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, 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 powder injection specifications of 18μg per dose, 20μg per dose and 30μg 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 (SEC-HPLC) was chosen. This method can on-line detect the concentration of NGF in a much higher speed and accuracy, and the most important property is that the detection separation and purification are at the same time. It will largely reduce the cost of qualify control in the practical production process.

1 Results
1.1 Selection of chromatographic conditions
The PBS buffer was chosen as mobile phase for that it is also used as the dissolvent of stock solution. When the PBS at the condition of pH=7.0 and 0.05 mol/L ion concentration, it shows the largest NGF peak area respond and highest column efficiency in the chromatogram (Figure 1 and Figure2).
 

Figure 1 Chromatogram of NGF under different pH condition

 

Figure 2 Chromatogram of NGF under different ion concentration condition

As a result, the chromatographic conditions are finally selected as follows: Chromatograph: Shimadzu LC-10AT VP Plus HPLC Chromatography; Column: Shodex PROTEIN KW-802.5 No.E703044; Guard Column: Shodex PROTEIN KW-G No.E712606; Column Temperature: Room Temperature; Mobile Phase: 0.05 mol/L PBS pH=7 (2.05 g Na₂HPO₄ + 1.29 g NaH₂PO₄•2H₂O + 4.4 g NaCl) / 500 mL; Detecting Wavelength: 280 nm; Flow Rate: 0.8 mL/min.

1.2 Manufacture of the standard curve
1.2.1 Standard curve for stock solution
The standard curve was manufactured using NGF standard by external standard method. After gradient dilution of NGF standard, various samples of various concentrations are detected by SEC-HPLC. We got chromatograms and the data of retention time, peak height, peak area and other parameters in every 40μL injection. Compiling the data of two parallel experiments in average, the standard curve was got in the linear range of 719 μg/mL to 44.9 μg/mL (has manufactured the standard curve in three different times, and reveals high repeatability that RSD <1.42%) (Figure 3; Figure 4 andTable 1).
 

Figure 3 The chromatograms of manufacturing the standard curve for stock solution

Figure 4 The standard curve for stock solution

Table 1 Data of the standard curve for stock solution

1.2.2 Standard curve for product
Using the external standard method as well, we prepared gradient NGF concentration of products based on the production formula by adding human serum albumin and mannitol into NGF standard (keep the same concentration of human serum albumin 1%V and mannitol 5%V as selling products) to manufacture the standard curve. Detected by SEC-HPLC, we got chromatograms and the data of retention time, peak height, peak area and other parameters in every 90μL injection. Compiling the data of two parallel experiments in average, the standard curve was got in the linear range of 399.4 μg/mL to 79.9 μg/mL (has manufactured the standard curve in three different times, and reveals high repeatability that RSD <1.42%) (Figure 5; Figure 6 and Table 2).

Figure 5 The chromatograms of manufacturing the standard curve for product

Figure 6 The standard curve for product

Table 2 Data of the standard curve for product

1.3 Detection of NGF stock solution
The NGF stock solution of production batch I010 and H015 were detected by SEC-HPLC based on the chromatographic conditions established above (three parallel experiments in average). The results were calculated through the standard curve and compared with that of BCA method (Table 3). There are tiny differences of results between the two methods, that proving of the reliability of SEC-HPLC quantitative method in detecting NGF stock solution.

Table 3 Compare of the SEC-HPLC results and BCA results

1.4 Detection of NGF product
A dose of Mouse Nerve Growth Factor (NGF) for Injection - Nobex® (18μg per dose) was dissolved in 0.2mL PBS buffer forming the NGF product sample that the calculated theoretical concentration is 90μg/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 91.2μg/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.
 

Figure 7 HPLC chromatogram of NGF standard

 

Figure 8 Mass spectrogram of NGF standard

3.3 Method
In order to prevent the protein denaturation and bringing in impurities, we chose SEC-HPLC other than the traditional RP-HPLC (Reverse Phase High Performance Liquid Chromatography ) method which will introduce in impurities such as acetonitrile and methanol. This work used Shimadzu Shodex KW-802.5 column that exclusion range of 6~15 kD.

Authors' contributions
Da Huang and Hongwei Ren designed research; Da Huang and Jianrong Zhang performed research; Da Huang analyzed data; Da Huang wrote the paper; and Yiming Xu, Liming Lu, Lingyan Ma, Yongchao Fu, Minxiang Li  and Lingyuan Xiong provided support data and advisory. The authors declare no conflict of interest. 

Acknowledgements
This work was supported by the Xiamen Bioway Biotech Co. Ltd., and Biochemistry & Molecular Biology College of Life Sciences, Peking University, China.

References
Levi-Montacini R., 1987, The nerve growth factor 35 years later, Science, 237(4819): 1154-1162 doi:10.1126/science.3306916 PMid:3306916

William C.M., Schenker A., Shooter E.M., 1976, Characterization and isolation of proteolytically modified nerve growth factor, Biochemistry, 15(25): 5543-5552