process validation protocol for methylcobalamin niacinamide and pyridoxine injection

SUPERSEDES PROTOCOL No.
DATE OF VALIDATION
VALIDATION BATCH NUMBERS
VALIDATION BATCH SIZE

PROTOCOL CONTENTS

S. NO.	TITLE
1.0	PROTOCOL APPROVAL
2.0	OBJECTIVE
3.0	SCOPE
4.0	RESPONSIBILITY
5.0	PRODUCT INFORMATION
6.0	PROCESS VALIDATION METHODOLOGY
7.0	REASON FOR VALIDATION
8.0	SITE OF STUDY
9.0	PROCESS VALIDATION PRE-REQUISITE
10.0	MANUFACTURING FORMULA
11.0	PROCESS FLOW DIAGRAM
12.0	PROCEDURE
13.0	REVALIDATION
14.0	VALIDATION STABILITY PLAN
15.0	REFERENCES
16.0	DOCUMENTS TO BE ATTACHED
17.0	NON COMPLIANCE, IF ANY
18.0	DEVIATION FROM PRE-DEFINED SPECIFICATION, IF ANY
19.0	CHANGE CONTROL, IF ANY
20.0	ABBREVIATIONS
21.0	REVISION HISTORY

1.0 PROTOCOL APPROVAL:

PREPARED BY:

DEPARTMENT	NAME	DESIGNATION	SIGNATURE/DATE
QUALITY ASSURANCE

REVIEWED BY:

DEPARTMENT	NAME	DESIGNATION	SIGNATURE/DATE
QUALITY ASSURANCE
ENGINEERING
PRODUCTION
QUALITY CONTROL

APPROVED BY:

DEPARTMENT	NAME	DESIGNATION	SIGNATURE/DATE
HEAD QUALITY ASSURANCE

2.0 OBJECTIVE:
• To validate the manufacturing and packing process of Methylcobalamin, Niacinamide & Pyridoxine injection as per

the Master Formula Record to establish documentary evidence that the process will consistently produce the product,

meeting its predetermined In process and Finished Product Specification by examining (total no. of 03 batches) consecutive production batches.
3.0 SCOPE:
• The Protocol covers all aspects of Process Validation of Methylcobalamin, Niacinamide & Pyridoxine Injection manufactured

as per the Master Formula Record in Ampoule Line (Injection Block) at A B C Pharma
• Three consecutive production batches of Methylcobalamin, Niacinamide and Pyridoxine Injection manufactured as per its

Master Formula Record are to be validated as per this protocol.
• Report generated after performing sampling and testing is to be utilized for finalization of Master Formula Record as well as Batch Manufacturing and Packing Record.
4.0 RESPONSIBILITY:
• The Validation Group, comprising of a representative from each of the following departments, shall be responsible for the overall compliance of this Protocol.

DEPARTMENTS	RESPONSIBILITIES
Quality Assurance	To Prepare, Review and Approval of Protocol Protocol Training To monitor all validation activities and ensuring the validation as per the protocol. To monitor protocol completeness and technical accuracy To review and compile the data and approve the validated procedure, if found as per acceptance criteria.
Production	· To review of protocol. · To schedule the validation activity. · To provide personnel to assist for execution of this protocol.
Quality Control	· To review of protocol · To provide all applicable analytical procedures and documentation necessary for execution of this protocol. · To test the sample collected and provide all Analytical Data.
Engineering	· To review of protocol. · Co-ordination, Execution and technical support in process validation activity.

5.0 PRODUCT INFORMATION:

Product Code
Generic Name	Methylcobalamin, Niacinamide and Pyridoxine IP
Label Claim	Each 02 ml contains
	Methylcobalamin IP………….1000 mcg
	Niacinamide IP……………….100 mg
	Pyridoxine IP…………………100 mg
	Water for Injection IP………….q. s.
Standard Batch Size:
Batch Size:
Pack Size:	02 ml
Primary Pack Description	02 ml Amber Glass Ampoules with green ring
Pack sizes	01 Ampoule per inner carton (Blister pack)
Shelf-life	18 Months
Storage Condition	Store Protected from Light and Moisture. Kept in cool and dry place
Product Description	Red colour Clear solution

6.0 PROCESS VALIDATION METHODOLOGY:
• Only after validation protocol approval, the protocol shall be executed.
• During the course of validation the documentation systems, laboratory controls, in process checks during production shall be evaluated.
• The process validation methodology consists of three basic parts critical process parameters monitoring, the routine in process and final

product release testing & additional validation sampling and testing.
• The type of validation to be carried out is Concurrent Validation.
• The validation exercise for three batches of production scale shall be done to assess the process consistency.
• QA departments shall take samples as per approved Process Validation Protocol.
• All parameters shall be recorded in relevant records (e.g. Process Validation Report, Formats and BMR & BPR etc.)
• QC shall analyze all validation samples and the raw data shall be recorded / attached with the report. Where applicable the graph

and data print outs of critical process parameters shall be obtained and attached.
• Process Validation data shall be recorded in the process validation report and the recorded data will be identified by date.
• All documents related to process validation including batch specific information, certificates for test instrument used for analysis and

certificates for critical instruments of the processing equipment shall be attached to Process Validation Report.
• A final report shall be prepared summarizing the data obtained from the validation batches, conclusions drawn and recommendations, if any.

7.0 REASON FOR VALIDATION:
• New Product manufactured at Ampoule Line in ABC pharma

8.0 SITE OF STUDY:
• Injection Block at ABC Pharma

9.0 PROCESS VALIDATION PRE-REQUISITE:
• The batches shall be manufactured as per the batch manufacturing record / process.
• Three batches shall be taken for process validation study.
• Three consecutive successful runs shall be considered for completion of validation activity.
• The equipments used for manufacturing and processing of these batches shall be as per list of equipments.

Critical equipment/instrument must be in validated/qualified status being used during process validation.
• The raw material used for manufacturing shall be from approved vendors and shall be released by quality control.
• Sampling for in-process samples shall be carried as per established sampling procedure and plan.
• Critical in-process control shall be evaluated with respect to the laid down specification.
• Finished drug product of these batches shall be analyzed as per laid down test procedures and predetermined specifications.
9.1 Verification of Documents:
• Master document shall be approved, signed and dated by appropriate persons prior to commencement of batch processing.
• Verify the following master documents and record the details in Process Validation Report
a. Master Formula Record
b. Batch Manufacturing Record
c. Batch packing record
d. Process Validation Protocol and Report
e. Raw Material Specifications and Standard Testing Procedures
f. Packaging Material Specifications and Standard Testing Procedures
g. Finished Product Specification and Standard Testing Procedures
9.2 Training Record of Validation Team:
• All the personnel involved in the manufacturing and Packing of Validation Batches, Sampling and Testing of Validation Samples

should be appropriately trained both in their job related activities and on the process validation protocol by Head-QA.
• Verify the Training Records of the persons involved in the process validation activity and record the details in Process Validation Report.

9.3 Equipment Qualification Verification:
Ensure all equipments to be used for the manufacturing must be qualified. The reference Qualification Documents shall be verified and

mentioned in the Process Validation Report. The list of major equipments used for manufacturing of Methylcobalamin,

Niacinamide and Pyridoxine Injection IP in Ampoule line mentioned below:

S. No.	Name of Equipment / Machine	Identification No.
1.	Manufacturing Vessel
2.	Manufacturing Vessel
3.	Collection vessel
4.	Collection vessel
5.	Ampoule Washing Machine
6.	Depyrogenating Tunnel
7.	Ampoule filling & sealing Machine
8.	Laminar Air Flow Unit (filling & Sealing)
9.	Laminar Air Flow Unit (Cooling zone)
10.	Laminar Air Flow Unit (Filtration Room)
11.	Laminar Air Flow Unit (Filtration Room)
12.	Autoclave
13.	Pure Steam Generation System
14.	Water System (WFI)
15.	Dynamic Garment Storage Cabinet
16.	Dynamic Pass Box
17.	Dynamic Pass Box
18.	Dynamic Pass Box
19.	Mobile Laminar Air Flow
20.	Mobile Laminar Air Flow

10.0 MANUFACTURING FORMULA:

10.1 Bill of Raw Materials:

Sr. No.	Ingredient	Pharmacopoeial Status	Label Claim	Theoretical Quantity (For 220 Liters)	Units	Overages	Source of API vendor
01.	Methylcobalamin	IP	500 mcg		Kg.	100 %
02.	Niacinamide	IP	50 mg		Kg.	10 %
03.	Pyridoxine Hcl	IP	50 mg		Kg.	10 %
04.	Benzyl Alcohol	IP	–		Ltr.	–
05.	Monothioglycerol	IP	–		Ltr.	—
06.	Di Sodium EDTA	IP	–		Kg.	—
07.	Sodium Hydroxide	IP	–		Kg.	—
08.	Water for injection	IP	–	Q.S.	Liter	—

10.2 Bill of Primary Packing Materials:

Sr. No.

Name of Material

Theoretical Quantity

(For 220 Liters)

Function

Unit

Source of PPM Vendor

02 ml Amber Glass Ampoules with green ring

100000 *

Primary

Packing Material

Nos.

* 5% excess quantity of material to compensate processing loss.

11.0 PROCESS FLOW DIAGRAM: (Manufacturing Process)

11.1 PROCESS FLOW DIAGRAM (Packing Process)

12.0 PROCEDURE:
12.1 SAMPLING METHODOLOGY:

S. No.	STAGE	DEPARTMENT	PROCEDURE
1.	Machine part Rinse/Swab	Production	Wash the Equipment’s as per the respective Cleaning Procedure.
		QA	Monitor the process as per the BMR and withdraw the samples as per Sampling Plan.
		QC	Timely complete the analysis and provide the results to QA
2.	Cleaning and Sterilization of Equipment’s	Production	Wash the Equipment’s as per the respective Cleaning Procedure and Sterilize the washed equipments at 121.4ºC for 30 minutes.
		QA	Monitor the process as per the BMR and cross verify Sterilization Charts.
		QC	Timely complete the analysis and provide the results to QA
3.	Water For Injection	Production	Take the WFI sample before bulk manufacturing from mixing tank. WFI Specification as pH – (5-7), Conductivity- NMT 1.3 µs/cm, BET- 0.25 EU/ml, Bio Burdon 10 CFU/100 ml, TOC NMT 500 PPB.
		QA	Monitor the process as per the BMR and cross verify Sterilization Charts.
		QC	Timely complete the analysis and provide the results to QA
4.	Preparation of Bulk Solution	Production	Prepare bulk Solution as per step mentioned in Batch Manufacturing record.
		QA	Monitor the process as per the BMR and withdraw the samples as per Sampling Plan.
		QC	Timely complete the analysis and provide the results to QA
5.	Filtration of Bulk Solution	Production	Perform the bubble point test of the membrane filter (pre integrity) Start the filtration from Mixing vessel to collection vessel by applying 0.2 micron filtered Nitrogen gas pressure. Perform the bubble point test of the membrane filter (post integrity).
		QA	Monitor the process as per the BMR & BPR and withdraw the samples as per Sampling Plan
		QC	Timely complete the analysis and provide the results to QA
6.	Ampoules washing	Production	Wash Ampoules sample collect during initial middle & end stage from washing area.
		QA	Monitor the process as per the BMR & BPR and withdraw the samples as per Sampling Plan
		QC	Timely complete the analysis and provide the results to QA
7.	Sterilization and Depyrogenated Ampoules	Production	Set the temperature of heater as per validated set parameters and sterile the ampoule through ampoule sterilization Tunnel. In order to sterilize the ampoules at NLT 310°C.
		QA	Monitor the process as per the BMR & BPR and withdraw the samples as per Sampling Plan
		QC	Timely complete the analysis and provide the results to QA
8.	Filling & Sealing	Production	Perform Filling and Sealing Operation as described in Batch Manufacturing Record. The syringe is calibrated for fill volume range of 1.05ml to 1.15ml after the uniformity is established, start the Filling and Sealing Operation.
		QA	Monitor the process as per the BMR & BPR and withdraw the samples as per Sampling Plan
		QC	Timely complete the analysis and provide the results to QA.
9.	Visual Inspection of the Filled Liquid Ampoules	Production	Perform Visual Inspection of the filled Ampoules for any defects e.g. Scratches, Black or White Particles, sealing defect, Fiber & glass defect Volume Variation, etc.
		QA	Monitor the process as per the BMR/BPR and withdraw the samples as per Sampling Plan
		QC	Timely complete the analysis and provide the results to QA.
10.	Packing	Production	After getting approval from IPQA Department, pack the Inspected Liquid Ampoules as per the packing style mentioned in the BMR/BPR.
		QA	Monitor the process parameters as per the BPR and withdraw the samples as per the sampling plan. QA shall also withdraw the samples for stability as per the sampling plan.
		QC	Timely complete the analysis and provide the results to QA.

12.2 PROCESS STEPS AND CRITICAL PROCESS VARIABLES FOR VALIDATION:

PROCESS STEP	CRITICAL PROCESS VARIABLES	JUSTIFICATION
Cleaning and Sterilization of Equipment’s	Cleaning of Machine parts/Equipments Sterilization Time (30min.) Sterilization temperature (NLT 121.4⁰C)	The Process of check & ensure for Mixing Vessel, Collection Vessel and Machine parts Clean and sterilization.
Preparation of Bulk Solution	Mixing Time Process Time Mixing Speed	The process of mixing shall be evaluated for homogeneity of the solution through the sampling of mixed materials from 2 locations (Top Layer & Bottom Layer) at different time interval i.e. After 05 minutes, after 10 minutes and after 15 minutes of mixing. Sample shall be collected from different locations and 1 composite sample. The Samples shall be submitted to QC As per bulk specification.
Filtration of Bulk Solution	Pre & Post Integrity of Filters Process Time.	The process of Filtration shall be evaluated through the sampling of filtered solution for determination of Sterility. Samples shall be collected by the trained Microbiologist and perform Sterility Test.
Ampoules washing	Recirculated water Pressure 1.5 to 2.0 Kg/cm2 Compressed Air Pressure 1.5 to 3.0 Kg/cm2 Purified Water Pressure 1.5 to 2.0Kg/cm2 WFI Pressure 1.5 to 2.0Kg/cm²	The process of Ampoules washing shall be evaluated through the sampling of ampoule washing at different stage mention below. 1. Initial 2. Middle 3. End
Ampoules Sterilization	Differential Pressure in (Pa) · DP of Drying zone 05-15 mm of water · DP of Sterilization zone 10-25 mm of water · DP of Cooling zone 05-15 mm of water Temperatures in °C a. Drying zone b. Sterilization zone c. Cooling zone Speed of Conveyor (mm/ min)	The process of Ampoules Sterilization shall be evaluated through the sampling of Ampoule Sterilization at different stage mention below. 1. Initial 2. Middle 3. End
Filling and Sealing	Presence of Particles Process Time Machine Speed a. Minimum Speed (144 Ampoules/min.) b. Optimum Speed (192 Ampoules/min.) c. Maximum Speed (240 Ampoules/min.)	The process of Filling and Sealing shall be evaluated through the sampling of Filled and Sealed Ampoules which is produced at different time points as mentioned below: 1. At the start of filling and sealing operation. 2. At the middle of Filling and Sealing Operation. 3. Towards the end of Filling and Sealing Operation. Samples shall be submitted to QC for testing as per finished product specification Sterility & BET.
Packing	Visual Inspection Overprinting details Packing Style	Observation of Visual Inspection shall be check as per BPR. During packaging process At Start, middle and towards end of packing parameter shall be check. Overprinting details Packing Style Check As per batch packing record

12.3 SAMPLINGs AND ANALYSIS PLAN WITH ACCEPTANCE CRITERIA:
Collect the samples at various intervals at different operations as per the Sampling Plan mentioned below

Sample	Location of Collection	Test to be performed	Acceptance Criteria	Responsibility
Machine parts Rinse/Swab (Whichever applicable)	From Equipment washing area	Description	Clear colourless free from particulate matter	QC/Micro
		Clarity	Should be clear
		pH	5.0 to 7.0
		Conductivity	NMT 1.3 µs/cm
		Previous product Residue	No residue of previous product
		Bio Burdon	NMT 10 cfu/100 ml
Wash water of Cleaning	From Manufacturing tank, Collection vessel	Description	Clear colourless free from particulate matter	QC
		Clarity	Should be clear
		PH	5.0 to 7.0
		Conductivity	NMT 1.3 µs/cm
		Previous product Residue	No residue of previous product
Water for Injection	Before batch manufacturing	Description	Clear colourless free from particulate matter	QC/Micro
		pH	5.0 to 7.0
		Conductivity	NMT 1.3 µs/cm
		BET	NMT 0.25 EU/ml
Bulk Mixing	From Manufacturing tank (After 05, 10, 15 minutes Mixing) Top + Bottom at 2880 RPM & Composite Bulk	Description	As per Specification	QC/Micro
		pH
		Assay
		Bio Burdon
Bulk sample before filtration (Covering Hold Time Study)	From Manufacturing tank Initial Zero Hour	Description		QC/Micro
		pH
		Assay
		Bio Burdon
	From Manufacturing tank After 24 Hours	Description		QC/Micro
		pH
		Assay
		Bio Burdon
	From Manufacturing tank After 48 Hours	Description		QC/Micro
		pH
		Assay
		Bio Burdon
Bulk sample after filtration (Covering Hold Time Study)	From Filtration area Initial Zero Hour	Description		QC
		pH
		Assay
		Sterility		Micro
		BET		Micro
	From Filtration tank After 24 Hours	Description		QC
		pH
		Assay
		Sterility		Micro
	From Filtration tank After 48 Hours	Description		QC
		pH
		Assay
		Sterility		Micro
	From Filtration tank After 72 Hours	Description		QC
		pH
		Assay
		Sterility		Micro
		BET		Micro
Washing Ampoules	Washed Ampoule (For PV batches initial, Middle, End)	Clarity		QC
Depyrogenated Ampoule	From Turn table in ampoule filling & sealing room (PV batches Initial, Middle, End of batch at the start of filling)	Sterility		Micro
Depyrogenated Ampoule	Composite Sample	BET		Micro
Filling & Sealing	Initial, Middle and End Stage of filling Duration	Description, pH, Assay, Fill Volume & Sterility		QC/Micro
Finished sample	Finish Stage	Testing as per finished product Specification		QC/Micro
Packaging of filled Ampoules	Initial, Middle and End Stage of Packaging Duration	Correctness of coding and packing		QA/Production

12.4 SAMPLING LOCATIONS:
MIXING VESSEL:

T = Top Layer
B = Bottom Layer
T + B = Composite

13.0 REVALIDATION:
Revalidation shall be considered and carried out when any of the following conditions occur:
• Periodical Validation
• Change in critical formulation component i.e. raw material
• Change in manufacturer or vendor of Active Pharmaceutical Ingredient
• Change in critical specifications of the product
• Change in manufacturing process which may affect the quality of the products.
• Change in the facility and /or plant (location or site)
• Any major change in equipment.
Note: In case of the requirements for revalidation, because of above mentioned reasons, the validation of the critical steps

shall be undertaken through addendum protocol to this protocol or a separate protocol.

14.0 VALIDATION STABILITY PLAN:
• The three validation batches shall be subjected to stability studies as per stability plan.
15.0 REFERENCES:
The Principle References are as following:
• Relevant Specifications and Standard Testing Procedures
• Relevant Standard Operating Procedures
• Relevant Qualification Documents
• Indian Pharmacopoeia
• United State Pharmacopoeia
• Supplementary Guidelines on (GMP): Validation;

16.0 DOCUMENTS TO BE ATTACHED:
• Records for all critical parameters with graphical representation, where applicable.
• Relevant Sterilization Charts
• Raw Data of Validation Testing.
• Certificate of Analysis of API.
• Certificate of Analysis of Finished Product.
17.0 NON COMPLIANCE, IF ANY:
All the Non-compliances of procedure, specifications, and sampling, analysis and documentation activities shall be monitored & recorded.

18.0 DEVIATION FROM PRE-DEFINED SPECIFICATION, IF ANY:
• In case of any deviation observed during process validation, inform to Head QA for necessary action.
• Document the deviation detail in observed deviation section.
• The Head QA will study the impact of deviation. If deviation is acceptable and it does not have an impact on operation as well as on performance of the machine & prepare final conclusion.
19.0 CHANGE CONTROL, IF ANY:
• If any change control is required during process validation, inform to Head QA for necessary action.
• Document the details observed.
• The Head QA will study the impact of change. If change is acceptable and it does not have an impact on operation as well as on performance of the machine & prepare final conclusion.

20.0 ABBREVIATIONS:

PVP : Process Validation Protocol
mg : Milligram
KG : Kilogram
Ltr. : Liter
QS : Quantity Sufficient
BMR : Batch Manufacturing Record
BPR : Batch Packing Record
QA : Quality Assurance
QC : Quality Control
LOD : Loss on drying
% : Percentage
NLT : Not Less Than
NMT : Not More Than
SOP : Standard Operating Procedure
°C : Degree Centigrade
RPM : Rotation per minutes
WFI : Water For Injection
Kg/cm2 : Kilogram per centimeter square
DP : Differential pressure
cfu : Colony Forming Unit
EU/ml : Endotoxin unit per milliliter
WHO : World Health Organization