“Single-time-point surface bioburden study at 96 hour dirty-hold time on amlodipine manufacturing equipment in ‘Class D’ facility (non-sterile)”

 

“Single-time-point surface bioburden study at 96 hour dirty-hold time on amlodipine manufacturing equipment in ‘Class D’ facility (non-sterile)”

 

Background

The objective of dirty equipment hold time study (DEHT) was to find the load of microorganism on the equipments used for manufacturing amlodipine tablets 5 mg in class D (non- sterile) manufacturing facility. Amlodipine tablets contains starch and microcrystalline cellulose as major excipients which may serve as excellent growth medium for growth as well as proliferation of microbes on dirty equipments.

Method

The dirty equipment hold time study was carried out for single product, amlodipine 5mg tablet and hold time of a 96 hours was selected for hold time study on dirty equipment. From 9 equipments, altogether 18 samples were sampled using sterile swab from 25cm² on single time point (96 hours) for study purpose. Details of criteria for sample site selection mentioned in Table 1.

Result

Microbial limit 100 cfu/25cm² was set as predefined pass/fail limit in analogy to the class D area in pharmaceutical manufacturing facility. The results of microbial sample were found below 100 cfu/25cm². Standard deviation of total aerobic microbial count was found to be 20.61(n=9) and total yeast and mould count 0.44 (n=9) respectively.

Conclusion

The result of study suggest that the load of microorganism were in predefined limit on amlodipine tablets manufacturing dirty equipment in controlled class D Environment but The load of microorganism was found to be very low, this might be due to controlled environment, low water activity on surface of equipments or low nutrient avaibality. Further multi time point studies are needed to establish hold time justification.

Key Words: Dirty equipment, Hold time study, Bioburden, TAMC, TYMC

 

Introduction

Dirty Hold Time refers to the maximum duration that equipment can remain idle after the completion of a manufacturing process and before the start of the cleaning procedure. During this period, equipment surfaces are exposed to risks such as: Drying and hardening of product residues, which can reduce cleaning effectiveness, microbial growth, particularly when residues retain moisture or contain nutrient-rich material and Chemical degradation, potentially altering residue characteristics and complicating removal [1, 8].

Qualification of the cleaning process is carried out using methods of a wet-swab sampling and analysis of controlled substances. Generally, washing process in the pharmaceutical plant begins immediately after the production batch. The postponement of cleaning activities on dirty equipments for several days can make more efficient use of working time and lead to cost reductions, but the residuals of the produced material, may become more difficult to clean after the delay [2, 3]. Dirty-hold time is part of the cleaning validation process from the beginning, but often overlooked [4, 5].
The API (active pharmaceutical ingredient), an excipient or the detergent are the controlled substances. For a single production line or the production device one product representing all products manufactured on this line can be determined based on a method of "worst case" [6]. The criteria for selecting the worst case are a complexity of the cleaning process, a solubility in a cleaning media, the greatest toxicity, the lowest therapeutic dose and the lowest limit (eg. from the therapeutic dose). Sampling is usually performed after a maximum number of produced batches. Process qualifications of the washing procedure are carried out for newly manufactured products, a new equipment, new systems CIP (Clean in Place) and recently used detergents [8, 10].

The work was aimed to study the Influence of microbial activity on dirty equipments used for manufacturing of amlodipine tablets 5 mg at 96 hours hold time.  

Material and Method

The study was designed as observational single-time point study on amlodipine tablets 5 mg manufacturing equipments. The major constituents of Amlodipine tablets include Amlodipine besylate, Starch, Microcrystalline cellulose, Dibasic calcium phosphate, sodium starch glycol late, magnesium stearate ,hypoxy methylcellulose, titanium dioxide, iron oxide red, iron oxide yellow, benzyl alcohol and poly ethylene glycol.

Table 1: Rationale of sampling site selection based on microbial risk

Equipment	Sampling Location	Rationale For selection
Saizoner Mixer Granulator (SMG)	Base of SMG	Rinse water and other residues may settle or remain in these areas due to insufficient water flow on machine
Fluidized Bed Dryer (FBD)	Inner wall of FBD dome	Rinse water and other residues may remain in these surfaces due to large surface area on machine
Binder Preparation Vessel	Base of vessel	Equipment used for preparation of starch paste which is difficult to clean and hence residues may remain in these areas
Square Bin	Inner wall near base of bin	Rinse water and other residues may settle or remain near the discharge of machine.
Mesh	Surface of mesh	Residues may remain clogged in the pores of mesh
Tablet Compression Machine	Surface of Turret	Complex equipment parts with an unusual design so chances of API and other residue accumulation on machine
Colloid Mill	Inner wall	Chances of dead lags and water retention  on machines
Coating Suspension Vessel	Sample from baffles of vessel	Rinse water and other residues may settle or remain in these baffles and discharge due to insufficient water flow from machines
Blister Packing Machine	Inner surface of hopper of hopper	Chances of API and other material residue on the surfaces of machine

 

 

 

 

 

 

 

 

 

The equipments used during the study were well-calibrated. Stensils used for measuring surface area was sterilized in autoclave at 121^◦C, 15psi for 15 minutes. Growth promotion test of media and sterility check of swab stick were done prior to conducting the test as per USP 61 [9]. Soyabean casein digest agar were used for isolation of Bacteria, incubation maintained at 35 ^◦C for 72 hours and Sabouraud chloramphenicol agar for isolation of fungi, incubation maintained at 25 ^◦C for 5 days. Statistical analysis were done using standard deviation on obtained data from all equipments.

The test was conducted in class D non- sterile pharmaceutical manufacturing facility. Nine equipments were included in study and altogether 18 samples were collected from difficult to clean area using sterile swab from 25cm² on single time point (96 hours) for study purpose The test areas of 5×5 cm² were measured with sterile stencils. The sterile swabs were moistened with sterile water and sample was collected from two different area of 25cm² from each dirty equipments. Altogether 2 samples from each equipments were collected, in unidirectional movements, first with 10 horizontal strokes followed by 10 vertical strokes from each equipments for test of total aerobic microbial count and total yeast and mould count. The swabs were dipped in a test tube containing 10 ml peptone water and transported to microbiology lab.

Each tube containing the swabs sample was shaken for 1-2 minutes. 1 ml of the sample solution was pipetted individually in 50 ml peptone water, mixed well by shaking and the whole content were filtered through membrane filter having pore size of 0.45 µm. The filter paper was transferred on Soyabean casein digest agar (SCDA) with sterile forceps and incubated at 35 ^◦C for 72 hours and the SDA chloramphenicol agar plates at 25 ^◦C for 5 days for test of total yeast and mould count.

Calculation of test result

2.1.1        TAMC or TYMC (cfu/ml

No. of colonies per ml × Dilution factor

Volume of Sample

Result

Table 2: Observation of Microbial Count

Equipment	Observations (Cfu/25cm2 area)
	Total Aerobic microbial count	Total Yeast and Mould count
Saizoner Mixer Granulator	30	1
Fluidized Bed Dryer (FBD)	10	1
Binder Preparation Vessel	20	2
Square Bin	50	1
Mesh	10	2
Tablet Compression Machine	50	1
Colloid Mill	50	1
Coating Suspension Vessel	70	1
Blister Packing Machine	40	1
Standard Deviation	20.61 (n=9)	0.44 (n=9)

 

 

 

 

 

 

 

 

 

 

 

Result and Discussion

Dirty equipment hold time study was done at class D non-sterile pharmaceutical manufacturing facility on amlodipine 5 mg tablets manufacturing equipments. The equipments were hold for 96 hours before sampling and sampling was done at single point of time (96 hours) from all equipments. (Table 1). The results of microbial sample were found below 100 cfu/25cm² (Table 2).The result is in agreement with similar study done by Singh SK ,2025[7].

The composition of amlodipine tablets contains starch and microcrystalline cellulose as major excipients which can serve as excellent growth medium for growth and proliferation of microorganism. The load of microorganism on dirty equipments hold for 96 hours was found to be very low, this might be due to controlled environment, low water activity on surface of equipments which hinder the rapid growth and proliferation of organism or due to low availability of nutrients on surfaces of equipment, the organism may have died. Compared to the bacterial load fungal load was very low on dirty equipments at 96 hours hold time (Table 2). Standard deviation of total aerobic microbial count was found to be 20.61(n=9) and total yeast and mould count 0.44 (n=9) respectively. Standard deviation was calculated on result of all equipments.

Conclusion

The dirty equipment hold time study was done on amlodipine tablets 5 mg manufacturing equipments to study the load of microorganism. Although the expectation of microbial load on dirty equipments was assumed to be found high but the results of microbial sample were found below 100 cfu/25cm² for total aerobic microbial count and total yeast and mould count in dirty equipments at 96 hours hold time. Many factors like low water activity, low nutrient availability for growth of organism may have contributed for the low microbial load on dirty equipments.

Limitation

1The study was done at single point of time and single product (Amlodipine), so further studies should be done on other products also to point out the real figure

2The limit 100cfu/25cm² was taken in analogy with class D area environment monitoring. It is difficult to fix limit of microbial load on dirty equipment so, further justification may be required regarding the limit.

References

1.      1.Cleaning validation TRS 937, Annex 4, 2006 and as cross-reference to TRS 970, Annex 2, 2012 (5)

2.    2.  Forsyth R J. Equipment hold-time for cleaning validation. Pharm Techno [online] 2008;2

3.      3.Fugate T. Hold time studies: A lost parameter for cleaning validation. J Val Techno 2007; 13 (3):206-209.

4.     4. Hall WE. Determining Appropriate acceptance criteria for cleaning programs in pharmaceutical facilities .J Val Technol 2004; 10(2):120-129.

5.    5.  J. Patera. Dirty-Hold Time Effect on the Cleaning Process Efficiency, Procedia Engineering 42 (2012) 431 – 436. doi: 10.1016/j.proeng.2012.07.434.

6.     6. LeBlanc D A. Equipment cleaning validation – Microbial control issues, J Val Techno 2002: 8(4):336-342.

7.      7. Singh SK. Microbial load assessment during dirty equipment hold time study, Nov.2025.DOI: 10.13140/RG.2.2.17667.36640.

8.      8.Supplementary guidelines on GMP: validation, non-sterile process validation. In: WHO Expert Committee on Specifications for Pharmaceutical Preparations: forty-ninth report. Geneva: World Health Organization; 2015: Annex 3 (WHO Technical Report Series, No. 992).

9.      9. United States Pharmacopoeial Convention. (2025). <61> Microbiological examination of

Non-sterile products: Microbial enumeration tests. In USP–NF 2025. Rockville, MD.

10.  Yang P. Cleaning validation assay – Qualification of cleaning procedures for a small-molecule residual active pharmaceutical ingredient (API). J Val Technol2004;10 (4):280-289.