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1.0 Objective:

The purpose of conducting the validation of disinfectant (70 % Isopropyl Alcohol) is:

  • To evaluate the efficacy of the disinfectant using defined organism to be studied as defined in the validation protocol.
  • To establish the shelf life of the disinfectant when prepared & stored as defined in the respective SOP

2.0 Scope:

This Protocol is applicable for evaluation of efficacy & establish shelf life of the 70% Isopropyl Alcohol

3.0 Responsibility:

            Microbiology Officer -

                        -     To prepare the validation protocol & summary report.

                        -     To perform the validation as per the approved protocol.

                        -     To summarize the validation results.

                        -     To report non-conformances,

                        Manager QA -

                        -     To review the validation protocol & summary.

                        -     To review the validation data.

                        -     To evaluate any non-conformance & subsequent CAPA.

                        Head  – QC -

                        -     To review the protocol.

                        -     To review & approve validation data & report.

                        -     To review or investigate any non-conformance/failures.

                     -     To ensure initiation & execution of CAPA.

                     -     To ensure execution of the protocol.

                        Head – QA

-                    To review & approve the protocol. 

  • To ensure implement the validation programme according to the protocol.

                       Head – Quality

-                    To review & authorize the protocol. 

4.0 Reason for validation / Re-validation:

       Validation / Re-validation shall be carried out in case of

  • A new hand disinfectant in place of Isopropyl Alcohol
  • In case of change in concentration or addition of any additive

5.0 Procedure:

Suspension method shall be used for determination of the disinfectant ( 70 % Isopropyl Alcohol ) antimicrobial efficacy, the shelf life of the disinfectant shall also be established. The disinfectant shall be prepared & stored as per current version of SOP No.HR-024. The Micro-organism taken to determine the efficacy of the disinfectant is based on the historical experience & as well as some available published literature. The list of microorganism, their MTCC no, growth media, incubation temperature & period are defined as under-

 5.1   List of Micro-organism:

Following micro organisms should be selected for the Validation

S. No.
  •  

MTCC No.
  •  

Incubation period
  1.  

E.coli

MTCC -1687
  1.  

48 Hrs
  1.  

Salmonella

MTCC-3858

SCDA

48 Hrs
  1.  

S. aureus

MTCC – 737

SCDA

48 Hrs
  1.  

P. aruginosa

MTCC-1688

SCDA

48 Hrs
  1.  

Candida albicans

MTCC-227

SDA

72 Hrs
  1.  

Aspergilus Brasiliansis

MTCC-1344

SDA

72 Hrs

SDA-Sabouraud dextrose Agar, SCDA- Soybean Casein Digest Agar

5.2         Preparation of Disinfectant:

Composition of the disinfectant & working concentration & method of preparations are defined under current version of SOP No. HR-024.

Disinfectant should be prepared freshly (Not more than 4 hrs old) to take for initial testing. Thereafter it shall be stored as defined on the label of reference SOP No. QA-055.

5.3         Frequency of testing:

The same preparation prepared in duplicate shall be tested for its efficacy in order to check its stability under defined conditions when prepared & stored as defined in the SOP No.HR-024. The testing frequency shall be Zero day (within 4 hrs. of the solution preparation), after two days, five days, seven days & nine days of preparation of the disinfectant solution.

The date of testing may be altered by +1 day after 5th day of testing. All disinfectant (70 % IPA)are prepared using purified water

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  1. OBJECTIVE:

The objective of this protocol is to verify the Antimicrobial effectiveness of sterile penicillinase enzyme used as neutralizing agent in Microbial limit test for Beta lactam Products.

  1. SCOPE:

The scope of this study is limited to products manufactured in Beta lactam product plant of Neopharma. This protocol is applicable to meet the requirements for verification of Antimicrobial effectiveness of sterile penicillinase enzyme. Penicillinase defined as a group of enzymes with varying specificity for hydrolysis of ß-lactam compounds. Sterile penicillinase enzyme acts as neutralizing agent in which inactivate beta lactam antibiotics.

 

  1. RESPONSIBILITIES:

Validation/Quality Assurance:

  1. Preparation of Protocol.
  2. Ensure that all pre-requisites have been met and protocol information is completed.
  3. Execution of Protocol.
  4. Compilation of results and close out of report.

 

Validation/Quality Assurance:

  1. To review the protocol and report
  2. Approval of the protocol and report

 

  1. TEAM MEMBERS:

 

S. No.

Department

Name

Sign / Date

1

Quality Control

 

 

2

Validation / QA

 

 

 

 

 

 

  1. BACKGROUND:

 

 

Penicillinase defined as a group of enzymes with varying specificity for hydrolysis of ß-lactam compounds. Penicillinase are enzymes produced by bacteria that provide multi-resistance to β-lactam antibiotics .Through hydrolysis, the lactamase enzyme breaks the β-lactam ring open, deactivating the molecule's antibacterial properties. This protocol is to verify the Antimicrobial effectiveness of sterile Penicillinase enzyme. To verify the antimicrobial effectiveness of penicillinase, the  study is initiated.

 

  1. METHODOLOGY:

 

 

The methodology for the Antimicrobial effectiveness of sterile penicillinase enzyme study is described here under. For the execution of study below aspects are considered.

  1.  Pre – requisites of the study
  2. Procedure

 

  1. Pre-requisites of the study:
  • Test tube /Bottles/Flasks
  • Sterile Petri plates
  • Inoculation loops
  • Laminar air flow /Bio-safety cabinet
  • Micropipette and Sterilized Tips
  • Incubators
  • Vortex mixer
  • Colony counter
    1. Culture media & microbial Culture Required:
  • Soyabean Casein Digest Broth (SCDB)
  • Soyabean Casein Digest Agar (SCDA)
  • Escherichia coli (ATCC No. 8739
  • Penicillinase Enzyme
    1.   Procedure:

7.3.1 Preparation of Culture Suspension

  • Prepare inoculum as per the current SOP
  • Escherichia coli (ATCC No. 8739) used for validation of TAMC [Select culture suspension to be inoculated into the test solution, so that the final concentration of the organism is not more than 100 cfu/ml].

 7.3.2 Preparation of sterile penicillinase enzyme Bacillus cereus:

  • 5gm of Potassium di-hydrogen phosphate and 11.0gms of di-potassium hydrogen phosphate and dilute with 900mL of water. Adjust the pH to 7.0 by adding diluted NaOH solution and make up to 1000mL. Sterilize the solution.
  • Weigh 18.03 mg of enzyme accurately to 100mL of sterile phosphate buffer to prepare 84units/mL of sterile Penicillinase enzyme.

 

  1. Sample Preparation:
  • Take 06 containers of 90 ml of Soyabean casein digest medium. Carry out the sample preparation as per the below table.

SCDM

Container number

Product

(g)

Organism Inoculated

(Not more than 100 cfu/ml)

Sterile penicillinase enzyme

( ml)
  1.  
  1.  

Escherichia coli (ATCC No. 8739)

0.5 ml
  1.  
  1.  

Escherichia coli (ATCC No. 8739)
  •  
  1.  
  1.  

Escherichia coli (ATCC No. 8739)

1.5 ml
  1.  
  1.  

Escherichia coli (ATCC No. 8739)

2.0 ml

4
  1.  

Escherichia coli (ATCC No. 8739)
  1.  

5
  1.  

Escherichia coli (ATCC No. 8739)
  1.  

6
  1.  

NA
  1.  

 

  • In 1-3 container add 10 g product, 100 Cfu of inoculum and different aliquots of sterile penicillinase (0.5, 1.0, 1.5 & 2ml) enzyme and shake the sample preparation to create a homogeneous solution or suspension.
  • In container-4 add only 10 g product and 100cfu of inoculum and shake the sample preparation to create a homogeneous solution or suspension.
  • Container-5 is positive control which contains only 100cfu of inoculum
  • Container-6 is negative control which contains only 90ml Soyabean casein digested media.
  • Pipette out 1 ml of the sample preparation into duplicates from each of the container number 1 to 6.
  • Add 20–25 ml of Soyabean casein digest agar medium, cooled to about 45°C to these plates.
  • Mix the plate contents well by rotating the plate clockwise and anticlockwise motion. Allow to solidify at room temperature.
  • Incubate the Soyabean Casein Digest Agar plates at 30-35oC for 3-5 days.
  • Once incubation completed plates were checked for antimicrobial effectiveness of penicillinase enzyme.
  • The particular dilution which shows the maximum activity shall be selected and same shall be checked for 1st, 7th, 14th ,21 and 28th day.
  • Antimicrobial effectiveness of penicillinase enzyme shall be checked for 1st, 7th, 14th ,21st and 28th day.
  1. TESTS AND ACCEPTANCE CRITERIA:
  • The recovery observed for of the test organisms (container 1-3) shall not differ by a factor greater than 2 from the calculated value for Positive control plates. (recovery rate should be between 50% - 200%).
  • No growth in the negative control must be obtained to establish the sterility of the media.
  • No growth in the Container-4 must be obtained to establish the effectiveness sterile penicillinase enzyme.
  • The recovery observed for any of the test organisms (container -5) shall not differ by a factor greater than 2 from the calculated value for Positive control plates. (recovery rate should be between 50% - 200%).
  1. APPENDIX

Sr. No.

Details

NA

NA

 

  1. ABBREVIATION
  1.  
  •  

Quality Control
  1.  
  •  

Standard operating Procedure
  1.  
  •  

Quality Assurance
  1.  
  •  

American Type Culture Collection.
  1.  
  •  

Not More Than

 

 

 

 

 


PERFORMANCE QUALIFICATION (PQ) PROTOCOL FOR FOURIER TRANSFORM INFRARED (FTIR) SPECTROMETER

1.0 OBJECTIVE

To verify and document that the FTIR spectrometer consistently performs to its intended use under actual conditions, and complies with GMP and relevant pharmacopoeial/regulatory standards including USP <857>, EP 2.2.24, and JP.

2.0 SCOPE

Covers performance verification of:

  • Wavenumber accuracy and precision
  • Resolution verification
  • Signal-to-noise ratio
  • Photometric accuracy and reproducibility
  • Detector linearity (optional based on software capability)
  • System suitability under normal use

3.0 ACCEPTANCE CRITERIA SUMMARY

Parameter

Reference Standard

Acceptance Criteria

Wavenumber Accuracy

USP/EP Polystyrene film

Within ±0.01 cm⁻¹ (USP), ±0.1% (EP)

Wavenumber Precision

Repeated scans

SD ≤ 0.005 cm⁻¹

Resolution Verification

1028 / 966 cm⁻¹ peaks

Ratio ≥ 1.5 (USP/EP standard)

Signal-to-Noise Ratio

Blank region ~2200 cm⁻¹

≥ 35,000:1 as per manufacturer specs

Absorbance Precision

Repeated scans

%RSD ≤ 1%

Photometric Accuracy

Certified reference material

Within ±0.01 AU (or ±1%)

Baseline Stability

Baseline drift

< ±0.01 AU across scan

System Suitability

Daily test with standard

All peaks reproducible, baseline stable

4.0 DETAILED TEST DESCRIPTIONS

4.1WAVE NUMBER ACCURACY TEST

4.1.1 Purpose:
Verify the instrument accurately reports IR peak positions in accordance with USP/EP/JP standards.

4.1.2 Reference:

  • USP <857>
  • EP 2.2.24
  • Certified polystyrene standard film (NIST traceable)

4.1.3 Procedure:

  1. Warm up instrument (≥30 minutes).
  2. Scan certified polystyrene film (resolution: 2 cm⁻¹).
  3. Identify and record peaks at:
    • 1601.4 cm⁻¹
    • 1583.0 cm⁻¹
    • 1154.5 cm⁻¹
  4. Repeat scan 3 times.
  5. Compare results with certified values.

4.1.4 Acceptance Criteria:

  • Each peak deviation ≤ ±0.01 cm⁻¹ (USP), ≤ ±0.1% (EP).

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PERFORMANCE QUALIFICATION (PQ) PROTOCOL FOR UV-VISIBLE SPECTROPHOTOMETER

1.0 OBJECTIVE

The purpose of this protocol is to establish documented evidence that the UV-Visible Spectrophotometer, installed in the Quality Control laboratory, consistently performs according to pre-defined acceptance criteria under actual operating conditions. This ensures the instrument is fit for its intended GMP use.

2.0 SCOPE

This PQ protocol applies to the UV-Visible Spectrophotometer:

  • Instrument Make/Model: [e.g., Agilent Cary 60, PerkinElmer Lambda 365, Shimadzu UV-1800]

  • Serial Number: [Serial Number]

  • Location: QC Laboratory, [Company Name], [Site Location]

  • Software: [e.g., Agilent UV-Pro 21 CFR Part 11 Version X.X, PerkinElmer UVWin, Shimadzu UVProbe]

  • Calibration Due Date: [______________]

  • Instrument Configuration: [e.g., 1 cm Quartz Cuvettes, Halogen Lamp, Deuterium Tungsten Source, etc.]

The PQ will cover performance verification in accordance with USP <857> and ICH Q2(R2), and ensure the instrument is suitable for assays, dissolution testing, and method validations.

3.0 RESPONSIBILITIES (RACI MATRIX)

Activity

Analyst

QC Reviewer

QA

Instrument Vendor

Execution of PQ tests

R

A

C

C

Review of data

C

R

A

-

Final approval

-

C

R

-

Maintenance (if needed)

C

C

C

R

      R = Responsible, A = Accountable, C = Consulted

4.0 REFERENCE DOCUMENTS

Title

Reference Code

USP <857> Spectrophotometry

USP

USP <1852>, <1853>

USP

ICH Q2(R2): Validation of Analytical Procedures

ICH

FDA Guidance for Analytical Instrument Qualification

FDA

Internal SOP: Analytical Equipment Qualification

SOP/QA/XXX

GAMP 5 Guidelines

ISPE

5.0 EQUIPMENT, MATERIALS & STANDARDS

Item

Grade

Certificate

Expiry

Supplier

Holmium Oxide Filter

NIST Traceable

COA-HOX-001

2 Years

[Name]

Potassium Dichromate

AR/USP

COA-K2CR2O7-023

[Date]

[Name]

Toluene/Hexane Mixture

HPLC/UV Grade

COA-THEX-005

[Date]

[Name]

1 cm Quartz Cuvettes

Matched

NA

NA

[Name]

1.2% KCl Solution

Prepared Fresh

NA

NA

In-house

6.0 ENVIRONMENTAL CONDITIONS

The following environmental parameters must be maintained and logged during PQ:

Parameter

Specification

Temperature

20–25 °C

Relative Humidity

≤ 60% RH

Vibration

Nil

Power Supply

230V ± 10%, 50 Hz

Record values during each test.

7.0 TEST PROCEDURES

7.1WAVELENGTH ACCURACY TEST

7.1.1Objective:

To verify that the UV spectrophotometer correctly measures standard wavelength values.

7.1.2Standard:

Holmium oxide filter or solution

7.1.3Procedure:

  1. Warm up the instrument for ≥30 minutes.
  2. Insert holmium oxide filter or scan 4% solution.
  3. Perform a full scan from 200–600 nm.
  4. Record absorbance peak positions.

Expected Peaks:

  • 241.1 nm
  • 287.1 nm
  • 361.5 nm
  • 536.2 nm

7.1.4Acceptance Criteria:

 ±1.0 nm of certified values.

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PERFORMANCE QUALIFICATION (PQ) PROTOCOL FOR GAS CHROMATOGRAPHY SYSTEM

1.0 OBJECTIVE

To confirm and document that the Gas Chromatography system performs reliably and consistently under actual laboratory conditions, complying with USP, EP, ICH, and cGMP guidelines.

2.0 SCOPE

This Performance Qualification (PQ) protocol applies to the Gas Chromatography (GC) systems utilized for the analysis of pharmaceutical products, active pharmaceutical ingredients (APIs), and raw materials.

It specifically covers the performance verification of the following equipment under actual laboratory operating conditions, ensuring compliance with GMP regulatory expectations (USFDA, EMA, WHO, PIC/S) and pharmacopoeia standards (USP, EP).

2.1 Equipment Details

Parameter

Description

Instrument Make

Agilent / Shimadzu / Thermo

Model

7890B / GC-2030 / Trace 1310

Detector

FID (Flame Ionization Detector)

Software

OpenLab CDS / LabSolutions / Chromeleon

Serial No.

[Insert Serial No.]

Column Installed

DB-5 / Rtx-5 / Equivalent

Calibration Due

  

3.0RESPONSIBILITIES

Role

Responsibility

QC Analyst

Execution of tests, recording data

QC Manager

Technical review and interpretation

QA

Protocol approval, deviation handling, and final verification

4.0REFERENCE DOCUMENTS

  • USP <621> Chromatography
  • EP 2.2.28 Gas Chromatography
  • ICH Q2(R1): Validation of Analytical Procedures
  • 21 CFR Part 211 and Part 11
  • Internal SOPs on GC operation, calibration, maintenance, and system suitability

5.0EQUIPMENT AND MATERIALS

  • Gas Chromatograph with injector and detector (e.g., FID)
  • Autosampler (if applicable)
  • Data system with validated software (21 CFR Part 11 compliant)
  • Columns: DB-5 or method-specific
  • GC grade gases: Helium, Hydrogen, Air
  • Standard solutions (e.g., BTEX, Grob test mixture, or method-specific)
  • Certified Reference Materials (CRMs)
  • Analytical-grade solvents
  • Calibrated glassware, syringes, and crimp-top vials

6.0PREREQUISITES

  • Completed IQ and OQ
  • Current calibration certificates
  • Environmental monitoring log (Temp 20–25°C, RH 40–60%)
  • Clean and stabilized column
  • Gas pressures verified and leak tested
  • Maintenance record checked

7.0PERFORMANCE QUALIFICATION TESTS AND PROCEDURES

7.1System Suitability Test (SST)

  1. Purpose:

To confirm that the GC system is functioning as expected, providing consistent results for quantitative analysis and meeting established performance standards.

  1. Procedure:
    1. Sample Preparation: Prepare a multi-analyte standard mix (e.g., BTEX, Grob test mixture, or method-specific) at a known concentration.
    2. System Setup:
  • Install the prepared sample in the GC system.
    1. Set the GC parameters as per validated method parameters:
  • Column: DB-5, 30 m × 0.25 mm × 0.25 µm
  • Carrier Gas: Helium at 1.0 mL/min
  • Split ratio: 10:1
  • Injector Temp: 250°C
  • Detector Temp: 300°C
  • Oven: 60°C (1 min) → 10°C/min → 250°C (5 min)
  • Ensure the system has been stabilized and leak-free.
    1. Injection:
  • Inject a fixed volume (e.g., 1 µL) of the multi-analyte standard mix.
  • Perform the injection in triplicate (minimum of three injections).
  • Ensure the system is in equilibrium before each injection (i.e., injector and detector temperatures are stable).
    1. Analysis:
  • Record the retention time (RT), area, and other critical chromatographic parameters.
  • Calculate the theoretical plates (N), tailing factor (TF), resolution (Rs), and repeatability.
    1. Repeat for multiple standard mixes to evaluate consistency across the operating range.
  • Acceptance Criteria:
  • Retention Time (RT): RSD ≤ 1.0%
  • Area RSD: ≤ 2.0%
  • Plate Count (N): ≥ 2000
  • Tailing Factor (TF): ≤ 2.0
  • Resolution (Rs): ≥ 1.5


Performance Qualification (PQ) Protocol For HPLC Chromatography System

1.0 OBJECTIVE

To confirm and document that the High-Performance Liquid Chromatography (HPLC) system operates reliably and consistently under actual laboratory conditions, meeting USP/EP/JP, ICH Q2(R1), and cGMP regulatory guidelines.

2.0 SCOPE

This protocol applies to the PQ of HPLC systems used in pharmaceutical Quality Control laboratories for analysis of raw materials, APIs, intermediates, and finished products. It is applicable to:

  • Make: Agilent / Shimadzu / Waters / Thermo

  • Model: 1260 Infinity II / LC-2030C / Alliance e2695 / Vanquish

  • Detector: UV/Vis / PDA / RI / FL

  • Software: Empower / LabSolutions / Chromeleon

  • Column Used: C18, 250 mm × 4.6 mm, 5 µm or method-specific

  • Serial Number: [Insert Serial Number]

​3.0  REFERENCE DOCUMENTS

  • USP <621> Chromatography
  • EP 2.2.29 HPLC
  • ICH Q2(R1): Validation of Analytical Procedures
  • 21 CFR Part 11 and Part 211
  • Internal SOPs (Operation, Calibration, Maintenance, SST for HPLC)

4.0  EQUIPMENT AND MATERIALS

  • HPLC system with autosampler, pump, column oven, detector, and software
  • Analytical column (C18, 250 mm × 4.6 mm × 5 µm)
  • Reference standard (e.g., Caffeine, Acetophenone, or method-specific)
  • Mobile Phase (as per method, HPLC grade solvents)
  • Calibrated volumetric glassware and filtration system
  • 0.45 µm syringe/nylon membrane filters

5.0  PREREQUISITES

  • IQ & OQ completion report
  • Valid calibration certificates
  • Stabilized system (Temp: 20–25°C; RH: 40–60%)
  • Clean and installed column
  • Mobile phase freshly prepared and filtered
  • No pending maintenance

6.0  PERFORMANCE QUALIFICATION TESTS

6.1 System Suitability Test (SST)

Purpose: Verify the operational readiness of the HPLC system.
Test Sample: Standard solution of known concentration (e.g., caffeine).
Procedure:

  • Equilibrate system with mobile phase for ≥30 minutes
  • Perform six replicate injections of the standard solution
  • Record retention time, area, theoretical plates, tailing factor, resolution

Acceptance Criteria:

  • %RSD of RT: ≤ 1.0%
  • %RSD of Area: ≤ 2.0%
  • Theoretical Plates (N): ≥ 2000
  • Tailing Factor: ≤ 2.0
  • Resolution: ≥ 2.0 (if two peaks present)

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