Shandong Kexing Bioproducts Co., Ltd.

No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,

SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Stability Study Protocol of EPO finished product

APPROVAL SECTION

Prepared by: Position Signature Date

Inspector

Checked by: Position Signature Date

Supervisor

Approved by: Position Signature Date

Manager
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
1. Purpose of the study
Investigate the rule of variation with time of Recombinant Human Erythropoietin Injection under the influence of
temperature and humidity, provide scientific evidence for the production, packing, storage and transport conditions
of drugs, and meanwhile establish through test the useful life of drugs.

2. Program of stability studies

The stability study uses three continuously batches of final product with the batch numbers in pre-filling syringe are
20131104, 20131105,20131106.The packages of the test-using products used for accelerated test and long-term test
is the same with the packages of the sales products, in a prefilled syringe and a paper carton.

3. Responsibilities
This report have been checked and approved by the Chiefs of Quality Control and Quality Assurance. The stability
studies performed by the quality control specialists, under the supervision of the stability specialist and the Chief
of the Quality Control Department.

4.Types of Stability Studies

Real-time stability study:
The samples will be stored in a stability chamber at 2- 8°C for 36months, protect from light, The parameters
evaluated and the acceptance criteria are shown in table1.

Accelerated stability study :

The samples will be stored in a stability chamber at 25℃±2℃ for 6 months. The parameters evaluated and the
acceptance criteria are shown in table 1.

6. Product and Package Description

Pre-filled syringe in commercial packing

7. Analytical Test Parameters

The following table shows the test methods and the acceptance criteria used on the stability study.

Table 1: Analytic methods and acceptance criteria for the stability study.

Method Index SOP No Acceptance Criteria

Standard operation procedure for Positive
Identification test SOP-06-013
spot immuno-staining determination
Standard operation procedure for
Appearance Clear，colorless liquid
appearance and eye able eyewink of SOP-06-006
Visible particle Conform to standard
injection determination
Standard operation procedure for >1.0ml
filling level of injection Filling quantity SOP-06-008
determination
Standard operation procedure for
pH SOP-06-012 6.4 – 7.4
pH of solution determination
Standard operation procedure for Content of sodium SOP-06-043 ≤190 mmol/L
rhEPO injection determination ion
Content of citrate ≤25 mmol/L
SOP for citrate acid base hydronium SOP-06-037
ion
Standard operation procedure for SOP-06-001
Human serum
protein concentrations 2.0 – 3.0 mg/ml
albumin content
determination by Lowry method
SOP-06-015 210-270mOsmol/kg
Osmometer Osmolality
Standard operation procedure for SOP-06-046 80% - 120% of the stated
Activity in vitro
rhEPO injection determination Value
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Standard operation procedure for 80% - 140% of the stated
Activity in vivo SOP-06-049
rhEPO injection determination Value
Standard operation procedure for
Sterility test SOP-06-010 Sterile
asepsis determination
Standard operation procedure for <2 EU/1000IU EPO
Residual bacterial
bacterial endotoxin by gel method SOP-06-003 <10EU/more than 5000IU EPO)
endotoxin
determination
Standard operation procedure for Abnormal toxicity Conform to standard
SOP-06-011
undue toxicity determination test

8. Stability Testing Schedules

Table 2: Stability Testing Schedule for Samples Stored at 2 to 8C.

Months Indexes to be evaluated according to Caption

protocol
0 1,2,3,4,5,6,7,8,9,10,11,12,13 1. Identification test
2. Appearance and Visible particle
3 2,4,8 3. Filling quantity
4. pH
5. Content of sodium ion
6. Content of citrate ion
7. Content of protein
8. Activity in vitro
9. Activity in vivo
10. Sterility test
11. Residual bacterial endotoxin
12. Abnormal toxicity test
13. Osmolality

6 2,4,8

9 2,4,8

12 1,2,3,4,5,6,7,8,9,10,11,12,13

18 2,4,8

24 1,2,3,4,5,6,7,8,9,10,11,12,13

30 1,2,3,4,5,6,7,8,9,10,11,12,13

36 1,2,3,4,5,6,7,8,9,10,11,12,13
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Table 3: Stability Testing Schedule for the Accelerated Stability Study at 25±2℃

Months Indexes to be evaluated according to Caption

protocol
0 1,2,3,4,5,6,7,8,9,10,11,12,13 1. Identification test
2. Appearance and Visible particle
3. Filling quantity
1 2,4 ,8 4. pH
2 2,4 ,8 5. Content of sodium ion
6. Content of citrate ion
3 1,2,3,4,5,6,7,8,9,10,11,12,13 7. Content of protein
6 1,2,3,4,5,6,7,8,9,10,11,12,13 8. Activity in vitro
9. Activity in vivo
10. Sterility test
11. Residual bacterial endotoxin
12. Abnormal toxicity test
13. Osmolality

10. Sample Volume Required to Perform Stability Studies

Table 3: Sample volume per batch to perform real time stability study

Index Sample Frequency Volume per Total

volume retest
Identification test 1ml 12 1ml 12ml
Appearance 1ml 12 0ml 0ml
Filling quantity
1ml 12 5ml 60ml
pH
Content of sodium ion 1ml 10 1ml 10ml
Content of citrate ion 1ml 10 1ml 10ml
Content of protein 1ml 10 1ml 10ml
Osmolality 1ml 10 1ml 10ml
Activity in vitro 1ml 12 1ml 13ml
Activity in vivo 1ml 10 1ml 10ml
Sterility Test 1ml 10 12ml 120ml
Residual bacterial endotoxin 1ml 12 1ml 12ml
Abnormal toxicity test 1ml 10 3ml 30ml
Total volume of sample per batch for real time stability studies : 297 syringes

Table 4: Sample volume per batch to perform accelerated stability study

Index Sample Frequency Volume per Total

volume retest
Identification test 1ml 5 1ml 5ml
Appearance 1ml 5 0ml 0ml
Filling quantity
1ml 5 5ml 25ml
pH
Content of sodium ion 1ml 3 1ml 3ml
Content of citrate ion 1ml 3 1ml 3ml
Content of protein 1ml 3 1ml 3ml
Osmolality 1ml 3 1ml 3ml
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Activity in vitro 1ml 3 1ml 3ml
Activity in vivo 1ml 3 1ml 3ml
Sterility Test 1ml 5 12ml 60ml
Residual bacterial endotoxin 1ml 3 1ml 3ml
Abnormal toxicity test 1ml 3 3ml 9ml
Total volume of sample per batch for accelerated stability studies : 120 syringes

Table 5: Manufacturing and expiry date with its respective batch size for each batch/lot
Manufacturing batch size for Stability study
Batch No. Specification Package Expiry date
date At 2-8℃ At 25℃
Pre-filled
20131104 3000IU/ml/PFS 2013.11 2016.10 29700 201310005
syringes
Pre-filled
20131105 3000IU/ml/PFS 2013.11 2016.10 28300 201310006
syringes
Pre-filled
20131106 3000IU/ml/PFS 2013.11 2016.10 28300 201310007
syringes

10. Data Presentation

The three batches of products were stored at 2℃--8℃, protecting from the direct irradiation of sunlight. The samples
would be evaluated at intervals of 0, 3, 6, 9, 12, 18, 24, 30 and 36 months respectively.
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Long-term stability available
Batch No.: 20131104 Specification: 3000IU/ml/PFS Storage condition: 2℃～8℃protect from light Packaging: Pre-filled syringes
Items Visible
particles Filling Residual Abnormal
Identifica Content of Content of Content of Activity in vitroActivity in vivo Sterility
Appearance pH quantity Osmolality bacterial toxicity
tion citrate ion protein sodium ion （IU/ml） （IU/ml） Test
endotoxin test
Standard Concl
Study 80%-120% 80-140% usion
210～ Conform
Clear, colorless Conform to ≤25 2.0-3.0 of Of <2EU/
Positive 6.9±0.5 >1.0 270mOs ≤190 mmol/L Sterile to
liquid standard mmol/l mg/ml the stated the stated 1000EPO
Frequency time mol/kg standard
value amount
2013 Colorless, Conform
0 Conform to 2962 3109 <2EU/1000E Qualifi
.11 Positive transparent 6.9 1.05 240 18 2.5 164 Sterile to
month standard 99% 104% PO ed
liquid standard
rd 2014 Colorless,
3 Conform to 2966 Qualifi
.02 － transparent 6.9 － － － － － － － － －
month standard 99% ed
liquid
th 2014 Colorless,
6 Conform to 2987 Qualifi
.05 － transparent 6.9 － － － － － － － － －
month standard 100% ed
liquid
th 2014 Colorless,
9 Conform to 3000 Qualifi
.08 － transparent 6.9 － － － － － － － － －
month standard 100% ed
liquid
th 2014 Colorless, Conform
12 Conform to 3106 3104 <2EU/1000E Qualifi
.11 Positive transparent 6.9 1.03 247 21 2.5 153 Sterile to
month standard 104% 103% PO ed
liquid standard
2015. 6.9 1.04 245 20 2.6
th Colorless, Conform
18 Conform to 155 3009 3233 <2EU/1000E Qualifi
Positive transparent Sterile to
month 05 standard 100% 108% PO ed
liquid standard

Note: Refer to rules for rounding off of numberical values

 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Batch No.:20131105 Specification: 3000IU/ml/PFS Storage condition:2℃～8℃protect from light Packaging: Pre-filled syringes

Items Visible Content

particles Filling Residual Abnormal
Identific Content of Content of of Activity in vitro
Activity in vivo Sterility
Appearance pH quantity Osmolality bacterial toxicity
ation citrate ion protein sodium （IU/ml） （IU/ml） Test
endotoxin test
Standard ion Concl
Study 80%-120% 80-140% usion
210～ Conform
Clear, colorless Conform to ≤25 2.0-3.0 ≤190 of Of <2EU/
Positive 6.9±0.5 >1.0 270mOs Sterile to
liquid standard mmol/l mg/ml mmol/L the stated the stated 1000EPO
Frequency time mol/kg standard
value amount
2013.1 Colorless, Conform
0 Conform to 3418 3363 <2EU/1000EP Qualifi
1 positive transparent 6.9 1.03 244 18 2.6 164 Sterile to
month standard 114% 112% O ed
liquid standard
rd 2014.0 Colorless,
3 Conform to 3323 Qualifi
2 － transparent 6.9 － － － － － － － － －
month standard 111% ed
liquid
th 2014.0 Colorless,
6 Conform to 3444 Qualifi
5 － transparent 6.8 － － － － － － － － －
month standard 115% ed
liquid
th 2014.0 Colorless,
9 Conform to 3000 Qualifi
8 － transparent 7.0 － － － － － － － － －
month standard 100% ed
liquid
th 2014.1 Colorless, Conform
12 Conform to 3099 3345 <2EU/1000EP Qualifi
1 positive transparent 6.9 1.03 247 20 2.5 153 Sterile to
month standard 103% 112% O ed
liquid standard
2015. 6.9 1.04 245 20 2.6
th Colorless, Conform
18 Conform to 158 3010 3233 <2EU/1000EP Qualifi
Positive transparent Sterile to
month 05 standard 100% 108% O ed
liquid standard
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
Batch No.:20131106 Specification: 3000IU/ml/PFS Storage condition:2℃～8℃protect from light Packaging: Pre-filled syringes

Items Visible Content

particles Filling Residual Abnormal
Identificati Content of Content of of Activity in vitroActivity in vivo Sterility
Appearance pH quantity Osmolality bacterial toxicity
on citrate ion protein sodium （IU/ml） （IU/ml） Test
endotoxin test
Standard ion Concl
Study 80%-120% usion
Clear, 210～ 80-140% Conform
Conform to ≤25 2.0-3.0 ≤190 of <2EU/
Positive colorless 6.9±0.5 >1.0 270mOs Of Sterile to
standard mmol/l mg/ml mmol/L the stated 1000EPO
Frequency time liquid mol/kg the stated amount standard
value
2013.11 Colorless, Conform Conform
0 3269 3470 <2EU/1000E Qualifi
positive transparent to 6.9 1.04 244 18 2.6 164 Sterile to
month 109% 116% PO ed
liquid standard standard
rd 2014.02 Colorless, Conform
3 3137 Qualifi
－ transparent to 6.8 － － － － － － － － －
month 105% ed
liquid standard
th 2014.05 Colorless, Conform
6 3168 Qualifi
－ transparent to 6.9 － － － － － － － － －
month 106% ed
liquid standard
th 2014.08 Colorless, Conform
9 3100 Qualifi
－ transparent to 6.9 － － － － － － － － －
month 103% ed
liquid standard
th 2014.11 Colorless, Conform Conform
12 3105 3053 <2EU/1000E Qualifi
positive transparent to 6.9 1.03 250 21 2.5 153 Sterile to
month 103% 102% PO ed
liquid standard standard
2015. 6.9 1.03 251 20 2.5 meet
th Colorless, Conform
18 157 3100 3252 <2EU/1000E Conform Qualifi
Positive transparent to Sterile
month 05 100% 108% PO to ed
liquid standard standard
Conclusion:
Recombinant human erythropoietin injection, 3000IU/ml/syringe are stored at the condition of 2-8℃, protect from light for 18 months the result complies with the standard
stated in "Chinese Pharmacopoeia" 2010 Edition Volume III.
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023

11. Accelerated stability

Three continuous production scale batches of final product, recombinant human erythropoietin injection, in
commercial packaging, stored at 25 ± 2 ℃, protect from light, are studied for accelerated stability. Testing intervals
are 0 months, 1 month, 2 months, 3 months; 6 months respectively, the period of validity of medicines are
determined by comparing the test results with the state in 0 months.
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023

Accelerating stability study

Batch No.: 20131104 Specification: 3000IU/ml/PFS Storage condition: 25±2℃ 60%±5%protect from light Packaging: Pre-filled syringes

Items Visible
particles Filling Content Activity in Residual Abnormal
Identific Appearanc Content of Content of Activity in vivo Sterility
pH quantity Osmolality of sodium vitro bacterial toxicity
ation e citrate ion protein （IU/ml） Test
ion （IU/ml） endotoxin test
Frequency Concl
Standard 80%-120% 80-140% usion
Clear, 210- Conform
Time Conform to ≤25 2.0-3.0 ≤190 of Of <2EU/
Positive colorless 6.9±0.5 >1.0 270mOs Sterile to
standard mmol/l mg/ml mmol/L the stated the stated 1000EPO
liquid mol/kg standard
value amount
2013.. Clear, Conform 6.9 1.05 240 18 2.5 Conform
2962 3109 <2EU/1000 Qualifi
Initial 11 Positive colorless to 164 Sterile to
99% 104% EPO ed
liquid standard standard
st 2013.. Clear, Conform 6.9 － － － － 2970 －
1 Qualifi
12 － colorless to － 99% － － －
month ed
liquid standard
nd 2014. Clear, Conform 6.9 － － － － 3045 －
2 Qualifi
01 － colorless to － 102% － － －
month ed
liquid standard
rd
3 2014. Clear, Conform 6.8 1.03 244 18 2.5 2936 3172 Conform
<2EU/1000 Qualifi
Month 02 Positive colorless to 164 98% 106% Sterile to
EPO ed
liquid standard standard
th 2014 Clear, Conform 6.9 1.03 249 19 2.5 2958 3099 Conform
6 <2EU/1000 Qualifi
05 Positive colorless to 160 99% 103% Sterile to
month EPO ed
liquid standard standard
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023

Batch No.: 20131105 Specification: 3000IU/ml/PFS Storage condition: 25±2℃ 60%±5%protect from light Packaging: Pre-filled syringes

Items Visible
particles Filling Content Activity in Residual Abnormal
Identifica Content of Content of Activity in vivo Sterility
Appearance pH quantity Osmolality of sodium vitro bacterial toxicity
tion citrate ion protein （IU/ml） Test
ion （IU/ml） endotoxin test
Frequency Concl
Standard 80%-120% 80-140% usion
Clear, 210～ Conform
Time Conform to ≤25 2.0-3.0 ≤190 of Of <2EU/
Positive colorless 6.9±0.5 >1.0 270mOs Sterile to
standard mmol/l mg/ml mmol/L the stated the stated 1000EPO
liquid mol/kg standard
value amount
2013. Clear, 6.9 1.03 244 18 2.6 Conform
Conform to 3418 3363 <2EU/1000 Qualifi
Initial .11 Positive colorless 164 Sterile to
standard 114% 112% EPO ed
liquid standard
st 2013.. Clear, 6.8 － － － － 3358 －
1 Conform to Qualifi
12 － colorless － 112% － － －
month standard ed
liquid
nd 2014. Clear, 6.9 － － － － 3210 －
2 Conform to Qualifi
.01 － colorless － 107% － － －
month standard ed
liquid
rd 2014. Clear, 6.9 1.03 247 17 2.6 3380 3394 Conform
3 Conform to <2EU/1000 Qualifi
02 Positive colorless 164 113% 113% Sterile to
month standard EPO ed
liquid standard
2014. 6.9 1.03 249 19 2.5 3428 3334
th Clear, Conform
6 Conform to 165 114% 111% <2EU/1000 Qualifi
Positive colorless Sterile to
month .05 standard EPO ed
liquid standard
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023

Batch No.: 20131106 Specification: 3000IU/ml/PFS Storage condition: 25±2℃ 60%±5%protect from light Packaging: Pre-filled syringes

Items Visible
Content Activity in Activity in Residual Abnormal
Identific Appearanc particles filling Content of Content of Sterility
pH Osmolality of sodium vitro vivo bacterial toxicity
ation e quantity citrate ion protein Test
ion （IU/ml） （IU/ml） endotoxin test Concl
Frequency
usion
Standard Clear, Conform >1.0 210- 80-120% 80-140% Conform
≤25 2.0-3.0 ≤190 <2EU/
Time Positive colorless to 6.9±0.5 270mOs of the stated of the stated Sterile to
mmol/l mg/ml mmol/L 1000EPO
liquid standard mol/kg value value standard
2013.1 Clear, Conform 6.9 1.04 244 18 2.6 Conform
3269 3470 <2EU/1000 Qualifi
Initial 1 Positive colorless to 164 Sterile to
109% 116% EPO ed
liquid standard standard
st 2013.1 Clear, Conform 6.9 － － － － 3358 －
1 Qualifi
2 － colorless to － 112% － － －
month ed
liquid standard
nd
2 2014.0 Clear, Conform 6.9 － － － － 3210 －
Qualifi
month 1 － colorless to － 107% － － －
ed
liquid standard
rd 2014.0 Clear, Conform 6.9 1.03 250 18 2.5 3380 3432 Conform
3 <2EU/1000 Qualifi
2 Positive colorless to 164 113% 114% Sterile to
month EPO ed
liquid standard standard
2014.0 6.9 1.02 249 19 2.5 3205 3134
th Clear, Conform Conform
6 160 107% 104% <2EU/1000 Qualifi
Positive colorless to Sterile to
month 5 EPO ed
liquid standard standard

Conclusion:
Recombinant human erythropoietin injection, 3000IU/ml/syringe are stored at the condition of 25±2℃ 60%±5% protect from light for 6 months the result complies with
the standard stated in "Chinese Pharmacopoeia" 2010 Edition Volume III.
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023
12. Analytical facility

Appeara Content Content Human

Identifica Bacterial
nce and Filling of of serum Osmolalit Activity Activity in Sterility Abnormal
pH tion endotoxin
Visible quantity sodium citrate albumin y in vivo vitro test toxcicity
test content
particle ion ion content
Test Protein
Standardi Differenti Sterility
Instrum Clarity photome electroph nucleic Osmome Microplat Reticulocyt Tube
zed pH meter al Testing Balance
ents detector ter oresis acid ter e reader e counter reader
syringe detector Equipment
analyzer

13.References
<<Chinese Pharmacopeia>> edition 2010

14
 Shandong Kexing Bioproducts Co., Ltd.
No.2666 CHUANGYE ROAD, MINGSHUI DEVELOPMENT ZONE, ZHANGQIU, JINAN,
SHANDONG –250200, CHINA
Tel: (+86 531) 83316484 Fax: (+86 531)83311023

15