Page 7 - BM 2011 Vol.2 No.1

Bioscience Methods
BM 2011, Vol.2, No.1 http://bm.sophiapublisher.com
- 4 -
Table 2 Data of the standard curve for product
Concentration (μg/mL) Peak
area 79.9 159.8 239.7 319.6 399.4
1st 89 749.5 197 126.6 324 580.6 431 244.6 656 574.1
2nd 64 289.0 202 969.3 331 340.0 476 448.5 508 261.5
Average 77 019.3 200 048.0 327 960.3 453 846.6 582 417.8
Table 3 Compare of the SEC-HPLC results and BCA results
Batch BCA Method
(μg/mL)
SEC-HPLC
Method (μg/mL) RSD (%)
I010 519 539 2.67
H015 462 472 1.51
1.4 Detection of NGF product
A dose of Mouse Nerve Growth Factor (NGF) for
Injection - Nobex® (18 μg per dose) was dissolved in
0.2 mL PBS buffer forming the NGF product sample
that the calculated theoretical concentration is
90 μg/mL. The sample was also detected by SEC-
HPLC based on the chromatographic conditions
established above (three parallel experiments in
average) and got the result of 91.2 μg/mL. The two
results are nearly the same with the RSD of 0.93%,
that proving of the reliability of SEC-HPLC
quantitative method in detecting NGF product.
2 Discussion
The method of using HPLC to separate and determine
protein has been proposed from the 1970s. And in
recent years, its quantitative measurement is getting
more and more attention by researchers. However,
there is almost RP-HPLC method seen in the
literatures. Practically, in the production process of
NGF, the quality control method without degenerating
the protein and bring in impurities is needed. We focus
on the SEC-HPLC because of its moderate condition
and the potential of on-line detecting.
As a routine monitoring method which requires simple,
speedy and accuracy, SEC-HPLC also provide the
same environment as the stock solution, which will
prevent the difficulty of changing dissolvant and break
in the continuous production process. Compared to
BCA method that must formulate standard curve for
every detecting, HPLC method can achieve the
advantage of on-line monitoring, which means you
don’t need to break in the production process but
detecting the concentration while the solution flow is
processing. Meanwhile, in the product quality control,
once of the HPLC detection can separate human
serum albumin and NGF as well as directly calculated
the concentration from the peak area of NGF, showing
the convenient and speedy of this method. This work
has set up the technical standards of SEC-HPLC
quantitative determination for NGF with the actual
test results corresponding with China Pharmacopoeia.
It’s believed that the SEC-HPLC quantitative
determination method will become the dominant
quality control process in the future. It will assuredly
save cost and raise the accuracy in the production
process of NGF.
3 Materials and Methods
3.1 Materials
This work uses the NGF stock solution and products
(Mouse Nerve Growth Factor for Injection - Nobex®,
Xiamen Bioway Biotech Co. Ltd., China) as subjects.
The NGF stock solution is from the production batch
of I010, H015. And the injection specification is 18 μg
NGF per dose with human serum albumin (20%
Human Serum Albumin, Octapharma Pharmazeutika
Produktionsges m.b.H) 1%V/mL and mannitol 5%V/mL,
after lyophilized into dry powder formulations.
3.2 Standard
NGF standard was purified from NGF stock solution
by Sephacryl S-100 column in PBS buffer. The purity
of standard is 99.1% after corroborated by HPLC
(Figure 7) and MS (Figure 8), and the result is
corresponded with China Pharmacopoeia (2010).And
the concentration of NGF standard is 719 μg/mL.