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N-Terminal Site-Specific Mono-PEGylation of Recombinant
Human Granulocyte Colony-Stimulating Factor
Yongjun Nie1, Xiaojun Tan2, Wenli Wang2, Xinchang Wang1, Junhui Chen1*
1
State Key Laboratory of Pharmaceutical Biotechnology, School of Lifesciences, Nanjing University,
China; 2Department of Biochemistry, School of Lifesciences, Nanjing University, China, Nanjing,
China, 210093
Abstract:
Granulocyte colony-stimulating factor (G-CSF) serves as one of the most important remedies in
situations of reduced numbers of circulating neutrophilic granulocytes after high-dose chemotherapy
or radiotherapy in clinical. Additionally, G-CSF has also been used to mobilize peripheral blood
progenitor cells for autologous stem cell transplantation. Despite of all its therapeutic values for
human beings, the usage of G-CSF has a primary limitation, its short half-life in the peripheral blood.
In order to solve this problem, we PEGylated the N-terminus of recombinant human granulocyte
colony-stimulating factor (rhG-CSF), in a site-specific manner with methoxy poly-ethylene glycol
(mPEG) propionaldehydes with an average molecular weight of either 5 kD or 20 kD, respectively,
through a reactive terminal aldehyde group. With a statistical L9 (34) orthogonal test, the optimal
conditions of the site-specific reaction were achieved, and under such conditions the
mono-PEGylated rhG-CSF (mono-PEG-G-CSF) accounts for 72.89% of the total protein in the
reaction system. We demonstrated that PEG-rhG-CSF had higher stability than unmodified rhG-CSF
while incubated with protease or human serum or placed in an extreme environment such as at an
extreme temperature or in a solution with extreme pH. Although the relative activity of modified
rhG-CSF in stimulating the proliferation of NFS-60 cells in vitro was down-regulated, due to the
blocking effects of the molecule of PEG, yet modified rhG-CSF increased the numbers of
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leukocytes in the blood of Kunming mice much more significantly than did unmodified rhG-CSF. In
conclusion, these results show that PEGylation not only enhances the stability of rhG-CSF, but also
distinctly increases its in vivo activity and thus this PEGylation promotes the clinical application of
rhG-CSF.
Key Words:
rhG-CSF; PEGylation; PEG; stability; orthogonal test
Author contributions: J. C., X. W., Y. N. designed research, Y. N., X. T. and W. W. performed
research, Y. N., X. T. analyzed data and X. T. wrote the paper.
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重组人粒细胞集落刺激因子的N端氨基定点PEG化修饰
聂永军1，谭小军2，王文丽2，王新昌1，陈钧辉1*
1
南京大学生命科学学院医药生物技术国家重点实验室；
2
南京大学生命科学学院生物化学系；
中国南京 210093
摘要：
粒细胞集落刺激因子（G-CSF）是现今临床上抗肿瘤化疗、放疗手术之后最重要的辅助药
物之一，它的主要功能在于刺激嗜中性粒细胞增殖以缓解由于化疗、放疗造成的免疫系统伤
害。此外，G-CSF在自体干细胞移植中也用于动员外周造血干细胞。尽管G-CSF具有重大的治
疗价值，然而在实际应用却受到体内半衰期过短、需要频繁重复注射的限制。为了解决这一
问题，我们利用两种不同分子量（5 kD 和 20 kD）的单甲氧基聚乙二醇丙醛（mPEG-PAL）
对重组人粒细胞集落刺激因子（rhG-CSF）的N端氨基进行了定点PEG化修饰。通过正交实验
得到最佳修饰条件，此时rhG-CSF的单修饰产率达到72.89%。我们发现，PEG化的rhG-CSF比
原先的rhG-CSF具有更高的体外稳定性；虽然PEG化使其体外活性受到一定限制，但是修饰后
rhG-CSF的体内活性得到了很大的提高，体内作用时间得到很大延长。因此，对于rhG-CSF的
N端氨基定点PEG化修饰可以显著提到rhG-CSF的临床应用价值。
关键词：
重组人粒细胞集落刺激因子（rhG-CSF）；聚乙二醇（PEG）； 聚乙二醇化；稳定性；正
交实验
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Introduction
Since the purification of granulocyte colony-stimulating factor (G-CSF) from human in 1979 [1],
researches on G-CSF have revealed it as the principal cytokine regulating neutrophil development
and function [2]. Because of its proliferation activity on granulocytic progenitors and its
mobilization activity on hematopoietic progenitor cells [3, 4], leading to increased numbers of
circulating neutrophilic granulocytes, recombinant human G-CSF (rhG-CSF) has become one of the
most important therapeutic agents for situations of neutropenia in cancer patients receiving
high-dose chemotherapy [5, 6] or in patients suffering from severe congenital disorders [7, 8]. In
addition, G-CSF has been shown to have distinct functions on resisting infections like foot
infections in diabetics [9], depending on the type of infection and its route delivery [1]. Despite of
such clinical value for human beings, the outpatient use of G-CSF is greatly limited by the short
half-life of G-CSF in vivo and administration of G-CSF 1 or 2 times per day causes many allergic
responses. Thus, a molecular with a longer half-time in vivo is required to lessen the number of
administrations.
One important method to handle the problem of the short circulating half-time of proteins or
peptides in vivo is to conjugate them with poly-ethylene glycol (PEG), a widely investigated
polymer used for covalent modification of biological macromolecules, especially for proteins and
peptides [10]. PEG is characterized by little toxicity [11] and no immunogenicity and antibodies to
PEG are generated in rabbits only if PEG is combined with highly immunogenic proteins [12].
PEGylation improves pharmacokinetics by increasing the molecular mass of proteins and peptides
and shielding them from proteolytic enzymes [13]. Early reports described the PEG conjugates of a
wide range of proteins, including human growth hormone, interferon, insulin, granulocyte-colony
stimulating factor (G-CSF), and interleukin II [14], but most of these proteins were not
site-specifically PEGylated. Only recently, PEGylation of proteins in a site-specific manner has
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been achieved by virtue of a special class of PEG derivatives under specific reaction conditions
[15~17]. Site-specific PEGylation of a certain protein provides us with opportunities to obtain
mono-PEGylated product, thus ensures the purity of the chemical component of the product, which
is rather crucial in pharmacy.
In this study, we PEGylated the N-terminus of rhG-CSF in a site-specific manner with two groups
of methoxy poly-ethylene glycol propionaldehydes (mPEG-PAL) with an average molecular weight
of 5 kD and 20 kD, respectively, through a reactive terminal aldehyde group. The optimal conditions
for the N-terminal mono-PEGylation were identified through a statistical L9 (34) orthogonal test and
the product were shown to be pure and unitarily modified. We demonstrated that PEGylation not
only enhances the stability of rhG-CSF, but also distinctly increases its in vivo activity and thus has
promoted its clinical applications.
Materials and methods
Materials
Recombinant human G-CSF, the growth-factor-dependent NSF-60 mouse cell line and the
Kunming mice were donated by ZhongKai Bio-pharma (Suzhou, Jiangsu, China). The 5 kD
mPEG- propionaldehyde was purchased from SunBio (Anyang city, Seoul, Korea); 20 kD
mPEG-propionaldehyde was purchased form Beijing KaiZheng Biotech (Beijing, China). Trypsin,
dimethyl sulfoxide (DMSO) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) were obtained from Amresco Inc. (Solon, OH, USA). RPMI 1640 medium and bovine calf
serum were obtained from Hyclone (Logan, UT, USA).
PEGylation of rhG-CSF with PEG-Propionaldehyde Derivatives
The optimal conditions of the site-specific reaction were identified with a statistical L9 (34)
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orthogonal test. The four major factors that determine the site-specific reaction are molar ratio of
mPEG propionaldehyde to rhG-CSF, pH, temperature and time, and Table 1 shows the three levels
of each factor. Reactions were terminated the reactions by adjusting pH to 3.5 with 1 M HCl, and
analyzed the samples with SDS-PAGE and Gelworks 1D intermediate software system.
Separation and Purification by ÄCTA FPLC
The reaction mixture was loaded onto a CM Sepharose FF cation exchange column (1.0 cm × 20
cm) preequilibrated with 20 mM sodium acetate, pH 4.5 (buffer A), at a flow rate of 1.0 mL/min
using a Pharmacia LCC 501 Plus FPLC system. The column was washed with 300 mL of buffer A
before a ladder gradient to 20%, 40%, and 60% buffer B (buffer A + 1 M NaCl) was applied about
5 column volumes per gradient. The fractions were analyzed by non-reducing SDS-PAGE and
Gelworks 1D intermediate software system. The samples containing mono-PEGylated and
unmodified rhG-CSF were collected sequentially. Samples were stored at -20 ℃ after dialysis and
lyophilization.
In vitro bioassay
The in vitro activity of the PEG conjugates was determined using the MTT cell-proliferation
assay with the G-CSF-dependent NFS-60 cell line [18]. The assay was based on protocols from the
American Type Culture Collection and Bowen et al [19]. The NFS-60 cells were maintained in
RPMI 1640 medium with 15% (v/v) bovine calf serum. Cells were split 3–4 days before each assay.
Seven 2-fold dilutions of the G-CSF or conjugates were prepared in RPMI 1640 supplemented with
bovine calf serum. Duplicate aliquots of 100 μL were prepared in 96-well plates. The NFS-60 cells
were washed three times in assay medium, and the cell concentration was adjusted to 2 × 105
cells/mL in assay medium. They were plated in a volume of 50 μL/well, resulting in 1 × 104
cells/well, giving a final volume of 150 μL/well. Cultures were incubated for 48 hours at 37 ℃ in
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a fully humidified atmosphere of 5% CO2 in air, then pulsed with 20 μL of 5 mg/mL MTT reagent
for the next 5 hours of incubation. When the purple precipitates were clearly visible under the
microscope, 100 μL of detergent (20 mM HCl, 10% (v/v) TritionX-100 in isopropyl alcohol) was
added and the absorbance in each well was measured at 570 nm in the microtiter plate reader. The
absorbance was plotted against the dilute multiple in the logarithm coordinate system. PEG-G-CSF
potency was calculated using the formula below (equation 1):
p s  pr 
Ds  Es
Dr  Er
(Eq. 1)
where Ps = potency of the sample (IU/mL), Ds = dilute multiple of the sample, Pr = potency of the
reference (IU/mL), Dr = dilute multiple of the reference, Es = dilution factor of 50% effective
activity of sample, and Er = dilution factor of 50% effective activity of reference.
Thermal and pH Stability of mPEG-G-CSF
PEG-G-CSF and G-CSF at concentrations of 20 μg protein/mL in phosphate buffered saline
(PBS) pH 7.6 were incubated at 4 and 60 ℃ for indicated periods. All the samples were filtered
through 0.22 μm filters and stored at –70 ℃. The activity of the samples was estimated in the
MTT cell proliferation assay.
The pH stability of the PEG-G-CSF was assessed by incubation at 37 ℃ at pH 2.2 and 11.0 for
periods of 1, 2, 4, 8, 16, and 32 hours. All the samples were filtered through 0.22 μm filters and
stored at –70 ℃. The activity of the samples was estimated using the MTT cell proliferation assay.
Stability in Trypsin and Human Serum
In the trypsin stability assay, 20 μg protein/mL of PEG-G-CSF or G-CSF were incubated at
37 ℃ with trypsin at a protein/trypsin ratio of 100: 1. Aliquots (100 μL) were taken at 0, 5, 10, 20,
40, 80 and 160 minutes.
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Human serum was prepared using the following procedure. Fresh human blood was incubated at
4 ℃ for 30 minutes, and then centrifuged at 3000 rpm for 15 minutes. The supernatant fluid was
stored in aliquots at –70 ℃ until the assay was performed. PEG-G-CSF and G-CSF at 200 μg/mL
in PBS, pH 7.6, were diluted to 20 μg protein/mL with human serum. The mixtures were incubated
at 37; aliquots (100 μL) were taken at 0, 1, 2, 4, 8, 16, and 32 hours.
All the 100 μL aliquots were diluted with 900 μL RPMI 1640 (without bovine serum) to quench
the reaction. The relative activity was analyzed in the MTT cell proliferation assay.
In vivo bioassay
The activity of the mono-PEG-G-CSF to increase leukocyte numbers was examined using
Kunming mice. Fifteen mice, 6–10 weeks old, were used for this study and were housed in
air-conditioned rooms with a 12-hour light and dark cycle. The G-CSF and PEG-G-CSF were
diluted to 25 μg protein/mL with normal saline. The in vivo bioactivity of the PEG-G-CSF
conjugates was assessed by administering a single intravenous dose of 5 μg/animal (n = 5). A
parallel group of mice (n = 5) received G-CSF as controls. Blood samples were collected at 0
(predose), 8, 24, 48, and 72 hours post-dosing for each group of mice. The granulopoietic activity
was determined by counting the number of white blood cells (WBCs).
Results
Mono-PEGylation of rhG-CSF under the optimal conditions identified through
the orthogonal tests
PEGylation of rhG-CSF is an organic reaction, which could be influenced by a host of factors.
The numerous combinations of different factors with different levels put the identification of the
optimal reaction conditions into a rather knotty procedure. To handle this problem, we employed our
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time into the statistical L9(34) orthogonal tests, the results of which was analyzed by SDS-PAGE.
The mono-PEGylation of rhG-CSF was better in Lane 5 and 7 (Figure 1A) and the ratios of
mono-PEGylated rhG-CSF were 52.67% and 66.52% in Lane 5 and 7 (Table 2), respectively. High
ratios of PEG to rhG-CSF brought about higher ratios of PEGylated rhG-CSF (Lane 3, 5, and 7 in
Figure 1A, less rhG-CSF left), but it also caused more multi-PEGylation of rhG-CSF (Lane 3 and 5
in Figure 1A, not very evident in Lane 7). The orthogonal analysis of the tests’ results indicated that
two factors, time and the molar ratio of mPEG to rhG-CSF, played the most important roles during
the reaction and the other two factors, pH and temperature, were much less pivotal in the process
(Table 2).
In Table 2, the maximal Kx of each factor also provides us with the best level of the factor, from
which we worked out the optimal conditions for the reaction: 48 hours (longer than 24 hours, so as
to let more PEGylation reactions happen); pH5.0; molar ratio of mPEG to rhG-CSF 10:1 (not larger
than 10:1 in order to prevent too much multi-PEGylation); 20 ℃. Under the optimal conditions, the
ratio of mono-PEGylated rhG-CSF turned out to be 72.89%, higher than that in Lane 5 and 7 in the
above orthogonal tests (Figure 1C), which demonstrated the feasibility of orthogonal tests dealing
with some experiments characterized by lots of combinations of different factors with different
levels.
Isolation and Purification of Mono-PEG-G-CSF
The desired products, the two kinds of mono-PEG-G-CSFs modified with mPEG-PAL of an
average molecular weight of 5 kD and 20 kD respectively, were clearly separated from unmodified
rhG-CSF and multi-PEGylated rhG-CSF by cation exchange fast protein liquid chromatogram
(Figure 2A and 2B). The purity of the two mono-PEG-G-CSFs after dialysis and lyophilization was
examined by cation exchange fast protein liquid chromatogram (2C and 2D) and further examined
by SDS-PAGE analysis (Figure 2E) and one single band was shown for each of the two products.
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The results proved that the products obtained were pure and the PEG-residue count was 1 for each
rhG-CSF molecular, suggesting that the product was unitarily PEGylated.
PEGylation Retains the Activity of rhG-CSF in vitro
The relative activities of mono-PEGylated rhG-CSFs in vitro were examined so as to judge
whether PEGylation removed the activity of rhG-CSF. Absolute activities of rhG-CSF and
PEGylated rhG-CSF were first detected through cell proliferation assay by MTT and the results
were transferred into relative activities, as shown in Figure 3. The relative activities of
PEG(5kD)-G-CSF and PEG(20kD)-G-CSF was down-regulated to about 67% and 40%, respectively,
of that of unmodified rhG-CSF. The inhibition effect of the PEG molecules on the contacts between
rhG-CSF and NFS-60 cells can be responsible for the down-regulation of the activity of modified
rhG-CSF, since the PEG molecules are on the surface of rhG-CSF. This suggested that PEGylation
did not destroy the biological activity of rhG-CSF.
PEGylation enhances the stability of rhG-CSF in vitro
According to Figure 4, both modified and unmodified rhG-CSFs show relatively low stability in
high temperature or high pH or in trypsin and relatively higher stability in low pH or human serum
within a short time scale (less than 32 h), and all of them remain good activity (~96% activity
remains after 64 h) in neutral environments in 37 ℃. Except the experiment in 37 ℃, all other
stability assays in vitro indicated that PEG(20 kD)-G-CSF was more resistant than PEG(5
kD)-G-CSF and much more resistant than unmodified rhG-CSF to different environmental
conditions.
Modified and unmodified rhG-CSFs were all stable in 37 ℃ within 64 h (A), but all of them
decomposed quickly in 60 ℃, with PEGylated rhG-CSFs in lower speeds than unmodified rhG-CSF
(B). After incubation in 60 ℃ for 8 h, 10% of G-CSF remained, yet 40% of PEG(20 kD)-G-CSF
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and 30% of PEG(5 kD)-G-CSF was left(Figure 4B). Hence, PEGylation increased the thermal
stability of rhG-CSF.
Recombinant human G-CSF was more or less of the same stability with PEGylated rhG-CSFs in
pH 2.2 (Figure 4C) and 7.0 (Figure 4A), but in pH 11.0 (Figure 4D) the half-life of rhG-CSF was
only about 6 h while those of PEG(5 kD)-G-CSF and PEG(20 kD)-G-CSF were elongated to 12 h
and more than 52 h, respectively. This indicated that PEGylated rhG-CSF had distinctly higher
acid-base stability than unmodified rhG-CSF.
After 32 h of incubation with human serum, the denaturalization ratio of rhG-CSF and PEG(5
kD)-G-CSF reached 20% and 15%, respectively, while that of PEG(20 kD)-G-CSF was only 7%
(Figure 4E). When incubated with trypsin for 10 h, over 75% of rhG-CSF in contrast to only 10% of
PEG(5 kD)-G-CSF and 4% of PEG(20 kD)-G-CSF was degraded (Figure 4F). Therefore,
PEGylation also significantly enhanced proteolytic stability of rhG-CSF.
Mono-PEG-G-CSF stimulates the proliferation of the leukocytes in the blood of mice
more significantly than unmodified rhG-CSF
To investigate whether mono-PEGylated rhG-CSF has higher activity than unmodified rhG-CSF
in vivo, the effects on leukocyte proliferation in the peripheral blood of Kunming mice after a single
injection of 5 μg for each of mono-PEG-G-CSF or rhG-CSF were tested. As shown in Figure 5, the
number of leukocytes in mice treated with rhG-CSF reached its highest point about 8 hours after the
injection, while in mice treated with mono-PEG(5kD)-G-CSF or mono-PEG(20kD)-G-CSF, the
time was prolonged to nearly 24 and 48 hours, respectively. Moreover, the peak point of leukocytes
in mice treated with each mono-PEGylated rhG-CSF went distinctly higher than that in mice treated
with unmodified rhG-CSF. These results adequately demonstrated that mono-PEG-G-CSF
stimulates the proliferation of the leukocytes in the blood of mice more significantly than
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unmodified rhG-CSF, suggesting that increased activity and prolonged half time of rhG-CSF in vivo
were achieved by PEGylation.
Discussion
PEGylation of proteins and peptides promotes pharmacokinetics by increasing the apparent sizes
of them, shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake by
the reticuloendothelial system, and preventing recognition and degradation by proteolytic enzymes
[10]. One of the most important progress of PEGylation is the introducing of sorts of PEG
functional groups used to attach the PEG molecule to the protein or peptide, which allows
site-directed PEGylation to be achieved under specific conditions. For instance, under acidic
conditions, the aldehyde of mPEG-PAL is highly selective for the N-terminal α-amine of G-CSF
because of the different pKa values of the primary amine residues in protein: pKa 7.8 for the
N-terminal α-amine compared to pKa 10.1 for the ε-amino group in lysine residues [15, 20].
The optimal conditions for the N-terminal site-specific PEGylation of rhG-CSF in this work were
obtained through a statistical orthogonal test. In Table 2, the maximal Kx of each factor tells us the
best level of the factor: Level 3 for time and molar ratio of PEG to rhG-CSF and Level 2 for pH and
temperature (i.e. 24 hours, pH 5.0, molar ratio of PEG to rhG-CSF 10:1 and 20℃), but the optimal
conditions we identified were not the same as what the maximal Kx suggested. As either the reaction
time or the molar ratio of mPEG to rhG-CSF rises from Level 1 to Level 3, the Kx of each factor
increased in a positive correlation. We prolonged the optimal reaction time up to 48 hours, doubled
the time of Level 3 (24 hours), looking forward to more PEGylation reactions, while confining the
best molar ratio of PEG to rhG-CSF to 10:1, so as to prevent too much multi-PEGylation of
rhG-CSF resulted from high ratios of PEG to rhG-CSF (Lane 3 and 5 in Figure 1, not so evident in
Lane 7).
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The apparent molecular weights of the mono- PEG(5 kD)-G-CSF and mono- PEG(20 kD)-G-CSF
we obtained were about 26 kD and 44 kD, respectively, according to data analysis of the results of
SDS-PAGE shown in Figure 4. However, the theoretical molecular weights of them, that is the sum
of the molecular weights of PEG (5 kD or 20 kD) and rhG-CSF (~19.6 kD) are 24.6 kD and 39.6 kD,
respectively. The reason why the apparent molecular weights of our products surpassed the
theoretical values was perhaps that the highly hydration of PEG leading to the PEGylated proteins
showing larger apparent molecular weights than unmodified proteins [21]. A highly hydrated crust
formed on the surface of the protein molecule decreased the mobility of the protein during
electrophoresis. Thus, PEGylated proteins as standards are required for the determination of the
molecular weight of PEG conjugates of proteins through SDS-PAGE analysis [22].
In vitro bioactivity assay showed that PEGylation decreased the activity of rhG-CSF (Figure 3)
and similar results were obtained by other studies [23, 24]. This in vitro activity decrease was
reasonable because the PEG molecule on the surface of rhG-CSF protein may to some extent
prevent the interaction between rhG-CSF and its receptors on the membrane of NFS-60 cells and as
a result affected the in vitro activity of rhG-CSF. Despite of the restriction of activity in vitro,
PEGylation significantly enhanced the in vivo activity of rhG-CSF (Figure 5). Internal environment
is largely different from that in vitro, with too many factors influencing the bioactivity of rhG-CSF.
The distinct increase of in vitro stability of rhG-CSF by PEGylation provides the possible
explanation of the higher activity of PEGylated rhG-CSF in stimulating the proliferation of the
leukocytes in the blood of mice. PEGylation protects rhG-CSF from being cleared up too quickly by
the body and thus PEGylation functions as a method to slowly release the bioactivity of rhG-CSF,
which overcomes the shortage of rhG-CSF in clinical use.
In summary, we PEGylated the N-terminus of rhG-CSF in a site-specific manner with
mPEG-PAL through a reactive terminal aldehyde group. The purity of the products are
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demonstrated by ÄCTA FPLC and SDS-PAGE analysis, further proving that rhG-CSF was unitarily
PEGylated and the PEG residue count for each molecule was 1, which is quite important for future
development and production of PEG-rhG-CSF as a clinical agent. The stability and activity of
rhG-CSF in vitro after PEGylation are enhanced to a certain extent. Moreover, promoted activity of
rhG-CSF on leukocyte proliferation in the peripheral blood of Kunming mice and significantly
prolonged circulating half-time of rhG-CSF in vivo are achieved by PEGylation.
Acknowledgements
The authors thank Suzhou ZhongKai Bio-Pharma. Co., Ltd. for the generous donation of
recombinant human granulocyte colony-stimulating factor. This study was supported by the
Research Fund for the 2006’ National Undergraduate Innovation Program.
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Figure 1. Mono-PEGylated G-CSF was obtained through orthogonal tests and SDS- PAGE. (A) SDS-PAGE
analysis of the orthogonal tests’ samples stained with Coomassie Brilliant Blue. Lane 1-9: nine samples in the
orthogonal tests corresponding to the test number. More mono-PEGylated rhG-CSF was achieved under the
optimal conditions (48 h, pH 5.0, molar ratio of mPEG to rhG-CSF 10: 1; 20 ℃) than under the conditions of Test
5 or 7 in the orthogonal tests: (B) SDS-PAGE analysis of the reaction sample under the optimal conditions stained
with Coomassie Blue; (C) The ratio of the mono-PEGylated rhG-CSF in the reaction sample under the optimal
conditions surpasses that in Lane 5 and 7 in (A).
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Table 1. Factors and Levels of the Orthogonal Tests
Level
1
2
3
Time
(h)
5
10
24
Factor
molar ratio
mPEG/rhG-CSF
1:2
2:1
10:1
pH
4.0
5.0
6.0
Temperature
(℃)
4
20
30
Table 2. Results of Orthogonal Testsa
test no.
1
2
3
4
5
6
7
8
9
K1c
K2
K3
K1/3d
K2/3
K3/3
Re
time
(h)
1
1
1
2
2
2
3
3
3
36.11
84.06
134.17
12.04
28.02
44.72
32.68
pH
1
2
3
1
2
3
1
2
3
82.67
96.57
75.1
27.56
32.19
25.03
7.16
molar ratio
mPEG/rhG-CSF
1
2
3
2
3
1
3
1
2
51.44
55.93
146.97
17.15
18.64
48.99
31.84
a
temperature
(℃)
1
2
3
3
1
2
2
3
1
84.75
90.09
79.5
28.25
30.03
26.5
3.53
RMPG b
(%)
0.21
8.12
27.78
15.94
52.67
15.45
66.52
35.78
31.87
The relative importance of the roles that the four factors played in the reaction was revealed by the results of the
orthogonal tests. b RMPG: the ratios of mono-PEGylated rhG-CSF which was calculated from the gel by Gelworks
1D intermediate software system. c The value of Kx (x=1, 2 or 3) was the sum of RMPGs of a certain factor at the
same level of x; the maximal Kx of a certain factor implied that x was the best level of the factor. d Kx/3 equals one
third of Kx. e The difference between the maximal Kx/3 of a certain factor and the minimal one of it gave the R
value, the magnitude of which was in accordance with how important the factor’s role in the reaction. Therefore,
the most important two factors were time and the molar ratio of mPEG to rhG-CSF, while the other two factors,
pH and temperature, were relatively not as crucial.
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Figure 2. Isolated and purified of mono-PEG-G-CSF. (A and B) The profile of cation exchange fast protein
liquid chromatogram (ÄCTA FPLC) of the PEGylated rhG-CSF: the average molecular weights of mPEG-PAL
used are (A) 5 kD and (B) 20 kD, respectively. (C and D) The profile of ÄCTA FPLC of the purified
mono-PEG-G-CSF: the average molecular weights of mPEG-PAL used are (C) 5 kD and (D) 20 kD, respectively.
(E) SDS-PAGE analysis of the purified mono-PEG- G-CSF stained with Coomassie Brilliant Blue.
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Figure 3. Relative activities of modified and unmodified rhG-CSFs. Absolute activities were tested by NFS-60
cells proliferation assay and the results were translated into relative activities of modified and unmodified rhG-CSF.
Values are means ± SEM from three independent experiments.
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Figure 4. Mono-PEG-G-CSF showed distinctly higher stability than unmodified rhG-CSF in different
conditions in vitro. (A and B) The effect of PEGylation on the relative susceptibility of rhG-CSF to temperature.
Modified and unmodified rhG-CSFs solutions were exposed at pH 7.0 in 37 ℃ for 64 h (A) or in 60 ℃ or 16 h (B)
and the relative activities remaining was detected at different time points. (C and D) The effect of PEGylation on
the relative susceptibility of rhG-CSF to pH. Solutions of rhG-CSF and PEGylated rhG-CSFs were regulated to
pH 2.2 (C) and pH 11.0 (D) and incubated in room temperature for 32 h and the relative activities remaining was
detected at 1 h, 2 h, 4 h, 8 h, 16 h and 32 h, respectively. (E) The relative stability of PEG(5 kD)-G-CSF, PEG(20
kD)-G-CSF and rhG-CSF in human serum. (F) The relative resistance of PEG(5 kD)-G-CSF, PEG(20 kD)-G-CSF
and rhG-CSF to trypsin proteolysis. Values are means ± SEM from three independent experiments.
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Number of leukocytes (μL-1)
药苑论坛交流论文
30000
G-CSF
PEG(5 kD)-G-CSF
PEG(20 kD)-G-CSF
25000
20000
15000
10000
5000
0
0
20
40
60
80
100
Time (h)
Figure 5. The activity of mono-PEG-G-CSF to increase leukocyte numbers in the peripheral blood of
Kunming mice is significantly higher than that of unmodified rhG-CSF. 15 mice were randomly separated into
three groups, each with 5 mice; each mice was injected with 5 μg rhG-CSF, PEG(5kD)-G-CSF or
PEG(20kD)-G-CSF, respectively; the numbers of leukocytes in the blood obtained from the caudal vein were
counted at 0 h, 8 h, 24 h, 48 h and 72 h, respectively. Values are means ± SEM from three independent
experiments.
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References
[1] Nicola NA, Metcalf D, Johnson GR, Burgess AW. Separation of functionally distinct human
granulocyte-macrophage colony-stimulating factors. Blood 1979;54:614–27.
[2] Panopoulos AD, Watowich SS, Granulocyte colony-stimulating factor: Molecular mechanisms
of action during steady state and ‘emergency’ hematopoiesis, Cytokine 2008;
doi:10.1016/j.cyto.2008.03.002
[3] Duhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D. Effects of recombinant
human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients.
Blood 1988;72:2074–81.
[4] Molineux G, Pojda Z, Hampson IN, Lord BI, Dexter TM. Transplantation potential of peripheral
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[5] Bronchud MH, Scarffe JH, Thatcher N, Crowther D, Souza LM, Alton NK, et al. Phase I/II
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[6] Morstyn G, Campbell L, Souza LM, Alton NK, Keech J, Green M, et al. Effect of granulocyte
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[7] Welte K, Zeidler C, Reiter A, Muller W, Odenwald E, Souza L, et al. Differential effects of
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参加大学生创新训练计划的心得体会
2006 年 12 月是个记忆犹新的月份，因为大学生创新训练计划开始实施，我们本科学生
将有机会更早地走进科研；这的确是个激动人心的消息。我们团队四人不约而同地走到了一
起。创新训练计划给了我们锻炼和提高创新能力、实践能力的机会。
因为专业基础，我们团队成员在生物化学方面有着深入的了解，并对生物技术制药方向
的研究有着浓厚的兴趣。在聂永军老师的引导下，我们认识了多肽与蛋白质药物研究专家
——陈钧辉教授；最终，陈教授成了我们的导师！
生物化学是一门基础学科，而我们着眼于运用基础知识来解决实际问题。我们选择了重
组人粒细胞集落刺激因子(rhG-CSF)，它是现今抗肿瘤化疗、放疗以及骨髓移植手术之后最
重要的辅助药物之一。但是，rhG-CSF 的临床应用存在体内半衰期短、需要多次注射的问题；
这给患者带来了很大的不便，而且重复注射也会引起不良反应。使用聚乙二醇（PEG）对蛋
白药物 G-CSF 进行修饰，可以使 G-CSF 的性质得到改善，以提高它的临床应用价值。
至此，我们项目的总任务，就是通过对现有重组人粒细胞集落刺激因子——rhG-CSF 的
修饰方法及 PEG 的应用进行概括与总结；根据已有文献提出我们自己的修饰策略，选用合适
的 PEG 对 G-CSF 进行定点单修饰，分离、纯化得到单修饰的纯品，提高 G-CSF 的体内外活性，
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药苑论坛交流论文
减少其免疫原性，延长其体内半衰期，从而克服它在临床应用中的不足。
创新训练计划项目的重点在于训练，我们项目的进展过程和多数项目一样，缓慢而艰难。
然而，我们坚持不懈，了解并体验了科学研究的整个过程，我们受到了严格的科研训练。我
们训练了实验技能、实验习惯、实验设计、实验总结、实验创新等等。我们项目的创新点，
在于我们选择了合适的修饰工具——单甲氧 PEG 丙醛，在于我们的实验设计灵活运用了统计
学方法，在于我们建立了微量反应体系！
经历了国家大学生创新训练计划，我们学会了查阅科学文献，尤其是外文文献。在查阅
文献的过程中，我们体会到阅读方法的重要性；高效的阅读使我们的工作事半功倍。
创新训练计划提高了我们的实验设计能力。这是一个突破，因为本科实验教学中所有实
验都是照书本按部就班去做的。但在这次训练中，我们亲自参与了实验设计，并尝试运用了
新的试验设计思路——统计学方法，我们取得了可喜的成功。
当然，锻炼得最多的还要属动手能力。熟练地使用各种实验仪器，准确而到位的技术操
作，都对我们的动手能力提出了更高的要求。
通过创新训练，我们深化了专业知识。
“纸上得来终觉浅，绝知此事要躬行”。生物是一
门实验学科。理论是基于实验的，通过实验我们才能对所学的知识进行重现和加深理解，并
探索新的知识。
创新训练计划培养了我们严谨、创新、实事求是的作风，这就是科学精神。它们体现在
整个科研过程的方方面面。
创新训练计划，增强了我们的团队合作意识。无论是在实验过程中，还是在项目申请和
文献查阅上，团队合作精神都得到了很好的锻炼；更是在这样的合作过程中，我们又一次深
深体会到“众人拾柴火焰高”的道理。
创新计划，让我们更加清晰地认识了，自己专业的研究热点和发展方向。我们深入了解
了生物化学、细胞与分子生物学的研究内容、研究方法、研究意义和专业的未来发展。这种
认识，对我们未来的选择，对我们人生的计划，都大有裨益。
我们相信，所有参与过、参与着或者即将参与大学生创新训练计划的同学，都会从训练过程
中学到很多，成长很多！
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