Protocol For EDTA - Additional Study - Process Evaluation Hold Time

ADDITIONAL STUDY PROTOCOL FOR DETERMINATION OF EDETATE
DISODIUM (DIHYDRATE) CONTENT IN PREDNISOLONE ACETATE

OPHTHALMIC SUSPENSION
USP 1.0% (5ML, 10ML AND 15ML)
Product Name & Strength Prednisolone acetate ophthalmic suspension USP 1.0% (5mL, 10mL,15mL)
Protocol Number ASP-US-EDTA-DGO014A-001-01 Supersedes ASP-US-EDTA-DGO014A-001-00
Effective Date Page No. 1 of 10
ADDITIONAL STUDY PROTOCOL FOR DETERMINATION OF EDETATE DISODIUM

(DIHYDRATE) IN PREDNISOLONE ACETATE OPHTHALMIC SUSPENSION USP 1.0%
5ML, 10ML AND 15ML
PROTOCOL No: ASP-US-EDTA-DGO014A-001-01
Prepared By
Name
Designation & Department
Sign & Date
Checked By
Name
Sign & Date
Approved By
Name
Sign & Date
Format No.: DG-ARD012/F04-00

1.0 Objective
The objective of the study is to perform the determination of edetate disodium dihydrate content in
prednisolone acetate ophthalmic suspension, USP 1.0% (5mL, 10mL and 15mL) during different
stages of manufacturing along with hold time study during process evaluation batches.
2.0 Scope
The protocol describes the study is to be carried out to perform edetate disodium (dihydrate)
content during different stages of in-process manufacturing and finished product on Prednisolone
acetate ophthalmic suspension USP 1.0% for 5mL, 10mL and 15mL. The study will be performed
at Steril-gene life sciences (P) Ltd, Pondicherry.
3.0 Product and Study design

The study protocol covers the following product as mentioned below,
1) In-process Stages:
Unfiltered excipient phase
#
Process step Sampling point Sampled quantity
Final (Excipient phase) unfiltered bulk -
Manufacturing of Bulk 10mL + 10mL
Top of the Mfg. tank
solution (Excipient
phase) Final (Excipient phase) unfiltered bulk -
10mL + 10mL
Bottom of the Mfg. tank
Bulk solution testing - Bulk solution testing from pressure
10mL + 10mL
Unfiltered Bulk Hold vessel -after 12 hrs. Hold
study (Excipient phase) # Bulk solution testing from pressure
10mL + 10mL
Bottom of vessel vessel -after 24 hrs Hold
Hold time data shall be generated on one batch only of any fill volume as composition, batch size
and equipment train remain same for all fill volume.
# For hold time study sample is transferred to appropriate pressure vessel and sampling is
performed as per defined time points.
Filter Flush analysis of excipient phase:

Process step Sampling point Sampled quantity

th
5 Flush sample 10mL + 10mL
Filter Flush
6th Flush sample 10mL + 10mL
th th
# 3.0 liters of 5 and 6 flush collected for excipient phase. Above mentioned quantity to be
collected from 3.0L flush samples.
Filtered excipient phase:
Filtered sample Bulk solution Testing – After final filtration; from
10mL + 10mL
(Excipient phase) 100 L Filtration vessel _ Initial
Bulk solution Testing After final filtration; from
10mL + 10mL
Bulk solution testing- pressure vessel 12 hrs Hold
Filtered bulk Hold Bulk solution Testing After final filtration; from
10mL + 10mL
study (Excipient pressure vessel 24 hrs Hold
phase) # Bulk solution Testing After final filtration; from
10mL + 10mL
pressure vessel 36 hrs Hold
# Hold time data shall be generated on one batch only of any fill volume as composition, batch size
# For hold time study sample is transferred to appropriate pressure vessel and sampling is
performed as per defined. time points
10mL +
Bulk suspension testing After bulk suspension Mfg. completion
10mL
After bulk suspension Mfg. completion- 12 10mL +
hours Hold 10mL
Bulk suspension testing Hold
time study # After bulk suspension Mfg. completion- 24 10mL +
hours Hold 10mL
From recirculation loop
After bulk suspension Mfg. completion- 36 10mL +
hours Hold 10mL
# Hold time data shall be generated on one batch only of any fill volume as composition, batch size
Finished Sample
Finished Product:

Finished product (5mL) Pooled sample 02 +02 bottles

Finished product (10mL) Pooled sample 01 + 01 bottle
Finished product (15mL) Pooled sample 01 + 01 bottle
Note: Label Claim for Excipient Phase to be considered as 0.181 mg/mL of edetate disodium
dihydrate and for Bulk sample and finished product to be considered as 0.127mg/mL of edetate
disodium dihydrate.
Specification Value:
Test In-process and Release specification
Assay of edetate disodium dihydrate Between 85.0% w/v and 110.0% w/v of
labelled amount of Edetate disodium dihydrate
4.0 Methodology
The method is validated at sterilgene. Report number AMVR/AD/23/015-00
A. Instruments and Apparatus Chemicals and Reagents:
1) HPLC equipped with UV/PDA detector.
2) Analytical balance
3) pH meter
4) Laboratory Sonicator
5) Standard laboratory clean and dried glassware
6) 0.45μ Nylon filter
7) 0.45 µ PVDF Membrane filter
B. Chemicals and Reagents:
1. Water : Milli-Q or equivalent

2. Tetra butyl ammonium hydrogen sulfate : AR grade or equivalent
3. Ammonia solution : AR grade or equivalent

4. Glacial acetic acid : AR grade or equivalent

5. Acetonitrile : HPLC grade
6. Cupric sulfate anhydrous : AR grade or equivalent
C. Reference standard Chemicals and Reagents:
1. Edetate disodium (Dihydrate) : Reference standard/ working standard with
known potency
D. Preparation of Solutions
Note:
 Reference standard/ working standard weight can be adjusted, but final concentration should
not get changed.
 Follow the instructions on Reference/Working standard COA or on the label.
Preparation of Mobile Phase:

Buffer Preparation:
Weigh and transfer about 8.5 g of tetra butyl ammonium hydrogen sulfate into a beaker containing
1000 mL of Milli-Q water. Add 4 mL of ammonia solution and mix well. Adjust the pH 4.5± 0.05
with glacial acetic acid. Filter the solution through PVDF 0.45 µm membrane filter.
Preparation of Mobile Phase A:
Use Buffer as Mobile Phase A
Preparation of Mobile Phase B:
Use Acetonitrile as Mobile Phase B
Preparation of Mobile Phase B:
Use Acetonitrile as Mobile Phase B
Preparation of Diluent:
Weigh and transfer about 100 mg of Cupric sulphate into 1000mL volumetric flask containing
500mL of water, sonicate for about 2 minutes to dissolve and make up to volume and mix well.
Preparation of Standard stock solution:
Weigh accurately and transfer about 25 mg of edetate disodium dihydrate reference standard/
working standard into a 50 mL volumetric flask. Add about 30 mL of diluent and sonicate for 5
minutes dissolve. Make up the volume up to the mark with diluent and mix well.
Preparation of Standard solution:
Further Pipette 5 mL of above solution into a 100 mL volumetric flask. Make up the volume up to
the mark with diluent and mix well. (Concentration: about 25 μg/mL of edetate disodium
dihydrate).
Preparation of Sample solution:
For Excipient Phase:
Note: Mix the sample properly for 15-30 seconds before use.
Weigh accurately about 3 gm of Sample Solution into 20 mL volumetric flask, add about 10mL of
diluent-2 and sonicate for about 2 minutes to dissolve and make up to the mark with diluent mixed
well. Filter the sample through 0.45 µ Nylon syringe Filter. Discard about 2mL of filtrate and then
use the filtered solution. (Concentration about 27μg/mL).
For Bulk Solution:
Note: Shake the sample bottle vigorously for about 15 to 30 seconds before use to avoid any
aggregation of particles at bottom of container. Keep the bottle aside for five minutes and allow
any frothing to disappear.
Weigh accurately and transfer 4.0 g of sample into a 20 mL volumetric flask. Make up to the mark
with diluent and mix well. Filter the sample through 0.45 µ Nylon syringe Filter. Discard about
2mL of filtrate and then use the filtered solution. (Concentration: about 25 μg/mL of edetate
disodium dihydrate).
For Finished product:
Fill volume No of bottles to be taken
For 5mL fill 2
For 10mL fill 1
For 15mL fill 1
Note: Shake the sample bottle vigorously for about 15 to 30 seconds before use. Transfer the entire
content into a suitable glass test tube, keep aside for 5 minutes and allow frothing to disappear.
Weigh accurately and transfer 4.0 g of sample into a 20 mL volumetric flask. Make up to the mark
with diluent and mix well. Filter the sample through 0.45 µ Nylon syringe Filter. Discard about
2mL of filtrate and then use the filtered solution. (Concentration: about 25 μg/mL of edetate
disodium dihydrate).
A. Operating Parameter (Chromatographic Conditions):
Column : X-Bridge Shield RP18 (150 mm X 4.6 mm, 3.5 µm)

Part No: 186003045
Flow rate : 0.8 mL/minute
Column oven temperature : 30°C
Wavelength : 254 nm
Autosampler temperature : 25°C
Injection volume : 25 µL
Run time : 20 minutes
Retention time : Between 4.0 to 6.5 minutes
Needle wash : Prepare a mixture of Water : Acetonitrile in ratio of
50: 50 (% v/v) and degas.
Gradient program:
Time (in minutes) Mobile phase A (%) Mobile phase B (%)

0.0 100 0
10.0 100 0
10.1 10 90
15.0 10 90
15.1 100 0
20.0 100 0
Procedure:
Setup the chromatographic system as described under chromatographic conditions. Equilibrate the
column with a gradient program or until a stable baseline is observed.
Solution Name Order of Injection
Diluent 1
Standard Solution 5
Sample Solution 2
Standard Bracketing Solution 1
System suitability requirements


At Ws 5 20 P 100 Mw1
= --------x--------x--------x------- x------- x------- x --------x D
As 50 100 Wt 100 LC Mw2
Where,
At Average peak area of Edetate disodium peak observed from sample solution
chromatogram
As Average peak area of Edetate disodium peak observed from five replicate injection of
standard solution chromatogram
Ws Weight of Edetate disodium (dihydrate) (reference/ working standard) in mg.
Wt Weight of sample (in gm)
P Potency of Edetate disodium dihydrate (% on as is basis)
LC Label claim of Edetate disodium (dihydrate) in prednisolone acetate ophthalmic
suspension USP 1%
Excipient Phase: 0.181 mg/mL
Bulk Sample and Finished product 0.127 mg/mL.
Mw1 372.24 [molecular weight of Edetate Disodium (dihydrate)]
Mw2 336.24 [molecular weight of Edetate Disodium]
D Weight per mL of sample (gm/mL)#
# weight per mL value shall be taken from in-process bulk samples.
5.0 Sample and Batch details
All batches manufactured for 5mL, 10mL and 15mL to be considered for edetate disodium
dihydrate analysis as per above sampling plan.
6.0 Evaluation and Interpretation of Results

Samples will be evaluated for information purpose only. Any OOS (out of specification) results
will be analyzed as per Steril-gene SOP.
7.0 Documentation
 Study Protocol
 Study Report

8.0 Reference
Not applicable
Revision History:
Version No. Supersedes Changes made
00 Nil Newly prepared

updated for filter flush study sample analysis.
01 ASP-US-EDTA-DGO014A-001-00 Locations of sample points and Removed
stability samples

Protocol For EDTA - Additional Study - Process Evaluation Hold Time

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Protocol For EDTA - Additional Study - Process Evaluation Hold Time

Uploaded by

Copyright:

Available Formats

ADDITIONAL STUDY PROTOCOL FOR DETERMINATION OF EDETATE

DISODIUM (DIHYDRATE) CONTENT IN PREDNISOLONE ACETATE

ADDITIONAL STUDY PROTOCOL FOR DETERMINATION OF EDETATE DISODIUM

PROTOCOL No: ASP-US-EDTA-DGO014A-001-01

Designation & Department

Sign & Date

Designation & Department

Sign & Date

Designation & Department

Sign & Date

Format No.: DG-ARD012/F04-00

3.0 Product and Study design

Filter Flush analysis of excipient phase:

Format No.: DG-ARD012/F04-00

Process step Sampling point Sampled quantity

Format No.: DG-ARD012/F04-00

Finished product (5mL) Pooled sample 02 +02 bottles

Finished product (15mL) Pooled sample 01 + 01 bottle

1. Water : Milli-Q or equivalent

Format No.: DG-ARD012/F04-00

4. Glacial acetic acid : AR grade or equivalent

Preparation of Mobile Phase:

Column : X-Bridge Shield RP18 (150 mm X 4.6 mm, 3.5 µm)

Time (in minutes) Mobile phase A (%) Mobile phase B (%)

System suitability requirements

Format No.: DG-ARD012/F04-00

Format No.: DG-ARD012/F04-00

6.0 Evaluation and Interpretation of Results

Format No.: DG-ARD012/F04-00

Version No. Supersedes Changes made

00 Nil Newly prepared

Format No.: DG-ARD012/F04-00

You might also like