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STABILITY
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3.2.S.7 STABILITY

3.2.S.7.1 STABILITY SUMMARY AND CONCLUSIONS

3.2.S.7.1.1 Accelerated and long term stability study
Three batches of Tacrolimus monohydrate 03509076, 03509077 and 03509078 were kept on
accelerated storage condition at 40 C 2 C / 75% RH 5% RH for 6 months and for long term
study at the storage condition 25 C 2 C / 60% RH 5% RH for 36 months and long term
stability studies in the month of January 2010.
USP Monograph of the Tacrolimus has become official from August 2012, hence in order to
confirm the compliance of the stability samples to the USP specification, above batches
03509076, 3509077 and 03509078 of 30 and 36 months stability age were analyzed as per
USP test procedure and found meeting the USP specification.

Container closure system for stability samples:

The stability samples are packed in food grade aluminum container which is closed with a
HDPE lid and a self locking cap to simulate market conditions and is labeled with following
details.
(a) Stability condition
(b) Name of sample
(c) Batch number
(d) Date when analysis is due

Study design, storage condition and testing frequency:

Tacrolimus stability samples are kept at the following conditions and analysis time interval is
indicated below.

Temperature Conditions Intervals in months

0 1 2 3 6 9 12 18 24 36
40±2°C/75±5%RH √ √ √ √ √ ---- ---- ---- ---- ----
25±2°C/60±5%RH √ ---- ---- √ √ √ √* √ √* √*

RH: Relative humidity, √ : Data Station

* Microbial Limit Test is done only at initial, 12, 24 and 36 months of the stability study.

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Tests, specifications and method of analysis of stability samples:

In January 2017, Specification and test procedure Tacrolimus is revised to comply with the
nd
USP monograph of Tacrolimus, published in USP 39, 2 supplement to incorporate
following changes.
Specification of description test is changed to ‘White crystals or white crystalline
powder’ from ‘White to off white powder’
Specification and testing procedure of solubility test is changed to “Very soluble in
Methanol, Freely soluble in N, N Dimethylformamide and in Ethanol (95 %) and
practically insoluble in water” from “Soluble in Acetone, Chloroform and Ethyl
acetate; insoluble in water”
Peak identification solution 1 and peak identification solution 2 is introduced in the
testing procedure of Organic impurities test.

In June 2017, specification of Tacrolimus is revised to remove Ascomycin 19-epimer from

the specification of organic impurities test as it is not present in Tacrolimus API to comply
with the USP monograph of Tacrolimus published in the USP 40.
Analysis of stability samples of the new batch(es) manufactured after June 2017 (one batch
every year) shall be carried out as per specification no. SPC/QC/FP/057. Specification and
test procedure used in the analysis of stability samples is described in module 3, updated
section 3.2.S.4.1 and 3.2.S4.2 respectively.

Conclusion:
The results of the stability studies are presented in section 3.2.S.7.3. The results of the
stability study of both accelerated and long term indicate that:
1. No change is observed in the description of the material from the initial at any storage
condition and time interval.
2. IR spectrum of the samples is found similar and concordant with the IR spectrum of in-
house Tacrolimus working standard. RT of the major peak in the chromatogram of
samples also corresponds to the in-house Tacrolimus working standard as obtained
under the assay test.
3. There is no increase in the water content indicating that the material is not hygroscopic.
4. The related substances complied with the limits and there is no increase in the level of
impurities, indicating the stability of the drug substance.
5. Content of tautomers are found within specified limit, indicating the stability of the drug
substance.
6. The assay is within the limits indicating the stability of the drug substance.

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Retest Period : Three years

Storage conditions : Store in airtight container up to 25 °C.

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3.2. S.7.1.2 STABILITY INDICATING METHOD

Stress testing was carried out to determine the suitability of HPLC procedure for Assay and
Organic impurities including peak purity of main analyte peak for its ability to detect the
degradation products and for its adoption as a stability indicating procedure.
Earlier the Tacrolimus monograph was not official in USP; hence forced degradation study
was carried out as per in-house testing procedure. We have adopted the USP procedure for
the testing of the drug substance, hence forced degradation study is again done to study and
verify the stability indicating method as given in USP.
One production batch number 03512048 of Tacrolimus manufactured in April 2012 was
subjected to forced degradation studies under following conditions.

Procedure followed for Stress conditions:

1. Heat Treatment:
About 0.5 g of Tacrolimus sample was spread as a thin layer in a Petri dish and heated
to 100-105˚C for a total duration of 24 hrs in a heating oven. Samples were drawn at 8,
16, 24 hrs and analyzed for Assay and Organic impurities. Peak purity of main
Tacrolimus peak was checked.
2. UV Light at 254 nm:
About 0.5 g of Tacrolimus sample was spread as a thin layer in a Petri dish and exposed
to UV light for a total duration of 24 hrs at ambient temperature. Samples were drawn at
8, 16, 24 hrs and analyzed for Assay and Organic impurities. Peak purity of main
Tacrolimus peak was checked.
3. Acid Treatment:
About 0.5 g of Tacrolimus sample accurately weighed was transferred to each three
flasks, dissolved and made up to 50 mL with 0.5 N HCl in diluent. All the three flasks
were heated to 60˚C under stirring. One flask each at an interval of 8, 16 and 24 hours
respectively were removed from heating, cooled, neutralized and made up the volume to
50 mL with diluent and analyzed for Assay and Organic impurities. Peak purity of main
Tacrolimus peak was checked.

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4. Alkali Treatment:
About 0.5 g of Tacrolimus sample accurately weighed was transferred to each three
flasks, dissolved and made up to 50 mL with 0.05 N NaOH in diluent. All the three
flasks were heated to 60˚C under stirring. One flask each at an interval of 8, 16 and 24
hours respectively were removed from heating, cooled, neutralized and made up the
volume to 50 mL with diluent and analyzed for Assay and Organic impurities. Peak
purity of main Tacrolimus peak was checked
5. Thermal Oxidation:
About 0.5 g of Tacrolimus sample accurately weighed was transferred to each three

flasks, dissolved and made up to 50 mL with 5 % H 2O2 in diluent. All the three flasks
were heated to 60˚C under stirring. One flask each at an interval of 8, 16 and 24 hours
respectively were removed from heating, cooled, neutralized and made up the volume to
50 mL with diluent and analyzed for Assay and Organic impurities. Peak purity of main
Tacrolimus peak was checked.

Results of Forced degradation study:

Result of analysis of assay and organic impurities of different stress conditions are tabulated
in Table 1 to Table 5 given on the following pages.

Conclusion:
Following conclusions can be drawn based on analytical data from the table presented.
(1) Tacrolimus drug substance shows little or no degradation when exposed to Heat
treatment at 105°C. Drug substance shows thermal stability.
(2) Product shows stability to UV light exposure. No degradation is observed when
exposed to UV light at 254 nm.
(3) Drug substance degrades completely in alkali treatment.
(4) The drug substance degrades considerably under acid treatment and in thermal
oxidation.
(5) Both method of analysis of assay and organic impurities are found stability indicating.

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Table1: Heat Treatment at 100 - 105 ºC temperature

Time in hours Initial 08 hours 16 hours 24 hours

Assay (On anhydrous and solvent free basis) 98.8 % 98.6 % 98.6 % 98.6 %
Chromatographic Purity RRT↓ (About) 92.4 % 91.0 % 91.2 % 94.1 %
Unknown impurity 0.29 ND BDRL ND ND
Unknown impurity 0.39 ND BDRL ND ND
Unknown impurity 0.41 ND BDRL ND ND
Tacrolimus open ring 0.53 2.4 % 2.5 % 2.5 % 1.7 %
Tacrolimus 19-epimer 0.63 5.2 % 6.2 % 6.3 % 4.0 %
Unknown impurity 0.70 BDRL BDRL ND ND
Ascomycin 0.88 0.07 % 0.06 % 0.06 % 0.08 %
Unknown impurity 0.90 BDRL BDRL BDRL BDRL
Desmethyl tacrolimus 0.94 0.06 % 0.07 % 0.08 % 0.14 %
Unknown impurity 1.08 ND BDRL ND ND
Tacrolimus 8- propyl analog 1.31 BDRL BDRL BDRL BDRL
Unknown impurity 1.42 BDRL BDRL 0.05 % 0.07 %
Unknown impurity 1.46 ND 0.16 % ND ND

BDRL = Below disregard limit ≤ 0.05 %

ND = Not detected

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Table 2: UV light at 254 nm, ambient temperature:

Time in hours Initial 08 hours 16 hours 24 hours

Assay (On anhydrous and solvent free basis) 98.8 % 98.9 % 98.7 % 98.8 %
Chromatographic Purity RRT↓ (About) 92.4 % 91.4 % 92.7 % 94.4 %
0.29 ND ND BDRL 0.09 %
Unknown impurity 0.33 ND ND BDRL BDRL
Unknown impurity 0.36 ND ND BDRL BDRL
Unknown impurity 0.37 ND ND BDRL 0.08 %
Unknown impurity 0.39 ND ND 0.06 % 0.13 %
Unknown impurity 0.41 ND ND BDRL 0.09 %
Tacrolimus open ring 0.53 2.4 % 2.5 % 2.1 % 1.5 %
Tacrolimus 19-epimer 0.63 5.2 % 6.1 % 4.8 % 3.2 %
Unknown impurity 0.70 BDRL BDRL ND ND
Ascomycin 0.88 0.07 % 0.06 % 0.07 % 0.07 %
Unknown impurity 0.90 BDRL BDRL BDRL BDRL
Desmethyl tacrolimus 0.94 0.06 % 0.08 % 0.06 % 0.06 %
Unknown impurity 1.08 ND ND 0.09 % 0.20 %
Tacrolimus 8 propyl analog 1.31 BDRL BDRL BDRL 0.05 %
Unknown impurity 1.42 BDRL BDRL BDRL 0.05 %
Unknown impurity 1.46 ND ND 0.15 % 0.18 %

BDRL = Below disregard limit ≤ 0.05 %

ND = Not detected

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Table 3: Acid treatment with 0.5 N Hydrochloric acid at 60°C temperature:

Time in hours Initial 08 hours 16 hours 24 hours

Assay (On anhydrous and solvent free basis) 98.8 % 72.3 % 61.0 % 42.4 %
Chromatographic Purity RRT↓ (About) 92.4 % 71.3 % 54.8 % 40.0 %
Unknown impurity 0.19 ND 0.11 % 0.16 % 0.23 %
Unknown impurity 0.21 ND 0.10 % 0.22 % 0.27 %
Unknown impurity 0.27 0.09 % 0.15 % 0.16 %
ND
Unknown impurity 0.28 ND 0.65 % 1.3 % 1.1 %
Unknown impurity 0.30 0.61 % 1.2 % 0.91 %
ND
Unknown impurity 0.37 ND ND 0.30 %
ND
Tacrolimus open ring 0.51 1.3 % 0.82 % ND
2.4 %
Ascomycin 19-epimer 0.52 0.06 % 0.13 % 0.27 %
ND
Unknown impurity 0.57 0.12 % 0.38 % 0.69 %
ND 0.17 %
Unknown impurity 0.60 0.08 % 0.22 %
ND 5.7 % 5.1 % 4.0 %
Tacrolimus 19-epimer 0.63
5.2 % ND ND 0.16 %
Unknown impurity 0.70
BDRL 0.24 % 0.43 % 0.44 %
Ascomycin 0.88
0.07 % BDRL BDRL BDRL
Unknown impurity 0.90
Desmethyl tacrolimus 0.94 BDRL BDRL BDRL ND
Unknown impurity 0.97 0.06 % 0.13 % 0.38 % 0.56 %
Unknown impurity 1.06 ND 0.17 % 0.76 % 0.91 %
Unknown impurity 1.12 ND 0.21 % 0.48 % 0.60 %
Tacrolimus 8-epimer 1.31 ND 2.5 % 4.7 % 4.0 %
Tacrolimus 8 propyl analog 1.35 BDRL 0.08 % 0.17 %
ND
Unknown impurity 1.40 BDRL
ND ND 1.4 %
Unknown impurity 1.42 ND 0.42 % 1.1 %
ND
Unknown impurity 1.44 0.08 % 0.64 % 0.64 %
BDRL
Unknown impurity 1.46 0.09 % 0.53 % 2.1 %
ND
Unknown impurity 1.48 0.12 % 1.5 % ND
ND 0.71 %
Unknown impurity 1.50 ND ND
ND 12.3 % 24.5 % 30.3 %
Unknown impurity 1.60
ND 1.9 % 3.7 % 7.6 %
Unknown impurity 1.65
Unknown impurity 1.68 ND 0.13 % 0.24 % 0.19 %
BDRL = Below disregard limit ≤ 0.05 % ND
ND = Not detected ND

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Table 4: Alkali treatment with 0.05 N Sodium hydroxide at 60°C temperature:

Time in hours Initial 08 hours 16 hours 24 hours

98.8 %
Assay (On anhydrous and solvent free basis) * * *
92.4 %
Chromatographic Purity RRT↓ (About) ND ND ND
2.4 %
Tacrolimus open ring 0.53 11.0 % 5.6 % 4.6 %
5.2 %
Tacrolimus 19-epimer 0.63 0.79 % ND ND
BDRL
Unknown impurity 0.70 ND ND ND
0.07 %
Ascomycin 0.88 ND ND ND
BDRL
Unknown impurity 0.90 ND ND ND
0.06 %
Desmethyl tacrolimus 0.94 ND ND ND
BDRL
Tacrolimus 8 propyl analog 1.31 ND ND ND
ND
Unknown impurity 0.20 4.0 % ND ND
ND
Unknown impurity 0.21 3.2 % 4.6 % 3.6 %
ND
Unknown impurity 0.22 9.2 % 7.7 % 10.6 %
ND
Unknown impurity 0.24 11.8 % 6.5 % 8.0 %
ND
Unknown impurity 0.25 6.6 % 5.0 % 5.9 %
ND
Unknown impurity 0.45 0.40 % ND ND
ND
Unknown impurity 0.47 3.3 % 2.1 % 2.0 %
ND
Unknown impurity 0.50 3.0 % 2.7 % 0.41 %
ND
Unknown impurity 0.58 8.8 % 4.0 % 4.1 %
ND
Unknown impurity 0.61 0.71 % 0.26 % ND
BDRL
Unknown impurity 1.42 ND ND ND
* Drug substance degraded completely within eight hours

BDRL = Below disregard limit ≤ 0.05 %

ND = Not detected

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Table 5: Thermal oxidation with 5 % H2O2 at 60°C temperature:

Time in hours Initial 08 hours 16 hours 24 hours

Assay (On anhydrous and solvent free basis) 98.8 % 80.0 % 64.7 % 49.8 %
Chromatographic Purity RRT↓ (About) 92.4 % 71.1 % 57.4 % 44.2 %
Unknown impurity 0.15 ND 6.2 % 10.1 % 15.0 %
Unknown impurity 0.28 ND 13.8 % 24.3 % 36.1 %
Unknown impurity 0.35 ND 0.17 % 0.25 % 0.23 %
Unknown impurity 0.38 0.09 % 0.11 % 0.13 %
ND
Unknown impurity 0.39 ND BDRL ND
ND
Unknown impurity 0.47 0.33 % ND ND
ND
Tacrolimus open ring 0.53 2.2 % 2.3 % 1.6 %
2.4 %
Unknown impurity 0.61 0.15 % 0.14 % 0.08 %
ND
Tacrolimus 19-epimer 0.63 7.0 % 5.8 % 4.3 %
5.2 %
Unknown impurity 0.67 0.05 % 0.07 % 0.08 %
ND BDRL
Unknown impurity 0.70 0.13 % 0.22 %
BDRL
Unknown impurity 0.79 ND 0.05 % ND
ND ND BDRL BDRL
Unknown impurity 0.83
Ascomycin 0.88 ND 0.04 % BDRL BDRL
Unknown impurity 0.90 0.07 % BDRL ND BDRL
Desmethyl tacrolimus 0.94 BDRL 0.06 % 0.11 % BDRL
Unknown impurity 0.97 0.06 % ND ND 0.14 %
Unknown impurity 1.12 ND 0.06 % 0.07 % 0.06 %
Unknown impurity 1.22 ND BDRL ND ND
Tacrolimus 8 propyl analog 1.31 ND BDRL BDRL BDRL
Unknown impurity 1.42 BDRL ND ND ND
Unknown impurity 1.53 BDRL ND BDRL 0.04 %
Unknown impurity 1.59 ND 0.07 % 0.12 %
ND
BDRL = Below disregard limit ≤ 0.05 %
ND
ND = Not detected
Relevant HPLC chromatograms for the Initial, Heat treatment, UV treatment, Acid treatment,
Alkali treatment & Thermal oxidation of 24 hours for organic impurities of forced
degradation studies are presented in the following pages.

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3.2. S.7.1.3 PHOTO STABILITY STUDY OF TACROLIMUS

Photo stability study was carried out to evaluate the effect of light exposure on the drug
substance Tacrolimus. The photo stability testing is also carried out to evaluate the
suitability of the primary and secondary packing to the light exposure. The analytical
method shall also demonstrate its ability to detect the degradation product arising out of
photo stability as a part of stress testing.
Photo stability study of Tacrolimus was previously done in November 2008, where in-
house analytical method was used. We have adopted the USP procedure for the testing of
the drug substance, hence photo stability study is again done to study and verify the
stability indicating method as given in USP.
Photo stability study of Tacrolimus is done as per the ICH guideline (Q1B) on Photo
stability testing of new drug substances and products.
One production batch number 03512047 manufactured in April 2012 was subjected to
both fluorescent and UV light exposure under following conditions.

(a) Equipment Name : Humidity cum photo stability chamber

(b) Equipment ID : CBL/QC/HC-04
(c) Condition : Temperature: 25 ± 2° C
Humidity: 50 ±5%RH
(d) Exposure period for : 19 hours (200 W-Hr./m2 at spectral Ultra-violet
light distribution of 320 – 400 nm.)
(e) Visual light (Fluorescent) : 210 hours (1.2 million lux hours) exposure
(f) Packing of sample:
• Drug substance exposed as such without any packing.
• Drug substance in market simulated pack. The material was packed in
appropriately labelled airtight food grade aluminium container, closed
with a food grade virgin material HDPE lid and a self locking cap.

Method of analysis and test results:

Method of analysis of samples is as per test procedure described in the module 3, section
3.2.S.4.2. Test procedure is found stability indicating.
Results of organic impurity and assay analysis before and after exposure of UV and
Visible light are given below:

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1. UV light exposure result of drug substance after 19 hours UV light exposure:

Initial Simulated Without

Tests Specification
Result market pack any pack
Organic impurities (By HPLC)
Tacrolimus open ring For record only 3.1 % 2.8 % 2.6 %
Ascomycin 19 - epimer NMT 0.1 % ND ND ND
Tacrolimus 19 - epimer For record only 7.7 % 6.9 % 6.3 %
Ascomycin NMT 0.50 % 0.07 % 0.07 % 0.06 %
Desmethyl tacrolimus NMT 0.1 % 0.06 % 0.05 % 0.06 %
Tacrolimus 8 - epimer NMT 0.1 % ND ND ND
Tacrolimus 8 - propyl analog NMT 0.1 % ND ND ND
Any individual unspecified NMT 0.1 % BDRL BDRL 0.23 %
impurity

Total impurities NMT 1.0 % 0.13 % 0.12 % 0.59 %

Assay of Tacrolimus (By HPLC)
Assay 98.0 to 102.0 % 99.5 %
99.3 % 98.0 %
(Calculated on the anhydrous of C44H69NO12
and solvent-free basis)

BDRL = Below disregard limit

ND = Not detected

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2. Fluorescent light exposure result of drug substance (after 210 hours exposure):

Tests Specification Initial Simulated Without

Result market pack any pack
Organic impurities (By HPLC)
Tacrolimus open ring For record only 3.1 % 2.8 % 2.2 %
Ascomycin 19 - epimer NMT 0.1 % ND ND ND
Tacrolimus 19 - epimer For record only 7.7 % 6.6 % 5.5 %
Ascomycin NMT 0.50 % 0.07 % 0.07 % 0.07 %
Desmethyl tacrolimus NMT 0.1 % 0.06 % 0.05 % 0.05 %
Tacrolimus 8 - epimer NMT 0.1 % ND ND ND
Tacrolimus 8 - propyl analog NMT 0.1 % ND ND ND
Any individual unspecified NMT 0.1 % BDRL BDRL 0.14 %
impurity

Total impurities NMT 1.0 % 0.13 % 0.12 % 0.54 %

Assay of Tacrolimus (By HPLC)
Assay 98.0 to 102.0 % 99.5 % 99.4 % 97.2 %
(Calculated on the anhydrous of C44H69NO12
and solvent-free basis)

BDRL = Below disregard limit

ND = Not detected

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Conclusion:
1. No significant change is observed in the physical appearance of the drug substance
under all photo stability conditions.
2. No significant change is observed in organic impurities and in assay when the drug
substance packed in simulated market pack is exposed to both UV light and
fluorescent light.
3. When the drug substance is exposed directly to UV light, an unknown impurity at
RRT 1.08 is increased from below detection to 0.23 %. Total impurities also
increased from 0.13 % to 0.59 %.
4. When the drug substance is exposed directly to fluorescent light, two unknown
impurities found at RRT 1.08 and 1.45 increased from below detection to 0.14 %.
Total impurities also increased from 0.13 % to 0.54 %.
5. A decrease in assay to 98.0 % is observed when the drug substance is exposed
directly to UV light.
6. A decrease in assay to 97.2 % is observed when the drug substance is exposed
directly to fluorescent light.
7. No significant change is observed in the drug substance packed in simulated
market pack under UV light and fluorescent light exposure, it confirms that the
packaging material used is suitable for packing and storage of drug substance.

Relevant HPLC chromatograms of organic impurities of initial, after exposure of UV

light and in fluorescent light both in simulated market pack and without market pack of
Photo stability study are presented in the following pages.

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3.2.S.7.2 POST-APPROVAL STABILITY PROTOCOL AND STABILITY

COMMITMENT:

STABILITY PROTOCOL

Study Storage Conditions Duration

Long Term Stability Studies
Number of batches
Packing of stability samples
25 C 2 C/60%RH 5%RH 36 months
One batch after every year.
The stability samples are packed in food grade aluminum
container closed with a HDPE lid and a self locking cap to
simulate the market conditions.

Testing Frequency Initial, 3, 6, 9, 12, 18, 24, 36 months

COMMITMENT:
1. A minimum one production batch, packaged in the market simulated condition will be
added to the long-term stability program every year and will be studied for the 36
months period.
2. If the result of stability study is found out of specification at any point of time, it will be
investigated and informed immediately to Regulatory Authorities and the customers.

Format for recording stability data of accelerated and long-term study is presented in the
following pages.

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3.2.S.7.3 STABILITY DATA:

The results of the stability studies of accelerated and long term conditions of following batch
numbers of Tacrolimus are given in the following pages.

S. No. Batch number Stability Condition Duration Status

1. 03507076,
03507077, 40°C±2°C,75%RH±5%RH 6 months Completed
03507078

2. 03509076,
03509077, 25°C±2°C,60%RH±5%RH 36 months Completed
03509078

The results obtained from study of both accelerated and long term stability study of the
batches do not show any degradation and meets laid down specification.

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