

Instruments:

1. UV-visible-spectrophotometer (UV-1601 Shimadzu)

2. Sophisticated Analytical Balance (AEX 200 LB Shimadzu)

Materials:

Amoxycillin Trihydrate IP was obtained as gift sample from Elysium Pharmaceuticals Ltd., Vadodara. Isopropyl Alcohol LR (S.D. Fine Chem. Ltd. Boisar, Mumbai), Vivid solution LR (Vivid Enterprise Ltd., Mumbai), Detergent solution LR, Teepol LR, Phenyl solution LR, Water for injection, Demineralized water and Raw water.

TABLE II: LIST OF MANUFACTURING EQUIPMENTS,

SAMPLING POINTS AND SURFACE AREA

Sr. no.	Equipment /Area	Sampling points	Surface area (sq cm)
1.	Octagonal Blender	O1: Blade O2: Inside surface O3: Outlet O4: Outlet cover	5,76,244.63
2.	Rapid Mixture Granulator (RMG)	R1: Chopper R2: Blade R3: Inside surface	1,76,320.81
3.	Cad mill	C1: Hopper C2: Blade	56,348.44
4.	Drum Blender	D1: Lid D2: Inside surface	800400. 52
5.	Sifter	F1: Outlet F2: Gasket F3: Periphery	41589. 64
6.	SA9 Filling machine	A1: Hopper A2: Platform	80087. 11
7.	Polishing machine	P1:Polishing Brush P2: Sorter Unit	96337 99
8.	Blister pack machine	T1: Hopper T2: Channel	160626 .32
9.	Manufacturing Area		15,86,145.50
10.	Capsule filling and polishing room		1147435.50
11	Blister pack room		541512.00
12.	Total surface area (Equipments and Department)		2147989.08

Cleaning Procedure and Validation Studies:

A cleaning validation master plan was prepared; it consisted of all necessary documentation for performing cleaning validation studies which included selection of dosage forms, selection of sampling points which are critical to clean and likely to contaminate the product, cleaning method, development and validation of analytical method for active, sampling method and recovery studies. ICH guidelines were followed for the preparation of cleaning validation protocol, containing the details of manufacturing process, the cleaning methods used, and responsibility of each person involved were clearly described⁷.

TABLE III: LINEARITY DATA

Concentration mcg/mL	Absorbance ± S.D. (n=3)
1	0.0230 ± 0.0001
2	0.0466 ± 0.0003
4	0.0930 ± 0.0001
6	0.1393 ± 0.0005
8	0.1816 ± 0.0005
10	0.2310 ± 0.0003

For the purpose of cleaning validation, a solid dosage form, Symoxyl (Amoxycillin Trihydrate) Capsule was selected. The product detail has been highlighted in Table 1.

Sampling point determination:

In present study various sampling points critical to clean and, likely to cross contaminate the other products in manufacturing area namely Capsule department were selected. The list of manufacturing equipments and sampling points has been shown in Table 2.

Surface area determination:

The total surface area of production department including equipments involved in Capsule manufacturing was calculated. Table 2

Cleaning methods:

At the point of change over of the product, cleaning of areas and equipments was undertaken as per the guidelines given in Standard Operating Procedures (SOPs) for cleaning.

TABLE IV: REPEATABILITY DATA (10 mcg/mL)

No. of determination	Absorbance	Mean ± S.D.	% RSD
1	0.2310	0.231 ± 0.0001	0.0821
2	0.2308
3	0.2313
4	0.2311
5	0.2308
6	0.2310

Assay method and its validation:

Fifty mg of Amoxycillin Trihydrate was dissolved in water for injection to produce 1000 mL. From this solution various dilutions were made to obtain solutions having concentrations 1, 2, 4, 6, 8 and 10 mcg per mL. Absorbance was measured at 229 nm of for these solutions against water for injection as blank. Linear relationship between concentration and absorbance was observed (R²=0.9997) which was in agreement with Beer’s Lambert law (Figure 1). The method was validating using parameters such as linearity, repeatability, precision, accuracy and limit of quantification and limit of detection as shown in Table 3-7.

TABLE V: PRECISION DATA

Concentration (mcg/ml)	Absorbance Intra-day (n=3)	% RSD	Absorbance Inter day (n=3)	% RSD
1	0.0234	0.114	0.0230	0.675
2	0.0467	0.812	0.0466	0.643
6	0.1395	0.337	0.1393	0.414
10	0.2313	0.263	0.2310	0.139

Recovery studies:

Swab Method:

Stainless steel plates were used in swab recovery test to simulate manufacturing equipments. One side of each plate was spiked with 1 mL solution of active having concentration 500 mcg/mL, 1000 mcg/mL, and 1500 mcg/mL, respectively. Simultaneously, blank was spiked on second set. The plates were allowed to dry completely for overnight at room temperature. A swab was moistened with water for injection and the spiked plate surface was swabbed both vertically and horizontally. The drug content from the swab was extracted and the final volume made to 100 mL with solvent. The absorbance was measured at 229 nm to estimate Amoxycillin Trihydrate. (Table 8)

Rinse Water Method:

In rinse method same procedure was followed up to drying step. It was rinsed with water for injection and transferred to 100 mL volumetric flask. Volume was made with water for injection. The absorbance was measured at 229 nm to estimate Amoxycillin Trihydrate. (Table 8)

TABLE VI: ACCURACY DATA

Amount of drug in sample (mcg/mL)	Amount of drug added (mcg/mL)	Amount recovered (mcg/mL)	Percentage recovery	Mean percentage recovery
6	0	5.93	98.83 %	99.54 %
6	2	8.00	100.0 %
6	4	9.91	99.1 %
6	6	12.03	100.25 %

Sampling Method:

Swab was prepared by tying 400 mg of absorbent cotton with thread on the wooden stick. The care was taken to avoid contamination during and after its preparation. Water for injection was used as recovery solvent for Amoxycillin Trihydrate. The tip of swab was thoroughly wetted with recovery solvent and the excess liquid was allowed to drain back into the container. This was achieved by quickly and lightly touching the tip of swab to the inside of container. The tip of swab was pressed firmly and evenly on the surface of the substrate slowly and deliberately in a back and forth motion. The entire 10 cm * 10 cm area was covered in 7-10 strokes. The swab was transferred on a funnel placed over 100 mL volumetric flask. Subsequently the applicator was removed and cotton was extract with the recovery of solvent till the volume reached 100 mL absorbance of solution was measured at 229 nm with a view to estimate Amoxycillin Trihydrate. (Table 9)

TABLE VII: LIMIT OF QUANTIFICATION AND LIMIT OF DETECTION DATA

Limit of Quantification	Limit of Detection
1 mcg/mL	0.33 mcg/mL

Acceptance criteria:

Each company is responsible for establishing limits that are practical and achievable and have scientific basis. Residue limits established as an acceptance criterion in this facility is 10 mcg/sq cm, recommended by USFDA guidelines⁸ and Lilly crieteria⁹.

RESULTS AND DISCUSSION:

Validation of assay method:

It was observed that the assay method for estimation of Amoxycillin Trihydrate is accurate and precise (% RSD < 2 %). Further the limit of detection and limit of quantification was found to be 0.33 mcg/mL and 1 mcg/mL respectively (Table 3-7).

FIG.1: CALIBRATION CURVE OF AMOXYCILLIN TRIHYDRATE

Recovery Studies:

Recovery studies were conducted for drug under study using swab and rinse water method for recovery of Amoxycillin Trihydrate (Table 8). Due to less suitability of rinse water method for certain manufacturing equipments, most encapsulation equipment and other equipment which can not been completely rinsed without risking damage to electrical components and instrumentation. The FDA guide to inspection for cleaning validation notes that “The firm should challenge the analytical method in combination with the sampling method(s) used to show that contamination can be recovered from the equipment surfaces and at what level, i.e., 50 % to 90 % recovery.” It has been reported that the recovery of greater than 80 % is good. If recovery is greater then 50 % it may be acceptable. However, if the recovery is less than 50 %, questions arise and the source of poor

recovery should be investigated. In our study good swab recovery was obtained with respect to Amoxycillin Trihydrate (91.84 %).

TABLE IX: AMOXYCILLIN TRIHYDRATE (CAPSULE) RESIDUE LEVEL AT DIFFERENT SAMPLING POINT

Acceptance limit (mcg/sq cm)

10 mcg/sq cm

Equipment

Sampling point

A mcg/sq cm

B mcg/sq cm

C mcg/sq cm

Mean mcg/sq cm

S.D.

Drum Blender

D1:Lid

D2:Inside

2.94

1.60

1.51

0.99

0.69

1.71

0.86

1.13

0.80

Sifter

F1:Outlet

F2:Gasket

F3:Periphery

2.42

3.76

2.12

1.29

1.38

1.08

0.73

0.86

1.23

1.95

1.35

1.21

1.59

0.67

SA9 Filling machine

A1:Hopper

A2:Platform

1.51

3.89

0.82

2.20

0.99

0.77

2.36

0.75

1.45

Polishing machine

P1:Polishing brush

P2:Sorter unit

3.37

2.16

2.77

0.90

0.77

2.30

1.02

1.36

1.08

Blister packing machine

T1:Hopper

T2:Channel

3.03

2.07

2.72

2.38

0.95

1.47

2.23

1.97

1.12

0.46

AHU

A1:Supply grill

A2:Return grill

15.15

11.16

6.58

6.36

2.25

2.81

7.99

6.77

6.56

4.19

Area

Door

Floor

Wall

Window

9.09

4.28

8.05

3.20

2.29

5.06

4.37

3.67

2.55

0.64

1.99

1.03

4.64

3.32

4.80

2.63

3.85

2.35

3.05

1.40

Acceptance limit: 2.66 mcg/mL

ND- Not detected

A. Symoxyl (Amoxycillin Trihydrate) Capsule: 250 mg to Gerfex (Cephalaxin) capsule: 250 mg

B. Symoxyl (Amoxycillin Trihydrate) Capsule: 250 mg to Gerfex (Cephalaxin) capsule: 500 mg

C. Symoxyl (Amoxycillin Trihydrate) Capsule: 250 mg to Gerfex (Cephalaxin) capsule: 500 mg

Concentration	Absorbance (Swab)	% Recovery (Swab)	Absorbance (Rinse water)	% Recovery (Rinse water)
5 mcg/mL	0.106	92.17	0.110	95.65
10 mcg/mL	0.210	90.90	0.225	97.39
15 mcg/mL	0.320	92.47	0.335	96.81

TABLE I: PRODUCT DETAILS AMOXICILLIN TRIHYDRATE SYMOXYL CAPSULE

Brand Name

Equipment

/Area