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Stability Studies of Two Different Polygelin (Haemaccel and

Gelofusine) According to ICH Guidelines
Mansoor Ahmad and Nudrat Adil

PDA J Pharm Sci and Tech 2013, 67 610-620

Access the most recent version at doi:10.5731/pdajpst.2013.00948

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RESEARCH

Stability Studies of Two Different Polygelin (Haemaccel and

Gelofusine) According to ICH Guidelines
MANSOOR AHMAD*, and NUDRAT ADIL
Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Karachi, Karachi-75270,
Pakistan PDA, Inc. 2013
ABSTRACT: The stability of polygeline-based blood plasma expanders Haemaccel and Gelofusine were examined in
context of their sensitivity to environmental factors, because drug stability is critical element in accurate and
appropriate delivery of drug therapy to patients. This study was initiated to specifically and critically assess stability
of Haemaccel and Gelofusine according to ICH guidelines with the aim of delivering safe, appropriate, acceptable,
and efficacious administration of drug product in any situation. This study revealed that Haemaccel and Gelofusine
are suitable for storage at different temperature and at different storage conditions until its expiry date, shelf life, or
utility time, for their quality, safety, suitability, acceptability, and efficacy.
KEYWORDS: Haemaccel, Gelofusine, Stability, ICH guideline, Acceptability.
LAY ABSTRACT: Stability studies of two different plasma substitutes, Haemaccel and Gelofusine, were examined
according to ICH guidelines for their expiry or utility time, because drug stability is very important element in
accurate, suitable, and correct delivery of drug therapy to patients. The aim of present study is to deliver the safe,
appropriate, acceptable, and right drug product in any situation. The results indicate negligible changes in different
parameters during stability study, except for pH and viscosity. This study shows that both drug products are suitable
for storage at different temperature and at different storage conditions till their expiry date or utility time, for their
quality, safety, suitability, acceptability, and strength.

Introduction
Proteins or polypeptides and polysaccharides containing drug products such as plasma substitutes are particularly sensitive to environmental factors; therefore,
these have become an important class of potent therapeutic agents in plasma substitutes in the last two
decades due to their unique physicochemical and biological properties. However, proteins are marginally
stable and highly susceptible to both chemical and
physical degradation (13). Therefore, stability issues
are very important in protein and polysaccharides containing plasma substitutes due to prolonged storage.
The aim of this study was to determine the stability of
Haemaccel and Gelofusine at different temperature

*Corresponding Author: Mansoor Ahmad, Research

Institute of Pharmaceutical Sciences, Department of
Pharmacognosy, University of Karachi, Karachi75270, Pakistan. herbalist53@yahoo.com
doi: 10.5731/pdajpst.2013.00948

610

and at different storage condition such as at room

temperature, at 40 C, in freezing conditions, and in
exposure to direct sunlight.
Materials and Methods
Materials
The studies were carried out at room temperature, at
40 C, in freezing conditions, and in exposure to direct
sunlight. Different batch no. E073, E074, E080, N036
of Haemacel and Gelofusine batch no. 4212E41 were
tested for stability studies (Table I). Haemaccel batch
no. E073 and E080, Gelofusine batch no. 4212E41
were kept in a Binder climate chamber (model
KBF720, Bohemia, NY, USA) for stability studies at
room temperature and at 40 C.
A pH meter WTW 525 (Wissenschaftich-Technische
Werksttten, Germany) was used for measuring pH
during stability studies of Haemaccel and Gelofusine.
Free amino groups were assessed using a Mettler
DL40RC Memo Titrator and a WTW 525 pH meter.
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Table I
Different Brands of Drug Products Used for the Study
No.

Brand

1
2
3
4
5

Haemaccel
Haemaccel
Haemaccel
Haemaccel
Gelofusine

Weight
500
500
500
500
500

mL
mL
mL
mL
mL

The relative viscosity of Haemaccel and Gelofusine

was determined at 35 1 C using an Ostwald viscometer (capillary viscometer, Schott AG, Germany).
Total nitrogen was determined by the Kjeldahl method
using a Buchi 426 digestion unit and Buchi 339 distillation unit during studies. Electrolytes were detected
by a previously calibrated flame photometer PFP7
(Jenway, England) against freshly prepared standard
solution. A PCLM3 chloride meter was used to measure chloride. A calibrated pyrometer (Ellab APT75)
was used to determine whether Haemaccel/Gelofusine
was pyrogen-free. Sterility was determined with previously cleaned and sterilized filter assembly.
Methods
The following physical, chemical, and microbial parameters were evaluated after completation of storage
time: appearance, pH, viscosity, free amino groups,
nitrogen content, electrolytes, loss in weight, pyrogen,
and sterility. All glassware and utensils were cleaned
with chromic acid and then rinsed with distilled water.
Before starting the stability studies tests, all relevant
instruments were cleansed and calibrated for accuracy.
Appearance was assessed visually for signs of precipitation or other evidence of alteration such as turbidity, haziness, or changes in color. A WTW 525 pH
meter was used for measuring pH during stability
studies of Haemaccel and Gelofusine.
The relative viscosity of Haemaccel and Gelofusine
was determined at 35 1 C using an Ostwald viscometer (capillary viscometer, Schott). The flow rates
of 5 mL distilled water and then 5 mL Haemaccel and
Gelofusine were determined, respectively.
Free amino groups were assessed using a Mettler
DL40RC Memo Titrator and a WTW 525 pH meter. In
this test a mixture of 40 mL Haemaccel /Gelofusine
and 1.6 mL 2N hydrochloric acid was titrated up to pH
Vol. 67, No. 6, NovemberDecember 2013

Batch No.

Manufacturer

E073
E080
N036
E074
4212E41

Sanofi-Aventis
Sanofi-Aventis
Sanofi-Aventis
Sanofi-Aventis
B. Braun

6.00 using 0.2N sodium hydroxide. The quantity of

sodium hydroxide consumed (M1) was recorded; 4 mL
of formaldehyde solution (Merck) previously adjusted
to pH 7.00 0.02 was then added. The pH value fell
sharply after 1 min. The titration was continued to pH
8.50 and the consumption of sodium hydroxide (M2)
was recorded. Free amino groups were calculated using the formula: (M2 M1)/5F mL of 1N sodium
hydroxide/40 mL of Haemaccel/Gelofusine, where F
is a factor of 0.2N sodium hydroxide.
Total nitrogen was determined by the Kjeldahl method
using a Buchi 426 digestion unit and Buchi 339 distillation unit during studies. Haemaccel/Gelofusine
(1mL) plus concentrated sulphuric acid (15mL;
Merck) and one Kjeldahl tablet, batch no. TP707758
(Merck) were digested for 30 min at 650 C in the
digestion unit. After digestion, the sample was cooled
and titrated using the distillation unit. Before titrating
the sample, the pH electrode in the distillation unit
was calibrated using buffer solution pH 4, batch no.
OC354850, and buffer solution pH 7, batch no.
OC354838.
Electrolytes were detected by previously calibrated
flame photometer PFP7 (Jenway) against freshly prepared standard solution. The preparation of test and
standard solution was as follows. The sample/test solution was 1 mLHaemaccel/Gelofusine in 100 mL
distilled water. To prepare the standard solution,
1000ppm 3.34 mL Na , batch no. FINA4HI (Jenway), 1000ppm 0.20 mL K, batch no. FIK2LI (Jenway) and 1000 ppm 0.25 mLCa, batch no.
FKA12MI (Jenway) were mixed in a 100 mL volumetric flask and remaining volume filled with distilled
water. A PCLM3 chloride meter was used to measure
chloride. Calibration and determination of chloride
was as follows: 5 mL acid buffer solution, batch no
1273 (Jenway), 10 mL distilled water and 0.3 mL or
10 drops of gelatin solution, batch no 2237 (Jenway)
were placed in plastic beaker with a stirrer bar and the
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beaker was placed on the instrument platform. The

20 L range was selected and 20 L of a standard
solution of chloride 100mmol/L, batch no. 0250135CI
(Jenway) was added and conditioning was performed.
After conditioning, a further 20 L of a standard
solution was added and then titrated; the reading was
close to 100mmol/L. After calibration the whole process was repeated in the same way, except 20 L
Haemaccel was added instead of standard solution and
the reading were recorded.
Loss in weight was determined by recorded initial
weight (W1) of Haemaccel and Gelofusine bottle when
the study was started and the different storage time
weight (W2) during the study1, 2, 3, 6, 12, 24, and
36 monthsat different temperatures: room temperature, at 40 C, and in a freezer (5 to 15 C) by
using the formula
(W1W2)/(W1) 100
A calibrated pyrometer (Ellab APT75) was used to test
whether Haemaccel/Gelofusine was pyrogen-free. In
the pyrogen test, three pretested rabbits each received
10 mL Haemaccel/Gelofusine per kilogram body
weight intravenously. Temperature rises were interpreted according to the British Pharmacopeia IV (BP).
Sterility was determined by a previously cleaned and
sterilized filter assembly according to the European
Pharmacopeia (Ph.Eur.) (2.6.1). About 200 mL of
Haemaccl/Gelofusine was filtered through the filtration assembly by using a suction pump. After filtration
the membrane filter (Cellulose nitrate, diameter: 50
mm, pore size: 0.45 micron, Ireland) was cut into two
equal halves and one half transferred into soya broth
medium and the other into thioglycollate medium.
Observation time of incubation for both soya bean
casein digest medium (at 20 25 C) and for fluid
thioglycollate medium (at 30 35 C) is 14 days.
Results and Discussion
Stability Results
Stability study of Haemaccel and Gelofusine at various storage conditions showed considerable change in
pH value and in viscosity. Different scientists also
reported change in pH value and in viscosity, such as
Gabr (1996) and coworkers, who reported decreased
value in pH (7.3 to 7.0) and in relative viscosity (2.18
to 1.98) of oxypoly gelatin (4). Theiercelin also re612

ported that storage temperature affected the viscosity

of gelatin, polyvinyl pyrrolidone, and dextran (28).
The appearance of Haemaccl/Gelofusine remained the
same during the stability study. Stability of Haemaccel
at room temperature for 3 years showed decreased
values of viscosity and pH from 1.76 and 7.13 to 1.68
and 6.89, respectively. Increased value of free amino
groups (from 0.525 to 0.591) and loss in weight (from
0.10% to 0.83%) and fluctuation value was observed
in nitrogen contents. Loss in weight gradually increased in this study. The results of pyrogen and
sterility were satisfactory (Table II). During stability
study at 40 C for 6 months, decreased value of
viscosity (from 1.77 to 1.65) and pH (from7.28 to
7.13) were recorded. The value of viscosity sharply
decreased, which was considerable.
Increased value of free amino groups (from 0.527 to
0.578)which is also reported by Thiercelin et al.
(31)and loss in weight (from 0.21% to 0.35%) and a
little decreased value of nitrogen contents (from 6.40
to 6.35) were observed. Results of pyrogen and sterility were satisfactory (Table III). On the other hand,
stability study of Haemaccel in sunlight (days and
night) for 3 months showed gradually decreased pH
(from 7.16 to 6.17) and viscosity (from 1.76 to 1.70).
Fluctuations were observed in nitrogen and chloride
values. Results of free amino groups were from 0.542
to 0.514, and loss in weight was from 0% to 0.087%.
Results of Na, K and Ca ions remained almost
the same, and results of pyrogen and sterility were
satisfactory (Table IV). The stability of Haemaccel in
freezing conditions showed a slight change in viscosity (from 1.77 to 1.76), a decreased value in pH (from
7.27 to 7.10), a little change in free amino groups
(from 0.544 to 0.562), and loss in weight (from 0.01%
to 0.03%); fluctuation was observed in nitrogen but on
the whole insignificant changes were observed in this
study (Table V). Stability study of Gelofusine at room
temperature over a 3 year period showed slightly
dropped values in pH (from 7.28 to 7.08) and viscosity
(from 2.10 to 1.81), increased values in free amino
groups (from 0.555 to 0.641) and loss in weight (from
0.02% to 1.07%), and decreased values observed in
nitrogen (from 7.39 to 7.29). Rate of changes in different parameters are less in Gelofusine as compared
to Haemaccel, which may be depend on concentration
differences (Gelofusine is 6% and Haemaccel is 3.5%)
and also the manufacturing process (succinic acid
anhydride is used in Gelufusine, whereas hexamethylene di isocyanate is used as a cross-linking
agent in Haemaccel). On the other hand, the stability
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Table II
Stability Study of Haemaccel at Room Temperature
STABILITY DATA
Preparation: Haemaccel Batch No. E073
_______________________________________________________________________________________________
Packing material: Polyethelene bottle
Storage: 36 months room temperature (average 28 C and 60% relative humidity)
Source: (Formerly SKW Gelatin & Specialties France) Rousselot Cedex, France

pH
(7.007.60)

Free Amino
Groups
(0.500.65)

Loss in
weight
(%)

Nitrogen
Content
(6.06.6)
(g/l)

Sterility
(Ph.Eur)

Pyrogen
(BP)

1.76

7.13

0.525

N/A

6.60

Complies

Complies

1.75

7.09

0.528

0.10%

6.43

N/A

N/A

Clear yellowish
solution

1.72

7.00

0.552

0.36%

6.41

N/A

N/A

Room temperature

Clear yellowish
solution

1.71

6.96

0.575

0.65%

6.61

N/A

N/A

Room temperature

Clear yellowish
solution

1.68

6.89

0.591

0.83%

6.40

Complies

Complies

Storage
Time
(month)

Appearance

Viscosity
(Relative)
(1701.80)

Storage
Condition

Room temperature

Clear yellowish
solution

Room temperature

Clear yellowish
solution

12

Room temperature

24
36

mL of 1N NaOH/40 mL of Haemaccel.

of Gelofusine at 40 C showed decreased values in pH

(from 7.28 to 7.08) and in viscosity (from 2.10 to
1.632), increased values in free amino groups (0.555
to 0.739) and loss in weight (from 0.109% to 1.23%),
and decreased values observed in nitrogen (from 7.39
to 7.29) (Tables VI and VII). Stability study of both
plasma substitutes at 40 C show that Haemaccel is
more stable in the parameters of free amino groups and
in loss in weight, as degradation rate is low as compared to Gelofusine.

Discussion
This study was carried out to determine whether different temperature and different storage condition
such as at room temperature, at 40 C, in freezing
conditions, and in exposure to direct sunlight would
alter the physical, chemical, and microbial parameters
of Haemaccl/Gelofusine during stability study. For
this purpose various different parameters (pH, relative
viscosity, free amino groups, nitrogen content, elec-

Table III
Stability Study of Haemaccel at 40 C
STABILITY DATA
Preparation: Haemaccel Batch No. E080
_______________________________________________________________________________________________
Packing material: Polyethelene bottle
Storage: 6 Months 40 C (Average 40 C and 75% relative humidity)
Source: (Formerly SKW Gelatin & Specialties France) Rousselot Cedex, France
Storage
Time
(month)

Storage
Condition

0
3
6

pH
(7.007.60)

Free Amino
Groups
(0.500.65)

Loss in
weight
(%)

Nitrogen
Content
(6.06.6)
(g/L)

Sterility
(Ph. Eur)

Pyrogen
(BP)

1.77

7.28

0.527

N/A

6.40

Complies

Complies

1.69

7.17

0.567

0.21%

6.36

N/A

N/A

1.65

7.13

0.578

0.35%

6.35

Complies

Complies

Appearance

Viscosity
(Relative)
(1.701.80)

Room temperature

Clear yellowish
solution

40 C

Clear yellowish
solution

40 C

Clear yellowish
solution

mL of 1N NaOH/40 mL of Haemaccel
Vol. 67, No. 6, NovemberDecember 2013

613

614

Clear yellowish

Room
temperature

Sun light

Sun light

Sun light

Sun light

Sun light

Sun light

Sun light

20

34

49

56

63

74

1.75
1.74
1.73
1.71
1.70

Clear pale
yellowish l

Clear pale
yellowish

Clear pale
yellowish l

Clear pale
yellowish

Clear pale
yellowish

Relative,
mL of 1N NaOH/40 mL of Haemaccel,
mmol/L

1.76

1.76

1.76

Viscosity
(1.701.80)

Clear yellowish

Clear yellowish

Appearance

Storage
Condition

Storage
Time
(Days)

6.17

6.32

6.40

6.42

6.60

6.73

6.85

7.16

pH
(7.007.60)

0.514

0.515

0.517

0.515

0.520

0.520

0.537

0.542

Free Amino
Groups
(0.500.65)

149

149

149

149

148

148

149

148

Sodium
(m.mol/lit)
(139-152)

5.0

5.0

5.0

5.1

5.1

5.1

5.0

5.0

Potassium
(m.mol/lit)
(4.65.6)

5.6

5.6

5.6

5.5

5.5

5.5

5.5

5.6

Calcium
(m.mol/lit)
(5.5-7.0)

143

143

143

144

144

143

144

137

Chloride
(m.mol/lit)
(130-160)

0.08 (7%)

0.06 (3%)

0.03 (4%)

0.03 (3%)

0.02 (4%)

0.00 (6%)

N/A

Loss in
weight (%)

6.30

6.25

6.24

6.26

6.25

6.22

6.441

6.30

Nitrogen
Content
(6.06.6)g/l

N/A

N/A

N/A

N/A

N/A

N/A

N/A

Complies

Sterility
(Ph.Eur)

N/A

N/A

N/A

N/A

N/A

N/A

N/A

Complies

Pyrogen
(BP)

Table IV
Stability Study of Haemaccel under Sunlight (Day and Night)
STABILITY DATA
Preparation: Haemaccel Batch No. N036
_________________________________________________________________________________________________________________________________
Packing material: Polyethelene bottle
Storage: Under sunlight
Source: (Formerly SKW Gelatin & Specialties France) Rousselot Cedex, France.

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Table V
Stability Study of Haemaccel in Freezing Condition (5 to 15 C)
STABILITY DATA
Preparation: Haemaccel Batch No. E074
_______________________________________________________________________________________________
Packing material: Polyethelene bottle
Storage: 36 months in freezing conditions (5 to 15 C)
Source: (Formerly SKW Gelatin & Specialties France) Rousselot Cedex, France

pH
(7.007.60)

Free Amino
Groups
(0.500.65)

Loss in
weight
(%)

Nitrogen
Content
(6.06.6)
(g/L)

Sterility
(Ph.Eur)

Pyrogen
(BP)

1.77

7.27

0.544

N/A

6.472

Complies

Complies

1.77

7.26

0.544

0.01%

6.35

N/A

N/A

Clear yellowish
solution

1.77

7.21

0.550

0.02%

6.40

N/A

N/A

Freezer

Clear yellowish
solution

1.77

7.16

0.553

0.02%

6.44

N/A

N/A

Freezer

Clear yellowish
solution

1.76

7.13

0.555

0.03%

6.41

N/A

N/A

Freezer

Clear yellowish
solution

1.76

7.11

0.561

0.03 %

6.36

N/A

N/A

Freezer

Clear yellowish
solution

1.76

7.10

0.562

0.03%

6.44

Complies

Complies

Storage
Time
(month)

Appearance

Viscosity
Relative
(1.701.80)

Storage
Condition

Freezer

Clear yellowish
solution

Freezer

Clear yellowish
solution

Freezer

15
24
30
36

mL of 1N NaOH/40 mL of Haemaccel

trolytes, pyrogen, and sterility) were considered. The

results demonstrated negligible changes during stability study except for pH and viscosity, which were
considerable. Gabr (1996) and coworker also reported
decreased value in pH (7.3 to 7.0) and in relative
viscosity (2.18 to 1.98) of oxypoly gelatin. In the same
way degraded gelatin solution also decreased its relative viscosity (1.79 to 1.58), but pH value slightly
increased (5.2 to 5.4) (4).
Different scientist studied the stability of different
drug products in different infusion solutions at different storage conditions, such as Jean-Daniel (2005) and
coworkers investigated the effect of freezing, longterm storage, and microwave thawing on the stability
of ketorolac tromethamine in dextrose 5% infusion.
They observed no color change or precipitation, but
pH value decreased slightly (5). Fischer (1997) and
coworkers determined stability of fosphenytoin sodium admixtures with NaCl 0.9% injection and dextrose 5% injection storage at room temperature for 30
days. They did not observe any visible precipitation or
change in color or clarity during stability study (6).
Mendenhall (1984) has reviewed stability aspects of
parenteral products and has shown that discoloration
Vol. 67, No. 6, NovemberDecember 2013

often is either photochemical or oxidative; sometimes

a cloud or precipitate may appear in drug products as
storage time progresses. This is most often due to
chemical changes in the system (7). There was not any
change observed in appearance of Haemaccel and
Gelofusine in this study during different storage conditions. Pale yellowish color was observed in Haemaccel during stability study in sunlight. But there is not
any change of color observed in Haemaccel and Gelofusine during different storage conditions: at room
temperature, at 40 C, and in freezing conditions (5
to 15 C). Neuwald reported water loss in Haemaccel during storage up to 3 years at temperatures between 20 and 60 C (8). In our study slightly
decreased values were observed in loss in weight in
both Haemaccel and Gelofusine at room temperature
and at 40 C. Viscosity of liquid usually decreases
with rise in temperature. The amount of such a decrease is often of order of 1% to 10% per degree
Celsius.
The opposite effect may occur in certain cases, such as
aqueous solutions of synthetic polymers like methylcellulose, which exhibit gel formation when temperature is increased (9). Theiercelin et al. (28) examined
the viscosity of plasma substitute solutions and found
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Table VI
Stability Study of Gelofusine at Room Temperature
STABILITY DATA
Preparation: Gelofusine Batch No. 4212E41
_______________________________________________________________________________________________
Packing material: Polyethelene bottle
Storage: 36 months room temperature (average 28 C and 60% relative humidity)
Storage
Time
(month)

Storage
Condition

pH

Free Amino
Groups

Loss in
weight
(%)

Nitrogen
Content
(g/L)

Sterility
(Ph.Eur)

Pyrogen
(BP)

2.10

7.28

0.555

N/A

7.39

Complies

Complies

2.06

7.24

0.558

0.02%

7.33

N/A

N/A

2.05

7.18

0.586

0.12%

7.25

N/A

N/A

2.01

7.14

0.561

0.109%

7.13

N/A

N/A

Clear yellowish
solution

1.96

7.12

0.581

0.25%

7.08

N/A

N/A

Room temperature

Clear yellowish
solution

1.93

7.11

0.585

0.432%

7.12

N/A

N/A

Room temperature

Clear yellowish
solution

1.88

7.10

0.616

0.487%

7.09

N/A

N/A

Room temperature

Clear yellowish
solution

1.87

7.10

0.624

0.682%

7.10

NA

NA

Room temperature

Clear yellowish
solution

1.81

7.08

0.641

1.07%

7.00

Complies

Complies

Appearance

Viscosity
(Relative)

Room temperature

Clear yellowish
solution

Room temperature

Clear yellowish
solution

Room temperature

Clear yellowish
solution

Room temperature

Clear yellowish
solution

12

Room temperature

3
6

18
24
30
36

mL of 1N NaOH/40 mL of Haemaccel

that storage temperature affected the viscosity of gelatin, polyvinyl pyrrolidone, and dextran solutions. In
further study Thiercelin et al. (29) observed the effect
of supersonic waves on viscosity of plasma substitute
solutions. They concluded that ultrasonic waves have
little or no influence on viscosity of solutions. The
present study also conforms the stated results of
Theiercelin that storage temperature affects the viscosity, as Haemaccel and Gelofusine also showed
decreased value of viscosity, at room temperature and
at 40 C on duration of 3 years, 6 months, and 30
months (in freezing condition) in Haemaccel, and 3
years at room temperature, 30 months at 40 C in
Gelofusine in this study respectively; but at storage in
sunlight quite little change of results was observed
during study of Haemaccel under sunlight for 30 days.
But it was also observed that two different type of
acetyl starch (AS299 and AS297) showed a constant
relative viscosity over a period of time of 140 days
(10). Siragusa in 1955(11) demonstrated that increased
viscosity makes an emulsion more stable. Hetastarch
has a viscosity of 4.5 kg.m1s1 and Haemaccel 1.23
kgm1s1or Pas. The present study also confirmed
the study of Siragusa, as viscosity of Haemaccel was
decreased. But on the other hand, stability study of
616

Haemaccel in freezing conditions (5 to 15 C)

showed slightly decreased value of viscosity during 3
years, which conformed to the claim of the manufacturer.
The most important factors that influence the rate of
drug decomposition in drug delivery systems are solution pH and temperature. The stability of many
disperse systems, and especially of certain emulsions,
is often pH-dependent. Therefore, drug reaction rates
are generally less at intermediate pH values than at
high or low ranges, and most drugs are sufficiently
stable in pH range of 4 to 8. For example, morphine
solutions do not decompose during 60 min exposure at
a temperature of 100 C if the pH is less than 5.50
(12). During the Cuban crisis, the American supplies
of liquid dextran solutions were examined and scientists found change in pH values, diminished vacuum,
and a flaky white precipitate for up to 10 years in
storage conditions (30). But Cadrobbi coworker reported that no color change or precipitation occurred
in sodium folinate in 5% dextrose for a period of at
least 30 days at 48 C, but the pH values of infusion
solution decreased slightly without affecting chromatographic parameters (13). The present study also
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Table VII
Stability Study of Gelofusine at 40 C
STABILITY DATA
Preparation: Gelofusine Batch No. 4212E41
_______________________________________________________________________________________________
Packing material: Polyethelene bottle
Storage: 30 Months at 40 C (Average 40 C and 75% relative humidity)
Storage
Time
(month)

Storage
Condition

pH

Free Amino
Groups

Loss in
weight
(%)

Nitrogen
Content
(g/L)

Sterility
(Ph.Eur)

Pyrogen
(BP)

2.10

7.28

0.555

N/A

7.39

Complies

Complies

2.04

7.23

0.573

0.109%

7.40

N/A

N/A

2.00

7.20

0.578

0.112%

7.35

N/A

N/A

2.00

7.18

0.584

0.258%

7.31

N/A

N/A

Clear yellowish
solution

1.893

7.16

0.611

0.359%

7.30

N/A

N/A

Clear yellowish
solution

1.632

7.08

0.739

1.23%

7.29

Complies

Complies

Appearance

Viscosity
(Relative)

Room
temperature

Clear yellowish
solution

40 C

Clear yellowish
solution

40 C

Clear yellowish
solution

40 C

Clear yellowish
solution

40 C
40 C

1
2

30

mL of 1N NaOH/40 mL of Haemaccel
fusine during storage of 3 years at room temperature
and at 40 C. Haemaccel storage in freezing conditions
(5 to 15 C) showed a little change in pH value.
Lebitasy et al. also reported slight change in pH values
(6.52 0.01 to 6.50 0.01) of calcium levofolinate
in 5% dextrose solution stored at 5 3 C for 1 month
(14).
On the other hand, Haemaccel stored in sunlight for 30
days showed a gradual decrease in pH values, which is
supported by the above study. Carboxylic acid ester,
amides, and imines are labile to hydrolysis. As polygeline is manufactured by bovine gelatin, it consists of
different polypeptide bonds, bound together to form
urea bridges by cross-linking with hexamethylene diisocyanate. It has different side groups, which are reactive groups such as hydroxyl groups, amino groups,
carbonyl groups/carboxyl; these groups react with isocyanate group of hexamethylene di-isocyanate.
Amino groups react to form urea derivatives, where as
they react with hydroxyl groups to form carbamic acid
esters. Free carboxyl groups react with free amino
groups to form peptide bonds, and esters groups react
in known manner with lysine- amino groups, forming cross-linking peptide bonds. Or, if amine and
carboxylic acid functional groups in amino acids join
together to form amide bonds, a chain of amino acid
units is formed called peptide, and connected by C-N
bonds (covalent) will produce water.
Vol. 67, No. 6, NovemberDecember 2013

As esters are rapidly degraded in aqueous solution and

polygeline (Haemaccel) also contain water for injection (WFI) or (make up with water for injection), so it
is possible that hydrolysis causes breaking of polypeptide chains because the presence of hydroxyl groups
produce free amino groups. In the same ways extreme
temperature/heat will result in the unfolding of polypeptide chains, leading to change in structure and
often a loss of function. Covalent interactions between
amino acid side chains of ploypeptide are lost, there is
breaking of C-N, and free amino groups are formed.
At low-pH levels, protein will denature due to loss or
gain of protons, and will lose their charge or become
charged. This will eliminate ionic interactions and
may cause breaking of linkages of polypeptide bonds.
Thiercelin et al. reported that high temperature during
storage of plasmagel caused a slight increase in fluidity and acidity. He recommended that it should be
checked periodically (31). The present study also
showed increased value of free amino groups in both
Haemaccel and Gelofusine during storage at room
temperature and at 40 C (Table II and III). The
amphiphilic nature of the protein molecules results in
their adsorption to a wide variety of surfaces and also
in their loss and destabilization (1520). Side chains
of tyrosine, phenylalanine, and tryptophan, as well as
peptide bonds in proteins, absorb ultraviolet light.
Both ionizing and nonionizing radiation can cause
protein inactivation (21).
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Haemaccel stored in sunlight for 3 months as a protein

formulation has poor photostability because many
amino acid residues are prone to photolytic degradation (22). Our study also showed increased value of
free amino groups because heat is probably the most
common cause of disruption or unfolding of proteins
natural secondary or tertiary structure, leading to denaturation (23). In the same way, a little change was
observed in Haemaccels free amino groups during
storage in freezing conditions up to 3 years.
Neuwald reported the results of Haemaccel storage up
to 3 years at temperature between20 and 60 C (8).
His findings showed variations in nitrogen values during storage. The present research also supports the
Neuwald (8) study, that is, fluctuation was observed in
nitrogen content during storage of Haemaccel at room
temperature, at 40 C, in sunlight, and in freezing
conditions. Gelofusine showed the same results as
Haemaccel, that is, fluctuation. Several important drug
interactions occur as a result of therapeutic agents
altering concentrations of electrolytes, such as potassium and sodium. When these drugs are included in
therapeutic regimen, it is important that electrolyte
levels be periodically monitored. Electrolytes also
play important role in the stability system. If there is
a greater concentration of electrolytes then there is a
greater stability of the system.
In this study, results of electrolytes values were almost
similar during stability study. The problem of pyrogenic reactions to intravenous injection had been observed as early as in 1911 when Wechselman noted an
increase in temperature and chills in patients receiving
injections of arsphenamine. In 1923, Florence Seibert
discovered that the drug fevers referred to by Wechselman were caused by bacteria-produced pyrogens.
Her studies were extended to the development of the
rabbit test for pyrogens, still the USP test method.
While looking for a quicker and simpler pyrogen test
for radiopharmaceuticals, Cooper and his associates,
in 1969, developed the limulus test, a test for bacterial
endotoxin using Limulus amebocyte lysate (24). The
rabbit or pyrogen test, along with a sterility test,
become the two most important tools of the pharmaceutical industry. The pyrogen test employing rabbits
is still in limited use; an endotoxin test using an
extract from blood cells of horseshoe crab is the
predominant pyrogen test today.
In the present study the pyrogen test was employed.
Pyrogen testing is incorporated as a released criterion
618

for the product, but unlike sterility, it is rarely used as

a test criterion after release of product. During storage
at room temperature, at 40 C for 6 months, and in
freezing conditions for 30 months, Haemaccel and
Gelofusine remained pyrogen-free.
For certain LVP solutions in plastics, a sterility check
is incorporated into the stability protocol to verify the
integrity of container at specific intervals. The sterility
test or procedure will detect susceptible areas of the
container, potential problem sitesfor example, loosening of latex plugs at medication site, air-inlet site, or
improperly sealed rubber closuresand caps of bottles, as well as improper molding of bottles. This study
revealed that during storage of 3 years at room temperature (Haemaccel and Gelofussine), 6 months and
30 months at 40 C (Haemaccel and Gelofusine respectively), and 3 years storage in freezing conditions
(Haemaccel), both plasma substitutes are maintained
their sterility.
Lundsgaard-Hansen and coworker reported that solutions of 5 6% dextrose, 0.9% saline, and Ringers
lactate are stable for up to 1 year or more, even if
stored at ambient temperatures. Dextrose solutions
may acquire a yellowish tinge (caramelization), but
this is clinically insignificant.
The dextrans and the gelatins are very sensitive to
temperatures exceeding 20 30 C. A degradation into
smaller molecules begins after 1 month of storage at
40 60 C and is very marked (approximately 30
60% decrease in the average RMM) after 5 6 months.
In clinical terms, this would shorten their intravascular
volume effect and accelerate their renal elimination.
In contrast, hydroxyethyl starch 450 shows no such
degradation after 6 months of storage at 40 60 C.
Although heat and pH are the main factors that can
cause aggregation (25), they can lead to precipitation
of protein (26). But prolonged storage under adverse
conditions may result in a crystalline precipitate or a
deep brown, turbid appearance in starch solution (12).
Long periods of storage or exposure to temperature
fluctuations may cause the formation of flakes in dextran solution (4). Acetyl starch is not stable in longterm storage because acetic acid is separated in large
amounts at 20 C and 40 C (10). In hot climates,
colloidal plasma substitutes should not be stored at
ambient temperatures for periods exceeding 1 month,
and they preferably should be stored at temperatures
below 20 C. If stored at refrigerator temperatures,
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they should be warmed prior to use to avoid hypothermia in the recipient (27).

7. Mendenhall, D. W. Stability of parenterals. Drug.

Dev. Ind. Pharm. 1984, 10, 12971342.

All of these observations and results are useful for

storage of these plasma substitutes at different temperature for their expiration time, shelf life, or utility
time restriction for hot and humid region of world.

8. Neuwald, F. Stability of Gelatin Plasma Substitute

Solutions during Storage with Reference to Military
Medical Use. Modified Gelatins as Plasma Substitutes. In Bibliotheca Haematologica; Karger: Basel/
New York, 1969; Vol. 33, pp 598 600.

Conclusions
This study revealed that Haemaccel and Gelofusine
are suitable for storage at different temperatures and at
different storage conditions until their expiry date,
shelf life, or utility time, for their quality, safety,
suitability, acceptability, and efficacy.
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