ISO 14644 Clean Room Classification

What is Clean Room :

A clean room is designed in such a way which is having the provisions to reduce or control particulate contamination and to maintain the environmental parameters such as temperature, humidity and Differential pressure.

Clean room is a controlled environment that has a low level of pollutants such as dust, airborne microbes, aerosol particles, and chemical vapors and has a controlled level of contamination that is specified by the number of particles per cubic meter at a specified particle size.

The High Efficiency Particulate Air (HEPA) filter is used to trap particles that are 0.3 micron and larger in size.

All of the air delivered to a clean room passes through HEPA filters, and in some cases where stringent cleanliness performance is necessary, Ultra Low Particulate Air (ULPA) filters are used.

Clean rooms can also use ultraviolet light to disinfect the air and UV devices can be fitted into ceiling light fixtures and irradiate air, killing potentially infectious particulates, including 99.99 percent of airborne microbial and fungal contaminants.

Where clean Rooms are Used:

Clean rooms are used in industries where small particles can adversely affect the manufacturing process. They vary in size and complexity, and are used extensively in industries such as pharmaceuticals, biotech, medical device, Operation theaters (OT) and life sciences, as well as critical process manufacturing common in aerospace, optics, military and Department of Energy.

History of Clean room :

The modern clean room was invented by American physicist Willis Whitfield and he was the employee of the Sandia National Laboratories, Whitfield created the initial plans for the clean room in 1960.

Personnel Contamination in Clean room :

The greatest threat to clean room contamination comes from the users themselves.

In the healthcare and pharmaceutical sectors, control of microorganisms is important, especially microorganisms likely to be deposited into the air stream from skin shedding. Studying clean room micro flora is of importance for microbiologists and quality control personnel to assess changes in trends.

In assessing clean room microorganisms, the typical flora are primarily those associated with human skin (Gram-positive cocci), although microorganisms from other sources such as the environment (Gram-positive rods) and water (Gram-negative rods) are also detected, although in lower number.

Clean room Air Flow Principles:

Clean rooms maintain particulate-free air through the use of either HEPA or ULPA filters employing laminar or turbulent air flow principles.

Laminar, or unidirectional, air flow systems direct filtered air downward in a constant stream and this systems are typically employed across 100% of the ceiling to maintain constant, unidirectional flow. Laminar flow criteria is generally stated in portable work stations (LF hoods), and is mandated in ISO-1 through ISO-4 classified clean rooms.

Classification of Clean Room:

The clean room classification standards FS 209E and ISO 14644-1 require specific particle count measurements and calculations to classify the cleanliness level of a clean room or clean area. Classification of area are given in many guidelines but the base of all other guidelines is ISO-14644-1.

As we know that there are 9 classes of clean rooms in ISO but in pharmaceuticals mostly ISO Class 5 to ISO Class 8 are used for 0.5 μm and 5.0 μm.

ISO-14644-1 defines the maximum concentration of particles per class and per particle size. The below table shows the different classes of clean rooms accordingly to ISO-14644-1 Standards.

EU GMP classification:

EU GMP guidelines are more stringent than others, requiring clean rooms to meet particle counts at operation (during manufacturing process) and at rest (when manufacturing process is not carried out, but room AHU is on).

Validation of HPLC Method For Cleaning Validation

1.0  Specificity

Demonstrate the separation of the analyte from available Diluent, and Blank (swab) with standard solution using the HPLC system.

Note:  No other peak should be observed in diluent and Blank (swab) at the retention time of analyte.

2.0 System Precision

The precision as measured by multiple injections of a homogenous standard solution indicates the performance of the HPLC instrument under the chromatographic conditions and day tested.  As a part of method validation, a minimum of 6 injections of the standard preparation with an RSD of ≤ 5.0 % is recommended.

3.0 Linearity

Inject in duplicate at least 5 different standard concentrations covering range LOQ to 150% of the target concentration into the HPLC system.  Plot average peak response versus the actual concentration. The Correlation coefficient should be greater than 0.99.

4.0 Limit of detection

The limit of detection shall be arrived by diluting the standard solution and injecting the diluted solutions. Estimate the concentration, which gives the ratio between 2 and 3 for signal over noise from the same run.  Run the concentration approximately above, and at below level that actually produces S/N ratio of 2 to 3.  The LOD is considered the concentration which produces peak with a S/N = 2 to 3.  Measure a series of diluted solution with a very low concentration and inject six replicate into the HPLC system. Estimate the concentration, which gives the response % RSD between 10.0% and 33.0%.

5.0 Limit of Quantification

Inject six replicate injections of standard solution to give about 3.0 times the LOD concentration. Relative standard deviation of response should be between 3.0 % and 10.0 %.

6.0 Stability of Standard solution

The stability of Standard solution should be established over the period time by injecting into the HPLC system at 0, 24, 36,48, 60 and 72 hours. The difference in assay from initial should be not more than 3.0%

 7.0  Percentage Recovery

Standard Solutions: Prepare standard solutions corresponding to approximately 50%, 100% and 150% of Acceptance Limit concentration as per the test procedure and inject each standard solution in triplicate.

Spiked Swab solution: Prepare spiked swab solutions corresponding to approximately 50%, 100% and 150% of Acceptance Limit as per  the test procedure and inject each spiked swab solution in triplicate.

Calculate the %Recovery of spiked swab using the following  formula. 

 % Recovery =  (Spiked swab Area / Average Standard Area) x 100

Average % Recovery should be not less than 80.0%. 

In case of discrepancy repeat the experiment. If these criteria are still not met, investigate the reason.

Validation Of Dissolution Method By HPLC

1.0 Sink Conditions for non-compendial Dissolution Method

Determine the solubility of active drug substance in the dissolution medium at room temperature.  In case of very soluble compounds demonstrate that two times the amount of active drug substance in one tablet or capsule will completely dissolve in the designated volume of dissolution medium at room temperature.  The room temperature solubility should be more than two times the final concentration of the solution of one tablet or capsule in the designated volume of dissolution medium. Otherwise determine the solubility at 37°C.  The 37°C solubility generally should be at least three (3) times that of the final solution of one tablet or capsule in the designated volume of medium.

2.0 System Precision

The precision is measured by multiple injections of a  homogenous standard solution indicates the performance of the HPLC instrument under the chromatographic conditions. As a part of method validation, a minimum of 6 injections of the standard preparation with an RSD of <2% is recommended.

 3.0 Linearity

Inject in duplicate at least 6 different standard concentrations covering range 25% to 150% of the target concentration into the HPLC system. Plot average peak response versus the actual concentration.  The response may be considered linear, if the correlation coefficient is derived from the least squares fit of the data is not less than 0.999. In case of discrepancy still exists, 0.999 correlation, investigate and explain the reason.

 4.0 Method Precision

Prepare six test preparations as per the test procedure.  Dilute as specified in the dissolution and inject into the HPLC system.  The Q value should meet the specifications.  The relative standard deviation should not be more than 2.0%.

5.0 Recovery and Accuracy of the Method

Recovery from the spiked Placebo: Prepare solutions in triplicate corresponding to approximately 80%, 100% and 120% of working concentration as per the test procedure by spiking placebo (amount of placebo should be equivalent to the weight of placebo present in the sample amount required for the sample preparation as per test method) with different amounts of the drug substance.  Assay the samples by following the test procedure and calculate the concentration found in % of working concentration.  Calculate the mean recovery, the Relative Standard Deviation for the 80%, 100% & 120% spiked placebo data.  The mean recovery should be between 97 – 103%.

In case of discrepancy repeat the experiment.  If these criteria are still not met, investigate the reason.

 6.0   Placebo Interference

  Inject placebo and ensure there is no interference of excipients.

 7.0    Ruggedness

 System to system variation

System to system variability study should be conducted on two HPLC and Dissolution systems (same or different manufacturer) using five assay preparations prepared as per manufacturing formula at different times under similar conditions.

Results should be within test specifications.  Make the system suitability comparison table and establish the limits, if it is not in USP.  The Q value should meet the specifications.  The relative standard deviation should not be more than 3.0% for both the systems.

Analyst to Analyst Variation

Analyst to Analyst variability study should be conducted by two analysts on the same HPLC system at different times under similar conditions using five different assay preparations prepared as per manufacturing formula.

Results should be within test specifications.  Make the system suitability comparison table and establish the limits, if it is not in USP.  The Q value should meet the specifications.  The relative standard deviation should not be more than 3.0% by both the analysts.

8.0 Robustness

Effect of variation in mobile phase composition

Two mobile phases should be prepared having different concentrations of the method organic phase composition and inject into the HPLC system

The system suitability values should be evaluated for peaks of interest using both the mobile phases and the results should be within the limits for mobile phase.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

Effect of variation of pH of buffer in mobile phase

Two mobile phases should be prepared having buffers with ± 0.5 of the method pH and inject into the HPLC system.

The system suitability values should be evaluated for peaks of interest using both the mobile phases having buffers with ± 0.5 of the method pH and the results should be within the limits for mobile phase.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

Effect of variation in flow rate

Prepare the solution as per the test procedure and inject into the HPLC system at different flow rates.

The system suitability values should be evaluated for peaks of interest with both the flow rates and the results should be within the limits

If any of the system suitability value is not within the limits narrow the range and establish the allowable range of variation.

Effect of variation in column temperature

Prepare the solution as per the test procedure and inject into the HPLC system at ambient ±5°C column temperatures.

The system suitability values should be evaluated for peaks of interest at both the column temperatures and the results should be within the limits.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

9.0 Stability of standard and test preparations on Auto injector

The stability of standard and test preparations on Auto injector should be established over the period time by injecting into the HPLC system at 0, 24, 48 hours. The difference in assay from initial should be 3.0%

Validation of HPLC method for Assay

The below mentioned parameters are required to be complies during validation of HPLC method for Assay test.

1.0 Specificity :

Demonstrate the separation of the analyte from Placebo.

Conduct the following forced degradation studies to obtain degraded sample, preferably 10 – 50% degradation and demonstrate the separation of the analyte from degradants.

Heat, if necessary, aqueous, neutral, acidic and basic solutions or suspensions followed by neutralization.

If the compound is known to be susceptible to oxidation, expose to oxidative conditions such as reflux under oxygen or heating aqueous solution containing up to 10% hydrogen peroxide.

For light sensitive compound expose the drug substances to intense ultraviolet radiation up to minimum of 7 days.

Demonstrate the separation of the analyte(s) and the internal standard, if there is any, from the components of placebo mixture.

For a multi-component product, placebo formulation containing only one component should be forced to degrade by dry and moist heat (105°C), to demonstrate the separation from possible degradants of the other component.

Peak Purity :

Using an HPLC system, peak purity can be evaluated and the instrument will give the purity value. 

Peak purity shall be check for the above samples and criteria is “Purity angle shall be less than purity threshold”.   

2.0 System Precision :   

The precision as measured by multiple injections of a homogenous standard solution indicates the performance of the HPLC instrument under the chromatographic conditions. As a part of method validation, a minimum of 6 injections of the standard preparation with an RSD of ≤ 2% is recommended.

Column Performance :

If an integrator is being used, collect at least one chromatogram of the Assay, System suitability, Standard Solution or a Resolution and calculate Column Efficiency (Theoretical Plates), Tailing Factor, Resolution and Capacity Factor.  Results should be within specifications.  Record the Retention Times for all components of interest.

3.0 Linearity :

Inject in duplicate at least 6 different standard concentrations covering range 80% to 120% of the target concentration into the HPLC system.  Plot average peak response versus the actual concentration.

The response may be considered linear, if the correlation coefficient is derived from the least squares fit of the data is not less than 0.999. In case of discrepancy still exists, 0.999 correlation, investigate and explain the reason.

4.0 Method Precision

Assay not less than 6 individual sample weights from a homogeneous blend preparations (as per manufacturing formula).  The relative standard deviation should not be more than 2.0%.

5.0 Recovery and Accuracy of Method

Recovery from the spiked Placebo: Prepare solutions in triplicate corresponding to approximately 80%, 100% and 120% of working concentration as per the test procedure by spiking placebo (amount of placebo should be equivalent to the weight of placebo present in the sample amount required for the sample preparation as per test method) with different amounts of the drug substance.

Assay the samples by following the test procedure and calculate the concentration found in % of working concentration. Calculate the mean recovery, the Relative standard Deviation for the 80%, 100% & 120% spiked placebo data.  The mean recovery should be between 97 – 103%

In case of discrepancy repeat the experiment. If these criteria are still not met, investigate the reason.

 6.0 Placebo Interference                                   

Inject placebo and ensure there is no interference of excipients.

7.0 Range

If the linearity and the accuracy of the method is acceptable and the Relative Standard Deviation of  the accuracy data does not exceed 2.0% then range of the method is the concentration range used for the accuracy of the method (75-125%).

8.0 Ruggedness

System to system variation :

System to system variability study should be conducted on two HPLC systems with two different columns (same or different manufacturer) using Six assay preparations prepared as per manufacturing formula at different times under similar conditions.

Results should be within test specifications. Make the system suitability comparison table and establish the limits. The relative standard deviation should not be more than 2.0% for both the systems.

Results should be within test specifications.  Make the system suitability comparison table and establish the limits, if it is not in USP.  The relative standard deviation should not be more than 2.0% for both the columns.

Analyst to Analyst Variation :

Analyst to Analyst variability study should be conducted by two analysts on the same HPLC system at different times under similar conditions using Six different assay preparations prepared as per manufacturing formula.

Results should be within test specifications. Make the system suitability comparison table and establish the limits, if it is not in USP.  The relative standard deviation should not be more than 2.0% by both the analyst.

Laboratory to Laboratory Variation :

Laboratory-to-Laboratory variability study should be conducted in different labs on different HPLC systems at different times under similar conditions using Six different assay preparations prepared as per manufacturing formula.

Results should be within test specifications.  Make the system suitability comparison table and establish the limits, if it is not in USP. The relative standard deviation should not be more than 2.0% in both the laboratories.

9.0 Robustness :

Effect of variation in mobile phase composition:

Two mobile phases should be prepared having different concentrations of the method organic phase composition and inject into the HPLC system.

The system suitability values should be evaluated for peaks of interest using both the mobile phases and the results should be within the limits for mobile phase.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

Effect of variation of pH of buffer in mobile phase :

Two mobile phases should be prepared having buffers with ± 0.5 of the method pH and inject into the HPLC system.

The system suitability values should be evaluated for peaks of interest using both the mobile phase.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

Effect of variation in flow rate :

Prepare the solution as per the test procedure and inject into the HPLC system at different flow rates (+ 10%).

Effect of variation in column temperature :

Prepare the solution as per the test procedure and inject into the HPLC system at ambient ± 5°C column temperatures.

The system suitability values should be evaluated for peaks of interest at both the column temperatures and the results should be within the limits.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

10.0 Stability of standard and test preparations on Auto injector

The stability of test preparations on Auto injector should be established over the period time by injecting into the HPLC system at 4 hours intervals upto 24 hours. The relative standard deviation of peak area for standard and test preparations should be not more than 2.0%.

Qualification of visual inspectors for media fill vial inspection

 1.        OBJECTIVE

To lay down a procedure for qualification of visual inspectors for media fill vial inspection.

2.         RESPONSIBILITY

2.1 Technical Assistant – To undergo the qualification test.

2.2   Microbiologist – Preparation of vials / check for qualification.

3.       PROCEDURE

3.1    Materials and Equipment Required

3.1.1 Sterile Vials

3.1.2 Rubber stoppers

3.1.3 Flip off seals

3.1.4 S.S Tray

3.1.5 Sealer

3.1.6 De-sealer

3.1.7 Gloves & Nose masks.

3.2    Media and Culture requirement

  • Sterile SCDM
  • Culture suspension – Less than 100 cfu/ml of E.coli and Staphylococcus aureus

3.3  Aseptically dispense the sterile SCDM into 150 no’s of vials and seal the vials using sterile  rubber stoppers and flip off seals.

Note:   The quantity of media dispensed should not be less than 50 % of the vial size.

3.4  Likewise dispense sterile SCDM in another 30 no’s of vials under the biological safety cabinet.

3.5 Make the prepared SCDM vials into two sets containing 15 nos. of vials for each set.

3.6 Set -1 vials shall be inoculated with 3 different inoculum size of E.coli (i.e., 0.25 ml, 0.5 ml, and   1.0 ml culture suspension) in each of 5 no’s of sterile SCDM vials.

3.7 Set -2 vials shall be inoculated with 3 different inoculum size of Staph. aureus (i.e., 0.25 ml, 0.5 ml,and 1.0 ml culture suspension) in each of 5 no’s of sterile SCDM vials

3.8 Seal both the Set-1 and Set-2 vials.

3.9 Incubate all the culture inoculated SCDM (30no’s) and sterile SCDM (150 no’s) vials at 30-35°C for 24- 48 hours. After the incubation period check the inoculated vials for turbidity and proceed for visual inspection test.

3.10 Qualification of the inspectors.

3.10.1   The inspector shall be given a set of 150 no’s of sterile media fill vials along with 30  no’s of turbid vials that are numbered serially from 1, 2, 3,.. up to 180, mixed and kept randomly in a S.S tray.

3.10.2 The vial inspection for turbidity shall be done in the black/white background under fluorescent light.

3.10.3 After completion of visual inspection, the inspector shall separate all the turbid vials from sterile vials and the same shall be verified by the microbiologist.

3.10.4 The details of turbid vials and sterile vials shall be entered in Annexure1.

3.10.5 Based on the assignment of the vials i.e. 100%, the inspector shall be qualified. If the visual inspector fails to separate all the turbid vials repeat the qualification test.

3.10.6 Re-qualification of the inspector shall be carried out once in a year.

3.10.7 Eye check of the inspector shall be carried out once in six months

4.         ABBREVIATIONS

    4.1        QA       –    Quality Assurance        

   4.2        QC       –     Quality Control 

   4.3        Cfu       –   Colony forming units

   4.4        SCDM  –   Soya bean casein digest medium

   4.5        no’s      –   Numbers

5.         REFERENCES

Nil

6.         ANNEXURES

6.1        Annexure-1       Vial inspection Qualification Record

Annexure-1

VIAL INSPECTION QUALIFICATION RECORD

Name of the inspector :           

Date of qualification test:

Type of vials:

S.NoNo .of Sterile vials observedNo .of Turbid vials observedQualifiedDisqualifiedTotal
        
        
        
        
        
        
        
        
        
        
Done by/Date  
Checked by/Date  

Flow chart of deviation in Pharmaceutical

Below mentioned are the flow chart for deviation and everybody working in pharmaceutical industry must know the basic of deviation and its steps from login to closer.

The person identifying the occurrence of deviation shall be termed as observer and the observer shall inform about the occurrence to the Initiator and deviation shall be initiated within 24 hours of the incidence or occurrence. Then immediate action shall be taken in consultation with concerned department head and with Quality Assurance. The deviation shall be reviewed by the HOD of respective department along with risk and impact assessment in order to know the impact on product quality then it shall be reviewed by QA and QA shall check the similar type of deviation repeated in the past 1 year and will allocate the deviation number and categorised the deviation as minor, major and critical etc. and same shall be approved by Head Quality assurance.

Investigation shall be carried out for the deviation to identify the root cause as per site or plant investigation Standard operating procedure by using various investigation tools then based upon the root cause Corrective action and Preventive action (CAPA) shall be proposed with target completion date along with CAPA number.

Then final conclusion shall be provided by Quality assurance department which includes batch acceptable or rejected etc.

Then same shall be forwarded to customer or Qualified person for approval or notification and if required for regulatory comments then disposition of material or batches which are affected shall be released to the next step or market shall be decided and then the deviation shall be forwarded to CQA for approval.

Deviation shall be closed by initiator after completion of proposed action plan along with supporting documents and CAPA shall be logged in CAPA form and in logbook.

Pl note that all the deviation shall be closed before release if the product or batch to the market.

Flow of deviation

Flow chart of Change Control

Below mentioned are the flow chart for change control and everybody working in pharmaceutical industry must know the basic of change control and its steps from login to closer.

As we know that if we want to change in any system, equipment, written procedure, documents etc we have to raise change control and it should be reviewed by the department head and same shall be submitted to QA and QA will review the same and allocate the number and will categorised the Change control like minor or major and select the reviewers for impact assessment and then the Change control shall be forwarded to regulatory and customer for impact assessment ( pl note that all the change controls shall not be forwarded to regulatory and customer). After that Quality assurance shall compile the actions of change control which includes the activities to be performed after approval of change control I.e documents to be prepared, validation to be performed, stability to be performed, training to be imparted, old documents to be made obsolete etc… based upon the description of change controls.

Then Head Quality shall review and approve or rejects the change control and after approval the same shall be forwarded to Corporate quality assurance for approval and same can be implemented.

After completion of all actions with in the target completion date the change control can be closed and initiator department has to closed the change control and finally same shall closed by Quality assurance.

Change control flow

Quality Risk Management (QRM) Report for Non Operational Area Shutdown

The scope of this Risk assessment report is to provide the procedure for Quality Risk Management study for the non operational area shutdown and we can save the utility bills of our organization and can implement the same at our respective sites without impacting the product quality.

The Approach adopted to carry out risk assessment is failure mode effect analysis (FMEA) and in this report we have to study various risk parts like Risk identification, Risk analysis, evaluation, Severity, occurrence, detection, critical control points, current control points and Risk priority number.

Risk Review :

As per the QRM execution for the process of non operational area shut down various risk were identified. All the identification parameter meets the current control points under acceptable mode. The risk level based on Risk Priority number (RPN) is found widely acceptable.

Conclusion :

Based on QRM execution and risk review it was concluded that the risk involve in shutdown of nonoperational area is widely acceptable.