Human growth hormone (hGH) is a pituitary-derived polypeptide with a wide range of biological functions including protein synthesis, cell proliferation and metabolism. The hormone consists of 191 amino acid residues folded into a four-helix bundle structure with two disulfide bridges [1, 2]. Recombinant human growth hormone (rhGH) is a type of GH produced by recombinant DNA technology which is a biosynthetic hormone identical to human growth hormone [3, 4].
Isopropyl-β -D-thiogalactopyranoside, abbreviated to IPTG, is a highly stable molecular biology reagent used as a molecular mimic of allolactose, a lactose metabolite that triggers the transcription of the lac operon. IPTG is commonly used in cloning procedures to induce the expression of cloned genes which are under the control of the lac operon [5, 6]. In the thioether group of IPTG (Fig. 1), the sulfur atom creates a chemical bond which is non-hydrolysable by the cell, preventing the cell from degrading the inductant. IPTG has also been widely used in the recombinant processing of rhGH [7, 8]. However, the toxicity of IPTG [9, 10] restricted the usage of this promoter system, especially in industry, therefore measuring its presence and concentration in the final pro-ducts is very important.
Several different techniques used for the analysis of thiocompounds [11], of which LC, CE and HPLC-MS-based methods [12-17] have attracted much attention in evaluating sulfur-containing compounds because of their good separation and detection ability [12, 13]. However, because of the absence of strong chromophores or fluorophores, the determination of thiocompounds with UV [18, 19] or fluorescence [20, 21] methods should be coupled with derivatization.
Over the past decade, pulsed electrochemical detection (PED), together with HPLC, has been accepted by many research groups [21-24] as a highly sensitive and selective method to detect thiols, disulfides and many other sulfur-containing compounds. In addition, both reduced and oxidized thiol moieties can be easily detected without the need of derivatization [12]. In this method, thio-compounds are detected via an oxide-catalyzed mechanism, in which surface oxide formation occurs simultaneously with the detection process. Thiocompounds can be detected over a wide range of pH conditions (i. e., pH 0-14), and except for pH, the response is relatively unperturbed by buffer composition. Furthermore, integrated pulsed amperometric detection (IPAD) employs more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection than those of other PED waveforms.
In this paper, IPAD following reversed-phase HPLC was applied to assay IPTG in the widely used biopharmaceutical hormone rhGH. Electrochemical characterization via cyclic voltammetry was performed to optimize the IPAD waveform, and mechanistic information was used to select the proper settings of the chromatographic solvent system. An effective method using reversed-phase HPLC was developed to separate IPTG in rhGH samples and then determined using PED.
All solutions were prepared from reagent grade chemicals. HPLC grade sodium acetate (NaOAc) was purchased from Fluka (Steinheim, Germany). HPLC-grade acetonitrile (ACN) was purchased from Fisher Scientific (Fair Lawn, USA). Mobile phases were filtered with 0.45 μ m Nylon-66 filters (Fisher Scientific). All solvents were freshly prepared daily with ultra-pure grade water (electrical resistivity of 18.2 MΩ·cm at 25 ℃).
IPTG (high purity grade) was obtained from Calbiochem (San Diego, California, USA) and stored in a refrigerator at 1-5 ℃. rhGH samples were provided by Zhangjiang Bio-Tech Co. Ltd (Shanghai, China) and stored in a freezer at -18 ℃. The samples were placed in plastic microcentrifuge tube (3 000 Da molecular weight cutoff, Millipore Corp., Bedford, USA).
HPLC-PED was performed on a Dionex liquid chromatography system (Dionex Corporation, Sunnyvale, USA) with an eluent degassing device module GP40 and a Dionex Model ED40 electrochemical detector, equipped with a gold working electrode, an Ag/AgCl reference electrode, and a titanium auxiliary electrode. Solutions were injected with an injection valve (Rheodyne Inc., Cotati, USA) fitted with a 25 μ L injection loop. Data collection and system control were accomplished using Peaknet software (Dionex, version 5.12) on a Dell OptiPlex Gn computer.
Separation of IPTG was achieved using an Acclaim 300 analytical column (C18, 3 μ m particle size, 150 mm×4.6 mm, Dionex). Unless otherwise specified, the mobile phase solvents were 'solvent A'=0.01 mol/L pH 5.45 NaOAc buffer, and 'solvent B'=ACN (A : B=90 : 10, v/v), delivered at a flow rate of 1.00 mL/min. All solvents were filtered, degassed, and kept under N2 (pressure about 68.95 kPa) at room temperature.
The rhGH sample was placed in a 1.5 mL-plastic microcentrifuge tube. The tube was microcentrifuged (fixed rotor speed 10 000 r/min) for about 20 min at 4 ℃. After which the supernatant was discarded. A selected residual volume in the tube was weighed and diluted with degassed, de-ionized water at 1 : 1 000 (v/v). This solution was then filtered through a 0.45 μ m filter and injected immediately after preparation into the chromatographic system. The same amount of deionized water as the sample was used as blank solution for the same preparation process and analysis.
Cyclic voltammetry has been performed for the selection of approximate IPAD waveform potentials. The current-potential (i-E) response in 0.01 mol/L pH 5.45 NaOAc buffer/ACN (90 : 10, v/v) degassed in the absence and presence of 100 mg/L IPTG is shown in Fig. 2. Under acidic conditions, the residual response displays an anodic peak at about +1 250 mV (see Fig. 2) during the forward scan as the formation of surface oxide takes place. On the reverse scan, a cathodic peak at about +400 mV (see Fig. 2) corresponds to dissolution of the surface oxide formed on the forward scan. Breakdown of solvent occurs at about +1 800 mV and -1 000 mV, which leads to the generation of O2 and H2 respectively. Reduction of dissolved O2 takes place during both the forward and reverse scans and commences at about +200 mV.
The IPAD waveform has been shown to be effective in electronically rejecting the "background" signal from oxide formation, which is an order of magnitude larger than the signal of the analyte. The detection sequence consists of a series of triangular potential scans to maximize the signal from the transient oxide intermediates of the oxide-catalyzed mechanism. Fig. 3 shows the optimized waveform for the detection of IPTG. The waveform starts at a potential lower than that required for oxide formation, scan to a maximum potential that covers oxide formation and analyte, and ends at a potential that is more negative than that required for cathodic dissolution of the formed oxide.
The start and end potentials are also more positive than that required for the reduction of dissolved O2. Hence, the contribution to the overall signal from the reduction of dissolved O2 is minimized. On-line degassing as part of the solvent delivery system can relax constraints on the start/end potential [25]. The detection steps in both waveforms are followed by a large negative potential pulse (-2 000 mV for 10 ms) to induce both cathodic cleaning of the electrode [26] and reduce any partly-soluble Au [27]. This potential pulse is necessary to extend the life of the electrode for months of continuous use. Then, a short positive potential pulse (+1 600 mV for 100 ms) is used to induce anodic cleaning of the electrode. This is followed by 600 ms at -200 mV to allow for pre-adsorption of the analyte. The Johnson group [28] has shown that amines can be pre-concentrated 10-fold with the use of an adsorption step in the potential-time waveform. PED at an Au electrode has proven to be selective for sulfur-containing compounds under mildly acidic conditions. The direct electrochemical detection of numerous polar aliphatic compounds is achieved at Au and Pt electrodes under the control of multistep potential-time waveforms. In this technique, the waveforms manage the sequential processes of sampling the faradaic signal, from the oxidation of analyte, followed by oxidative cleaning and reductive reactivation of the electrode surfaces [28]. In agreement with these findings, LaCourse et al.[29] demonstrated that the optimum response for sulfur-containing compounds is obtained by maximizing the time of the adsorption step without negatively affecting the chromatographic integrity of the peaks.
Separation of IPTG was achieved by using an Acclaim 300 C18 analytical column with 0.01 mol/L pH 5.45 NaOAc buffer/ACN (90 : 10, v/v). The C18 phases have very high surface coverage, resulting in high capacity. This C18 column works at a pH between 2 and 8. Under such mildly acidic conditions, PED at an Au electrode has proven to be selective for sulfur-containing compounds [29-34]. In the work of LaCourse et al. [20], PED at pH 4.54 is specific to the detection of the sulfur-containing compounds IPTG. We studied the pH value of the buffer solution over the range of 4-6 and found that the separation (determination) peak of IPTG is slightly variable in these mobile phases. At pH 5.45, IPTG was well separated and the corresponding response was highly sensitive. Therefore, the pH 5.45 phosphate buffer was chosen as the mobile phase. Fig. 4 shows the chromatograms of IPTG standard in 0.01 mol/L NaOAc buffers at pH 5.45 and pH 4.54, respectively. IPTG was eluted at 2.87 min, with an oxygen peak observed at about 3.1 min. Dissolved O2 was found in both mobile phases, but the presence of O2 in the pH 5.45 solvent is a little later than that of pH 4.54 solvent. Consequently, the IPTG standard peak can be easily separated and determined at this pH value.
The linear dynamic range of the HPLC-PED response for IPTG was determined. The response was linear over the range from 0.1 to 10 mg/L. The regression equation was Y=0.0180X+0.126 7(where Y is the integral area, X is the mass concentration (mg/L)), and the correlation coefficient (r) was 0.997 2 (n=7). The limit of detection (S/N=3) for 25 μ L injections of IPTG was estimated to be 1 μ g/L (0.1 pmol).
The optimized method was utilized to determine IPTG in rhGH samples. Chromatograms of rhGH products are presented in Fig. 5 obtained with 0.01 mol/L pH 5.45 NaOAc buffer/ACN (90 : 10, v/v). The method of standard addition was performed to assay the recoveries of IPTG and the results were summarized in Table 1. Good separation and high sensitivity for IPTG were obtained.
Pulsed electrochemical detection following HPLC allows for the simple and direct detection of numerous sulfur-containing compounds. In agreement with the past efforts, IPAD is well-suited for the detection of thiocompounds such as IPTG. The IPAD waveform, which needs only to be optimized for a particular pH, allows for the direct detection of the analytes at low levels with superior limits of detection. Through the use of an acidic mobile phase (pH=5.45) under reversed-phase conditions, good selectivity is achieved for IPTG. The reported method shows good stability and reproducibility. The linear ranges cover over three orders of magnitude and the limits of detection can reach 1 μ g/L (0.1 pmol) for IPTG. The system performance was excellent for the rapid determination of rhGH samples. The high selectivity and good sensitivity of this approach assures that the matrix components of the assay do not interfere with the determination. The assay is also useful, efficient and low-cost in monitoring disulfide formation kinetics and assuring quality control for biopharmaceuticals.
2017,
Vol. 35 Issue (3): 308-313
PDF
2017,
Vol. 35 Issue (3): 308-313
PDF