Introduction
Human growth hormone (hGH) is a
responsible protein for a wide range of growth-promoting
effects in the body. Somatropin, the major component of growth
hormone produced by the human pituitary gland, is a
single-chain peptide composed of 191 amino acids and two
disulfide bridges with a molecular mass of approximately 22000
daltons (1, 2). Since 1980's, the availability of recombinant
DNA (rDNA)-derived hGH in sufficient quantities and high purity
allowed scientists to prepare various pharmaceutical
formulations for the fast increasing clinical applications (1).
Like most large proteins, growth hormone
readily undergoes various chemical and physical instability
reactions (3). The predominant degradative reactions of hGH are
deamidation, oxidation, amino-terminal degradation and physical
instability, which produce dimers or more aggregated forms. A
number of publications have referred to the various routs
through which hGH is degradated and described the mechanisms
and kinetics of its degradation (1-7).
The development of stability-indicating
analytical methods is critical for manufacturing, formulation
and storage of protein drugs. Complex structures of proteins
make their characterization difficult. Meanwhile, small
structural changes in a protein can influence the
physicochemical properties as well as its activity and potency
(8). Therefore, rapid, accurate and reliable methods are
necessary for the characterization and purity determination of
proteins (8, 9).
Reversed-phase high performance liquid
chromatography (RP-HPLC) is the most frequently used
chromatographic technique for the analysis of peptides and
intact proteins, especially for the determination of their
purity (8) as well as for the analysis of peptide fragments
(peptide mapping) obtained after enzymatic digestion or
chemical cleavage (8, 10, 11). It is notable that dimers and
more aggregated forms of proteins are studied by other methods
such as size exclusion chromatography, which is also applied
for the protein assay (1, 3).
Several different RP-HPLC methods for the
analysis of hGH and its variants and degradation products have
been reported in the literature (1, 6, 12-14) and also in the
European Pharmacopoeia (EP) (2) and US Pharmacopeial forum (7).
According to the EP monograph, the suggested run time for each
analysis is around 55 min. The retention time of the native
somatropin is about 33 min and desamido-somatropin appears
after 28 min (2). Other reported methods are able to separate
different variants within at least a 25 min-run time (6,
12-14).
The polymeric reversed-phase packing
materials, specifically the poly (styrene-co-divinylbenzene)
sorbents, have been introduced as excellent alternatives to the
alkyl-bonded silicas for RP-HPLC of some drugs and proteins.
Extended column life, reduced analysis time and inherent
stability to a wide range of pH are some of the claimed
advantages of these columns (15).
The aim of this study was to develop a
rapid and sensitive RP-HPLC method for conducting stability
studies on the recombinant hGH (rhGH) degradation products,
using a polymeric column.
Experimental
Materials
Somatropin coded NIBSC-98/574
(Hertfordshire, UK), accepted by the Expert Committee on
Biological Standardization of WHO in 2001 as the 2nd
International standard of hGH, was obtained in vials containing
1.95 mg of Somatropin and 26.5 mg of excipients (glycine,
mannitol, lactose and sodium bicarbonate). Norditropin® vials
(NovoNordisk, Denmark), a pharmaceutical dosage form of
Somatropin (1.3 mg) formulated with glycine, mannitol and
sodium bicarbonate, was also used. Water was purified with a
Millipore system (Bedford, USA). HPLC-grade acetonitrile and
trifluoroacetic acid (TFA), and all other analytical
grade reagents were obtained from Merck Chemical Co.
(Darmstadt, Germany).
Methods
Sample preparation
Stock solution with a concentration of 1
mg/ml was prepared by adding an adequate amount of purified
water to each vial. Solutions with lower concentrations were
prepared by serial dilution of the stock solution. All protein
solutions were stored at 4°C and used within
24 h.
Deamidation and Oxidation of hGH
Somatropin/desamido-somatropin mixture (1
mg/ml) was prepared according to the EP monograph (2) by adding
0.1 mg sodium azide to each ml of a freshly-prepared hGH
solution, and allowing the solution to stand at
room temperature for 24 h. This sample was then maintained at
2-8°C and used within 24 h.
The oxidation was performed by adding 2 or
20 mL
hydrogen peroxide (30%, v/v) to each ml of a hGH solution
(1mg/ml) and incubating at 2-8°C, overnight. The obtained
solution was used within 24 h (6).
Instrumental and chromatographic
conditions
A computer-controlled Shimadzu HPLC system
(Japan) consisting of two pumps (LC-10 ADvp), a system
controller (SCL-10Avp), a degasser (DGU-14A), a diode array
detector (SPD-M10 vp) and a column oven (CTO-10A vp) was used.
The separation was performed on a PRP-3 polymeric
reversed-phase column (10 mm, 300°A, 150*4.1 mm I.D.) (Hamilton,
Switzerland). A proper guard column (12-20 mm, 20*2.3 mm I.D.)
(Hamilton, Switzerland) was also applied. Mobile phase was
passed through the column with a flow rate of 1 ml/min as a
linear gradient of 0-60% of solution A (0.1% v/v
TFA/acetonitrile) in solution B (TFA/Water, pH=2.00) within 5
min followed by holding for 5 min. Mobile phase solutions (A
and B) were prepared freshly for each day. During separation,
the column temperature was set at 35°C. The injection
volume of the sample was 20 mL. Detection was carried out by measuring UV
absorbance at 215 nm.
Method validation
Validation of the method was performed in
terms of selectivity, linearity, intra- and inter-day
variations and percentage of recovery.
Selectivity of the method was assessed by
separate injection of each excipient within the sample (as
described above) and observing any interference with somatropin
peak. Selectivity assessment was also extended to the
evaluation of any interference from degradation (deamidation
and oxidation) products of the protein. Precision of the method
was evaluated by repeated analysis of solutions containing
known concentrations of somatropin. Linearity was studied over
a range of 0.05-1 mg/ml at 6 different concentration levels of
somatropin, each prepared freshly before injection.
Inter- and intra-day variations were
determined at three protein concentration levels, namely 0.1,
0.5 and 1 mg/ml.
Results And Discussion
Mobile phase constituents and pH have been
shown to influence the reversed-phase HPLC separation of
proteins (5). Modifiers are substances that are added to the
mobile phase, usually in a relatively low concentration, and
interact with both the stationary phase and sample constituents
in order to alter retention. Trifluoroacetic acid (TFA) is the
modifier most frequently used for peptide separation in
RP-HPLC, usually at a concentration of 0.1%. It has the most
important effect of improving peak shapes and exerts its
effects by pairing with positively charged and polar groups on
peptides and proteins to mask these sites from polar
interactions and bringing them to the reversed-phase
surface. The UV absorbance spectrum of TFA occurs below 200 nm
and thus creates minimal interference with the detection of
peptides at low wavelengths (16). The inherent stability of the
applied polymeric column in low pH extreme allowed taking
benefit of TFA as a modifier in the mobile phase. On the other
hand, the polymeric support is pressure stable and cross-linked
to prevent shrinkage or swelling when the mobile phase is
changed (15). This helped to run a rapid linear gradient from 0
to 60% of acetonitrile in water within 5 min. Thus, compared to
the EP method, the proposed method allowed a shorter analysis
time for hGH by reducing the run time from 55 min to 12 min.
The average retention time of the native somatropin peak (I)
was 8.7 min (Figure 1). Meanwhile, a shorter column length and
larger particle size of the packing material, along with higher
flow rate of the mobile phase contributed to shorter run time.
It is also evident that the solution-driven conformational
changes resulted from the addition of organic modifiers like
1-propanol (in EP method) or acetonitrile (in the present
method), significantly influence the retention behavior of the
protein. For example, 1-propanol at a concentration of >10%
induces the formation of a molten globule state, which is an
intermediate in the unfolding pathways of a globular protein
and provides additional hydrophobic surfaces for interaction in
RP-HPLC (17).
Under the chromatographic conditions
described, no interferences due to the presence of excipients
used within the samples were observed. As shown in Table 1, the
coefficients of variation for repeated analysis of freshly
prepared samples containing known concentrations of protein
were less than 6%, which is acceptable for the routine
measurement of somatropin.
The calibration curve for the
determination of somatropin in freshly prepared samples was
linear over the studied range and the equation intercept was
not statistically different from zero. The correlation
coefficient of the standard curve was 0.9980 and the
corresponding equation was: Y=2X-0.014, where Y is the peak
area of somatropin and X is the somatropin concentration.
Moreover, baseline separation of deamidated and oxidized forms
of hGH demonstrates selectivity of the method used.
Figure 2 shows separation of native
somatropin and the deamidated form, in which retention of the
latter was 7.2 min. The earlier elution of this form is
suggested to occur as a result of its higher polarity.
The chromatograms of hydrogen
peroxide-incubated hGH samples presented two peaks (peaks III
& IV in Figure 3) at 1.6 and 5.3 min. Oxidation of
methionine residues of proteins generally results in the
formation of more polar compounds. Therefore, the species that
contains the oxidized methionine group is eluted prior to the
non-oxidized species (8). Identity of the oxidized forms of the
protein (peaks III & IV) was revealed by rendering the
oxidation reaction under higher concentration of H2O2 (as
described in the Experimental section). Larger amounts of H2O2 led
to the acceleration of oxidation reaction, which resulted in an
increase in the peak heights of corresponding oxidized
products.
Conclusion
In this work, a selective, rapid and
reproducible HPLC method, based on reversed-phase polymeric
column, was presented for the identification of somatropin and
its degradation products. The method is potentially applicable
for the quality control of this protein during purification,
formulation or stability studies.
Acknowledgement
This work was supported financially by
Research Deputy of Shaheed Beheshti University of Medical
Sciences (SBUMS). The authors also wish to express their thanks
to Food and Drug Quality Control Laboratories of the Ministry
of Health of Iran and Traditional Medicine and Materia Medica
Research Center (TMRC) in SBUMS for their cooperation.
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