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Bioscience Methods
BM 2011, Vo.2, No.1 http://bm.sophiapublisher.com
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Research Article Open Access
Quantitative Determination for Nerve Growth Factor Concentration in Commercial
Product by SEC‐HPLC
Da Huang1,2 , Jianrong Zhang1 , Yongchao Fu1 , Liming Lu 2 , Yiming Xu1,2 , Lingyan Ma1 , Minxiang Li1
Lingyuan Xiong1 , Hongwei Ren 1,2
1. Xiamen Bioway Biotech Co. Ltd., Xiamen, 361009, P.R.China
2. Biochemistry & Molecular Biology College of Life Sciences, Peking University, Beijing, 100871, P.R. China
Corresponding author email: renhw@pku.edu.cn; Authors
Bioscience Methods 2011, Vol.2 No.1 DOI:10.5376/bm.2011.02.0001
Received: 23 Nov., 2010
Accepted: 12 Apr., 2011
Published: 18 Apr., 2011
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article as:
Huang et al., 2011, Quantitative Determination for Nerve Growth Factor Concentration in Commercial Product by SEC-HPLC, Bioscience Methods,
doi:10.5376/bm.2011.02.0001
Abstract The commercial product, Mouse Nerve Growth Factor (NGF) for Injection－Nobex®, is consisted of NGF and human
albumin. These two components are both proteins and make it difficult to determine the concentration of NGF by conventional
methods. In order to establish a new efficiency quality control process for NGF, this work choose Size Exclusion High Performance
Liquid Chromatography (SEC-HPLC) to detect stock solutions and products under screened chromatographic conditions: using
Shodex KW- 802.5 column, at room temperature with the 0.05 M PBS pH=7 buffer as mobile phase, 0.8 mL/min flow rate and
detected under 280 nm wavelength. The standard curves with correlation coefficient of 0.999 82 (for stock solutions) and 0.999 97
(for products) were obtained by external standard method. The NGF concentrations tested in HPLC were compared with which in
BCA for the same batches, and the RSD values were less than 3%, which corresponded with China Pharmacopoeia (2010). The
results show that SEC-HPLC method is sensitive, accurate and reliable for NGF on-line quality control and has the potential for the
quantitative determination of other mixed protein preparations.
Keywords Size Exclusion High Performance Liquid Chromatography (SEC-HPLC); NGF; Quantitative Determination
Background
Nerve Growth Factor (NGF), which has the properties
of regulating the neuronal differentiation and
development, repairing the neuronal damage and
regenerating the neurocyte, admittedly, is one of the
significant nutritional activity factors for the nervous
system [Levi-Montacini R., 1987]. This protein factor
consists of 3 subunit compositions (subunit of α, β,
and γ), forming a α2βγ2 structure with the β subunit as
the active group. The β subunit, which has the
molecular weight of 26 kD and isoelectric point of 9.3,
is a homodimer connected of two 118 amino acids
chains by non-covalent bond. The monomer of β subunit
is approximately 13.6 kD, and different monomers
may have 8 amino acids dissimilarity from the post-
transcriptional modification (William et al., 1976).
At present, the nerve growth factor is clinically using
for the treatment of nervous system damage as the
national first-class new drug (accorded with SFDA,
China). It uses human serum albumin and mannitol as
excipient by adding to the NGF stock solution in the
production process and manufactures three dry pow-
der injection specifications of 18 μg per dose, 20 μg
per dose and 30 μg per dose after lyophilization. To
the practical production process, the most important
quality control aspect lies in monitoring the
concentration of NGF in the stock solution and
products. Current protein concentration detecting
methods, such as Lowry method, Kjeldahl nitrogen
determination, BCA method and ELISA method, have
various difficult in monitoring NGF, especially that
they cannot distinguish the protein excipient of human
serum albumin from the target protein of NGF in a
quantitative process (in products, the concentration of
human serum albumin is much higher than that of
NGF). In order to settle these aporias, the method of
Size Exclusion High Performance Liquid Chromatography