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Rezk et al.: Journal of AOAC International Vol. 100, No.

4, 2017  1023

DRUG FORMULATIONS AND CLINICAL METHODS

Selective Determination of Human Growth Hormone


(Somatropin) in the Presence of Its Chemical Degradation
Products
Mamdouh R. Rezk
Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr El-Aini St, 11562 Cairo, Egypt
Marwa F. Mohamed1 and Faten Abdelaziz Fathalla
National Organization for Research and Control of Biologicals, 12654 Giza, Egypt
Mostafa A. Shehata
Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr El-Aini St, 11562 Cairo, Egypt

A rapid and sensitive HPLC method was developed in protein drugs (7, 8). rhGH consists of a single polypeptide
and validated for selective determination of the chain (191 amino acid residues) with two disulfide bridges with
human growth hormone (hGH) somatropin in the a mass of 22 kda (9; Figure 1). Like most large proteins, GH
presence of its deamidated and oxidized degradation undergoes various chemical and physical instability reactions
products. Reversed-phase chromatography (10). The predominant degradation pathways of hGH include
with an acetonitrile and ammonium bicarbonate deamidation, oxidation, and aggregation. Deamidated and
mobile phase in gradient elution mode was used. oxidized forms are known to be the primary degradation
A short run time of 15 min allowed rapid and products. Oxidation of methionine to methionine sulfoxide is a
cost-effective analysis, with an average retention common chemical reaction known to occur in proteins (10, 11).
time of 7.4 min for native hGH, 6.2 min for its Somatropin has a tendency to form aggregates, resulting mainly
deamidated form, and 4.3 and 6 min for its oxidized in a dimer but also in oligomers of higher molecular mass.
variants. The method was validated for selectivity, To date, the structures of the dimer and related substances of
linearity, and intra- and interday variations according higher molecular mass have not been clearly elucidated (12).
to International Conference on Harmonization However, different covalent and noncovalent aggregates have
guidelines. The proposed method was successfully been described for pituitary hGH.
applied for rapid evaluation of the quantity of Separation of somatropin from its chemical degradation
hGH during downstream processing, formulation, products has been described (7, 8, 10, 13–18); however, all
and storage. of these methods have a long retention time (RT) and are,
therefore, time-consuming and expensive. Currently, the
European Pharmacopoeia and the United States Pharmacopeia

I
n the last several decades, proteins have become an recommend the use of reversed-phase (RP)-HPLC for the
important class of therapeutic drugs in the pharmaceutical quantification of related proteins (19, 20). Both official methods
market based on recombinant DNA technology. Human have a very long run time, about 55 min, thereby also making
growth hormone (hGH) is secreted from the anterior pituitary these methods expensive (19, 20). Separation of somatropin
gland under hypothalamic control. It is responsible for a wide from its degradation products has also been achieved by using
range of growth-promoting effects in the body (1). Recombinant a polymeric poly(styrene-co-divinylbenzene) column (21);
hGH (rhGH) is used to treat growth failure in children and however, this method has a narrow linearity range and uses
metabolic dysfunction in adults with GH deficiency (2, 3). expensive materials. Thus, there is a need for a simple and
In the development of protein pharmaceuticals, regardless of reliable method for the selective determination of hGH in the
therapeutic potential, the challenge remains their susceptibility presence of its oxidized, deamidated degradation products
to physicochemical degradation (4). Protein drugs are sensitive and placebo components with a short analysis time. Attempts
to various environmental factors, such as temperature, light, were made in this study to develop a fast, sensitive, and
oxidizing agents, pH, freezing, shaking, and shear stress (5). stability-indicating LC method for the determination of hGH in
These factors influence proteins during the production process the presence of its variants during downstream purification in
of the active substance, as well as during the manufacture and the manufacturing process, in biopharmaceutical formulations,
storage of the drug product. Residual moisture and excipients in and under different storage conditions.
a formulation can also be included as factors that may impact the
chemical stability of proteins in the solid state (6). Development Experimental
of suitable methods of analysis that allow for the identification,
separation, and quantification of impurities is required for the Reagents and Materials
physicochemical characterization and QC of the attributes
(a)  rhGH standard.—1.95 mg/vial, obtained from NIBSC
Received June 20, 2016. Accepted by JB December 5, 2016.
(Hertfordshire, United Kingdom).
1
Corresponding author’s e-mail: marwafayez54@yahoo.com (b)  Somatropin lyophilized formulation.—1.6 mg/vial,
DOI: 10.5740/jaoacinct.16-0194 obtained from the local Egyptian market.

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1024  Rezk et al.: Journal of AOAC International Vol. 100, No. 4, 2017

Figure 1.  The primary structure of hGH. Arrows indicate the major degradation sites for deamidation and oxidation.

(c)  Somatropin liquid formulation.—3.33  mg/mL, obtained Instruments


from the local Egyptian market.
(d)  Acetonitrile.—HPLC grade, obtained from Thermo (a)  HPLC system.—Chromatographic separation was
Fisher Scientific (Waltham, MA). performed on an Agilent 1260 HPLC series system (Böblingen,
(e)  Ammonium hydrogen carbonates, EDTA, and hydrogen Germany) with a diode-array detector (DAD).
peroxide.—Obtained from Sigma-Aldrich (St. Louis, MO). (b)  Water purification system.—Milli-Q UF-Plus.
(f)  O-phosphoric acid.—85%, obtained from Merck (c)  pH meter.—HANNA (Wave Scientific, Egypt).
(Darmstadt, Germany).
(g)  Ultra-pure water.—Obtained using a Milli-Q UF-Plus Chromatographic Conditions
system (Millipore, Germany).
(h)  hGH stock solution.—1 mg/mL, prepared by dissolving The column temperature was maintained at 35°C, whereas
lyophilized standard containing 1.95 mg somatropin in the autosampler temperature was adjusted to 4°C. The mobile
1950 µL purified water. Solutions with lower concentrations phase was pumped at a flow rate of 1 mL/min in gradient mode
were prepared with the appropriate dilutions of stock solution. as shown in Table 1. UV detection was performed at 210 nm
The prepared solutions were stored at 4°C and used fresh with an injection volume of 10 µL. For the analysis of forced
within 24 h. degradation samples, the photo-DAD was used in scan mode
(i)  Lyophilized somatropin formulation.—1 mg/mL, obtained with a scan range of 190–400  nm. Peak homogeneity was
by reconstituting the somatropin lyophilized formulation expressed in peak purity and obtained directly from the spectral
(1.6 mg/ vial) in 1600 µL purified water. analysis report.
(j)  Liquid somatropin formulation.—1 mg/mL, obtained Preparation of degradation products.—Forced degradation
by diluting the liquid formulation (3.33 mg/mL) with purified of the somatropin reference standard and the drug product was
water. carried out to obtain the degradation products.
(k)  Solution A.—10% acetonitrile, containing 10.5  mM
ammonium bicarbonate buffer and 0.105  mM EDTA, pH 7; Table 1.  Gradient elution profile at a flow rate of 1 mL/min
then filtered through a 0.45 µm membrane and sonicated for
10 min prior to use. Phase
(l)  Solution B.—80% acetonitrile, containing 6  mM Time, min A, % B, %
ammonium bicarbonate buffer and 0.060  mM EDTA, pH 7;
0 50 50
then filtered through a 0.45 µm membrane and sonicated for
10 min prior to use. 10 40 60
(m)  Ammonium bicarbonate.—0.3 M solution, pH 9. 12 50 50
(n)  Analytical column.—YMC C18 (50 × 2.1 mm id; Yoko,
15 50 50
Japan) with a 300 Å pore and 5 µm particle size.

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Rezk et al.: Journal of AOAC International Vol. 100, No. 4, 2017  1025

(a)  Preparation of oxidized degradation product.—The Results and Discussion


reference standard was reconstituted using purified water to
yield a concentration of 1 mg/mL. Oxidative degradation was Assay of Somatropin in Biopharmaceutical Market
obtained by adding 20 µL hydrogen peroxide (30%, v/v) to each Formulations
milliliter of reference standard solution. The obtained solution
was used within 24 h. Somatropin was assayed in its biopharmaceutical formulations
(b)  Preparation of deamidated degradation product.—The
by the proposed method. Figure 2 shows the chromatograms
reference standard was reconstituted using 0.3  M ammonium
of pure somatropin, lyophilized and liquid formulations. The
bicarbonate (pH 9) to yield a concentration of 1 mg/mL. The
recoveries of hGH in its lyophilized and liquid formulations
resulting solution was incubated at 37°C for 24 h, then stored at
were found to be 101.02 ± 0.82 and 101.4 ± 0.21, respectively.
−20°C until analysis time.
The obtained results indicated the suitability of the method for
Method Validation the routine analysis of somatropin in different biopharmaceutical
formulations.
Serial concentrations of somatropin reference standard
solutions in the range of 5–1500 µg/mL were chromatographed Degradation of Somatropin
using the previously described chromatographic conditions.
Method validation was carried out according to International (a)  Chemical degradation.—(1)  Deamidation.—The hGH
Conference on Harmonization guidelines. molecule contains 9 asparagines and 13 glutamine residues.

Figure 2.  HPLC chromatograms of somatropin in its (A) pure form and (B) lyophilized and (C) liquid formulations.

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1026  Rezk et al.: Journal of AOAC International Vol. 100, No. 4, 2017

It has various reactive sites for deamidation. The major (b)  Physical degradation.—As with many other globular
deamidation site is at Asn-149 (10, 17). Deamidation depends proteins, hGH is susceptible to aggregation, resulting in the
on temperature and ionic strength (15). Deamidation is the formation of either covalent or noncovalent aggregated species.
result of storing lyophilisate at a high temperature or exposing These aggregates can be produced by temperature, freezing, or
it to pH 8 for several days (8, 16). Deamidation proceeds via a mechanical agitation (22, 23).
known cyclic imide intermediate, resulting in the formation of
Aspartic or isoaspartic acid upon hydrolysis. The formation of Method Optimization
cyclic imide depends on the neighboring C-terminal amino acid.
This explains the ease with which Asn-149 is deamidated, as Different stationary phases (C4, C8, and polymeric RP
the neighboring C-terminal amino acid is serine (14). In case of columns) and mobile phases (containing buffers such as
rhGH liquid formulation, the deamidated rhGH is predominant phosphate, borate, and tris-hydrochloric acid) with different pH
and overshadows Met-14 sulfoxide rhGH. values and organic modifiers (2-propanol and 1-propanol) were
(2)  Oxidation.—hGH contains three methionines at positions tested. A YMC C18 column, with a large pore size of 300 Å
14, 125, and 170. The kinetics of oxidation of the methionines and a particle size of 5 µm, performed better and had more
to form methionine sulfoxide were previously studied (18). The symmetrical peak shape compared with other columns.
most susceptible point of oxidation is methionine in position Separation of somatropin from its degradation products was
125 (9). Oxidation and deamidation sites are illustrated in achieved by applying the chromatographic conditions described
Figure 1. Sulfoxidation is the primary degradation pathway in in Experimental section. Figure 3 shows separation of deamidated
solid-state hGH. and oxidized somatropin from intact somatropin. A short run time

Figure 3.  HPLC chromatograms showing the separation of somatropin from its (A) desamidosomatropin and (B) oxidative degradation
products.

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Rezk et al.: Journal of AOAC International Vol. 100, No. 4, 2017  1027

of about 15 min was advantageous compared with a longer Table 3.  Intra- and interassay precision and accuracy
run time of about 55  min as presented in the official methods results of somatropin in its lyophilized formulation
and in Mohammadpanah et al. (24). An even longer run time Concn, mg/mL
of 140 min was obtained by another reported method (25). To Assay Na Addedb Found Recovery, %
reduce expenses and develop a reliable method, a short column,
Intra-assay 3 0.2 0.202 101.00
packing material, and composition of the mobile phase should be
3 0.4 0.410 102.50
considered. The developed method showed a wider range (i.e.,
5–1500 µg/mL) with good sensitivity compared with the method 3 0.6 0.610 101.66
developed by Nafissi Varcheh et al. (21), which had a range of   Mean ± SD 101.72 ± 0.75
50–1000 µg/mL. Their method used an expensive polymeric   RSD, % 0.74
column and a mobile phase with a very low pH value, which Interassay 6 0.2 0.204 102.00
could affect the stability of the somatropin samples. Instead, the
6 0.4 0.402 100.50
developed method used a YMC C18 column with a smaller particle
6 0.6 0.593 98.83
size and, hence, better resolution, in addition to a mobile phase
with pH 7.0, which would not affect the stability of the analyzed   Mean ± SD 100.44 ± 1.58
samples. The developed method showed complete baseline   RSD, % 1.57
separation between somatropin and its degradation products. a
  N = Number of replicates.
The proposed method could be used as a stability-indicating b
  Added to 0.4 mg/mL somatropin in the lyophilized formulation.
assay for the determination of somatropin in the presence of
its degradation products (i.e., deamidated and oxidized).
In addition, selective determination of somatropin in its (d)  Precision.—Inter- and intraday precision was tested and
biopharmaceutical formulations and in stability studies is also acceptable results obtained, as shown in Tables 3 and 4.
now feasible. (e)  Robustness.—The robustness of a method is the
It has been found that aggregation changes the immunogenic method’s ability to remain unaffected by small deliberate
properties of pituitary hGH (26) because antibodies can detect changes in parameters. To evaluate the robustness of this
differences between monomeric and dimeric somatropin (27). method, different experimental conditions were intentionally
Selective determination of somatropin in the presence of its altered and the chromatographic resolution between hGH and
aggregated form has been reported by Rezk et al. (28). its degradation products evaluated. The effect of flow rate was
evaluated at 1 ± 0.1 mL/min. The effect of column temperature
Method Validation on the resolution was studied at 35 ± 2°C. The robustness of
the method was assured by a small change in RT; however, the
(a)  Linearity.—Linearity was tested by injecting serial hGH peak retained its shape and resolution.
concentrations of hGH standard solutions in the replicate. (f)  System suitability parameters.—System suitability
The calibration graph was obtained by plotting the peak area parameters were tested by calculating the tailing factor,
against the drug concentration. The method was linear in the selectivity factor, theoretical plates, and resolution. The results
range of 5–1500 µg/mL somatropin. Regression parameters are are presented in Table 5.
presented in Table 2.
(b)  Specificity.—Selective determination of somatropin in the Indications of Stability
presence of other components or its degradation products was
tested. Peak purity was evaluated using a DAD. The peak purity
The proposed method was able to determine the intact drug
was found to be 999 for the drug substance peak at 210 nm. There
in the presence of up to 90% of its oxidative degradates and
was no interference from formulation excipients or degradation
products, as can be seen by the hGH peak in Figure 3.
(c)  Accuracy.—Known amounts of pure somatropin were
added to the drug product in the lyophilized and liquid formulations Table 4.  Intra- and interassay precision and accuracy
at three different levels. Spiked samples were then analyzed and results of somatropin in its liquid formulation
the percentage recoveries calculated (Tables 3 and 4). Concn, mg/mL
Assay Na Addedb Found Recovery, %
Table 2.  Regression parameters for the determination of Intra-assay 3 0.2 0.204 102.00
somatropin by the proposed method 3 0.4 0.404 101.00
Parameters Somatropin 3 0.6 0.620 103.33
Regression   Mean ± SD 102.11 ± 1.16
 Slope 0.3096   RSD, % 1.14

 Intercept 11.192 Interassay 6 0.2 0.203 101.50

  Correlation coefficient 0.9995 6 0.4 0.405 101.25


6 0.6 0.609 101.50
Range, µg/mL 5–1500
  Mean ± SD 101.41 ± 0.144
Average RT, min 7.4 ± 0.24
  RSD, % 0.142
LOD, µg/mL 2 a
  N = Number of replicates.
LOQ, µg/mL 5 b
  Added to 0.4 mg/mL somatropin in the liquid formulation.

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1028  Rezk et al.: Journal of AOAC International Vol. 100, No. 4, 2017

Table 5.  System suitability parameters for the determination (7) Karlsson, G.r., Gellerfors, P.r., Persson, A., Norén, B., &
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Stabilizers, Parenteral Drug Association, USA
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