The effect of temperature on the

stability of in-use insulin pen

Tanawan Kongmalai, M.D.

Lukana Preechasuk, M.D.,
Sarawut Junnu, B. Sc,
Siriphan Manocheewa, Ph.D.,
Chatchawan Srisawat, M.D., Ph.D,
Apiradee Sriwijitkamol, M.D. Mahidol university
Faculty of Medicine Siriraj Hospital
Siriraj Hospital, Mahidol university
 Mahidol university
Faculty of Medicine
Siriraj Hospital Disclosure

• Funding
o Grant from faculty of Medicine Siriraj Hospital,
Mahidol University, Grant Number (IO)
R016032029”

• Supported study drugs

o Eli Lily
o Cosma medical
o Sanofi
Mahidol university
Faculty of Medicine
Siriraj Hospital Insulin

• Complex labile protein

• Stability in 2-8 ºC

• Degraded by1

– Deamidation 10 ºC

– Polymerization > 10 ºC

1. Moslemi P et al. J Pharm. Biomed. Anal. 2003; 33: 45-51

 Mahidol university
Faculty of Medicine
Siriraj Hospital Introduction
• Therefore, the manufacturers recommend to
keep an in-use insulin at room temperature
not exceeding 25-30 ๐C.

• The potency of in use insulin vial was

decreased by 14 and 18% after kept it at 32
and 37°C for 28 days.1

1. Vimalavathini R et al. Indian J Med Res. 2009; 130; 166-69

Mahidol university
Faculty of Medicine
Siriraj Hospital Thailand
• In 2016
o Mean temperature throughout
the year was 34.0 ºC
o Highest temperature in
summer was 44.6 ºC
o Temperature higher than 35 ºC
more than a hundred day

https://www.tmd.go.th/programs/%5Cuploads%5Cweatherclimate%5Caprilbreakrecord.pdf
 Mahidol university
Faculty of Medicine
Siriraj Hospital
Stability of common insulin in Pens

Insulin Pens Stability Unopened and Opened at Room Unopened at

Stored in the Refrigerator or Refrigerator Room
Temperature temperature
(days) (days)
Intermediate-acting Insulin (NPH)
Humulin N pen The expiration date on the label 14 14
Novolin N pen The expiration date on the label 14 14

Long-acting Insulin
Lantus Solostar The expiration date on the label 28 28

Levemir FlexPen The expiration date on the label 42 42

Novolog Mix The expiration date on the label 14 14

70/30 Flexpen

Stability of Common Insulins in Pens and Vials. available at http://www.diabetesincontrol.com

Mahidol university
Faculty of Medicine
Siriraj Hospital
 Mahidol university
Faculty of Medicine
Siriraj Hospital Objective

To investigate the effect of temperature on the

stability of basal insulin after opening for 28 days.

• Insulin pen is widely use nowadays.

• Basal insulin commonly kept in room temperature

at home after opening.
 Mahidol university
Faculty of Medicine
Siriraj Hospital Materials and methods
• Experimental study
• Study drugs

Long acting insulin Intermediate acting insulin

Long acting insulin NPH-1 penfil
NPH-2 penfil
NPH-3 penfil

Two batches of each insulin were used to minimize the

variation of the manufacture’s production.
 Mahidol university
Faculty of Medicine
Siriraj Hospital Materials and methods
• To investigate the effect of different temperature
on stability of insulin in cartridge.

3rd-31st July 2017

Unopened insulin
Refrigerator (2-8 º C)
Room temperature Refrigerator (2-8 ºC) Incubator (37 ºC)
(Controlled group)

We had developed the 3 different conditions resemble

the daily usage of insulin-treated diabetes patients
 Mahidol university
Faculty of Medicine Temperature and humidity
Siriraj Hospital
measurement

• Easy log-USB-2-LCD+, RH/Temperature data logger

• The insulin pens and the logger were kept together in
the same environment.
• The temperature and humidity were recorded every 1
hour in each condition.

(LASCAR electronics, USA)

 Mahidol university
Faculty of Medicine
Siriraj Hospital Sample size calculation
• Sample size was calculated for each insulin pen.
– alpha = 0.05
– The power (1-β)= 90%

The estimated required sample size

is 5 per each insulin per condition.

The total sample of insulin pens will be 80.

 Mahidol university
Faculty of Medicine
Siriraj Hospital
Study design
Two batches of each
commercial insulin pens

Experimental groups Controlled group

Mixed 20 times and 2 units of Unopened insulin
insulin were discarded everyday

Room temperature Refrigerator (2-8 ºC) Incubator (37 º C) Refrigerator (2-8 ºC)

28 days

Ultra-high performance liquid chromatography (UHPLC)

Mahidol university
Faculty of Medicine Ultra-High Performance Liquid
Siriraj Hospital
Chromatography (UHPLC)
 Ultra-High Performance Liquid
Chromatography (UHPLC)
• The United States Pharmacopeial Convention, Revision
Bulletin, Official February 1, 2013.
• Using Agilent 1290 Infinity LC system with flow rate
0.75 ml/min, infection volume 2 l, run time 7 min
HPLC UHPLC
 Mahidol university
Faculty of Medicine
Siriraj Hospital Statistical analyses
• Data analysis
– SPSS program for windows, version 18.0
• The main statistical comparisons:
– Concentration of insulin was reported as mean.
– Independent t-test to detect difference between
groups.

Statistical significant: defined as P < 0.05

 Mahidol university Average temperature and humidity during
Faculty of Medicine
Siriraj Hospital the study period (3rd-31st Jul 2017)

36

34

32
MEAN (CELCIUS)

30

28

26

24

22
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
DATE

6.00-19.00 19.00-6.00

Variable Mean (range) 6.00 - 19.00 19.00 – 6.00

Temperature (ºC) 29.7 (25.5-37.1) 31.1 (27.1-37.1) 28.0 (26.0-31.0)
Humidity (%) 74.6 (50.5-89.5) 70.65 (50.5-86.2) 79.26 (67.5-87.8)
 Mahidol university
Faculty of Medicine
Siriraj Hospital
Chromatograms of insulin

Long acting insulin

1 Peak, Retention time 1.571 mins
Calculated AUC

Intermediate: 2 peaks
o Retention time: 0.94, 1.56 mins
Long-acting insulin

Non-Insulin Insulin

NPH-1 NPH-2 NPH-3

 Mahidol university Area under the curve (AUC) of each insulin
Faculty of Medicine
Siriraj Hospital after stored in different temperatures for 28 days

700

600 568.8 567.2 563.3 575.4

500
AUC

400
343.3 333.6 334.5 339.7
308.8 313.3 313.1 310.9 322.2 315 318.1 317.4
300

200

100

0
Long-acting insulin NPH-1 NPH-2 NPH-3
Control Room temperature (30 ºC) Refrigerator (2-8 ºC) Incubator (37 ºC)

Lantus NPH-1 NPH-2 NPH-3

P value 0.827 0.060 0.282 0.088
 Mahidol university Percent change from baseline of AUC of
Faculty of Medicine
Siriraj Hospital insulin after stored in different conditions
3 3
Humidity in each condition
1.5 1.5
75% 50% 38%
1.1
0 0
-1 -0.3 -2.6 -2.8 -1

‐1.5 ‐1.5

‐3 ‐3
Long-acting insulin NPH-1
P value 0.827 0.205 0.187 P value 0.060 0.051 0.409
3 3

1.5 1.5
1.4 1.5
0.7
0 0
-1.3 -2.2 -1.5

‐1.5 ‐1.5

‐3 ‐3
NPH-2 NPH-3
P value 0.282 0.100 0.584 P value 0.088 0.315 0.265
 Mahidol university
Faculty of Medicine
Siriraj Hospital Discussion
• Previous study had proved that temperature had
affected on stability of insulin1,2.
• Refrigerate an opened insulin was not
recommended by the manufactures because of
lacking cycling stability data3,4.

Evidence based recommendation should be

advised to storage insulin properly and maintain
adequate glycemic control.1. Vimalavathini R et al. Indian J Med Res. 2009; 130: 166-9
2. Storvick WO et al. Diabetes. 1968; 17: 499-502
3. https://www.lantus.com. Lantus-solostar-pen-guide.pdf
4. http://www.medicines.org.uk.Insulatard+Penfill.
Mahidol university
Faculty of Medicine
Siriraj Hospital Discussion

Room temperature as high as 37 ºC and

cyclic temperature had no effect on the
stability of an in-use basal insulin in pen
after opening for 28 days.
 Mahidol university
Faculty of Medicine
Siriraj Hospital Discussion
The present study Vimalavathini et al.1 Matthew A silva et al.2

Insulin product Pen Vial Vial

Basal insulin (long and Regular insulin Humulin (70/30),
Type of insulin
intermediate insulin) Biphasic insulin (30/70) Levemir
Sample size 5 Not mention 3
Temperature - Refrigerator - 5 ºC, 25 ºC, 26 ºC, - Refrigerator
condition - RT (30 ºC) - 32 ºC, 37 ºC (2-8 ºC)
- Incubator (37 ºC) - RT (22-24ºC)

Withdrawn insulin Everyday Every 7 days 28, 35, 42 days

Study duration 28 days 28 days 42 days
Insulin analysis UHPLC HPLC HPLC
No statistical significant Decrease 14, 18% of RI AUC decrease at 35
Results change in AUC of and 11, 14% of biphasic and 42 days after
insulin in all conditions insulin stored at 32, 37 ºC stored at RT

RT = Room temperature 1. Vimalavathini R et al. Indian J Med Res. 2009; 130: 166-9
RI = Regular insulin 2. Silva MA et al. J Pharm Pract Res 2013; 43: 37-40
 Mahidol university
Faculty of Medicine
Siriraj Hospital Discussion

Strength of the study Limitation

• First investigation in • Uncontrolled factors in

Thailand. room temperature
• Insulin pen. condition.
• UHPLC
• Study design resemble
real life practice of
insulin-treated diabetes
patients.
 Mahidol university
Faculty of Medicine
Siriraj Hospital
Conclusion

Temperature, as high as 37 ๐C, and the variability of

temperature range from 4 ๐C to room temperature
had no effect on the stability of in-use insulin pen.
 Mahidol university
Faculty of Medicine
Siriraj Hospital Future directions
• The trend toward different in stability of in-use insulin
between clear insulin analog and turbid NPH insulin.
• Greater difference of AUC in room temperature,
refrigerator condition. Humidity might effect its stability.

• The quantity was not changed but the quality might be

affected.

Need further investigation

Thank you for your attention