Analysis and Preparation of Linaclotide by High Performance Liquid Chromatography

Linaclotide is a guanylate cyclase-C (GC-C) agonists, which is aimed for treating constipation type of irritable bowel syndrome (IBS-C) and chronic idiopathic constipation (CIC) [1]. One third of IBS patients are IBS-C around the world. These patients have had influenced greatly in the quality of their lives, and had given a large burden to the society. Linaclotide have brought expectation for these patients since it was approved by FDA in 2012. Linaclotide and its active metabolite bind to GC-C, which is found on the intestinal epithelium, to stimulate the release of intracellular and extracellular cyclic guanosine monophosphate (cGMP) [2]. The rise of intracellular cGMP concentration promotes the intestinal fluid secretion, strengthen the gastrointestinal peristalsis, and lead to an increased frequency of defecation. The rise of extracellular cGMP concentration can reduce the sensitivity of the nere, which reduces patient's pain, so as to relieve the symptoms of abdominal pain and so on. Other medicines, which treat constipation, generally do not reduce adverse reactions in patients with abdominal distension, abdominal pain, etc.


Introduction
Linaclotide is a guanylate cyclase-C (GC-C) agonists, which is aimed for treating constipation type of irritable bowel syndrome (IBS-C) and chronic idiopathic constipation (CIC) [1]. One third of IBS patients are IBS-C around the world. These patients have had influenced greatly in the quality of their lives, and had given a large burden to the society. Linaclotide have brought expectation for these patients since it was approved by FDA in 2012. Linaclotide and its active metabolite bind to GC-C, which is found on the intestinal epithelium, to stimulate the release of intracellular and extracellular cyclic guanosine monophosphate (cGMP) [2]. The rise of intracellular cGMP concentration promotes the intestinal fluid secretion, strengthen the gastrointestinal peristalsis, and lead to an increased frequency of defecation. The rise of extracellular cGMP concentration can reduce the sensitivity of the nere, which reduces patient's pain, so as to relieve the symptoms of abdominal pain and so on. Other medicines, which treat constipation, generally do not reduce adverse reactions in patients with abdominal distension, abdominal pain, etc.
Linaclotide [3] is a peptide with 14 amino acids (CCEYCCNPACTGCY) and three disulfide bonds (1-6, 2-10, 5-13). The molecular weight is 1526.8 [3]. The structure was shown in Figure  1. Solid phase peptide synthesis was applied to synthesise linaclotide. Impurities in crude product mainly include incompletely synthesised peptides, incorrectly-formed disulfides of peptides, etc. Therefore, it is crucial to set up HPLC methods for the analysis and the preparation of linaclotide.

Materials
Linaclotide were synthesised by Fmoc solid phase peptide synthesis in our laboratory. Acetonitrile was obtained from Anaqua Chemicals Supply (Houston, TX, USA). Formic acid was obtained from Aladdin (Shanghai, China). Phosphoric acid and Sodium dihydrogen phosphate were purchased from XiLong Chemical Company (Shantou, Fujian, China). Distilled water was purified with Millipore's Milli-Q analytical deionization system (Bedford, MA, USA).

Sample preparation
Accurately weighed crude linaclotide product was dissolved in mixed solvents, which was water: acetonitrile=2:1 (v/v), with 0.1% formic acid. The sample solution and mobile phases were filtered through a 0.22 μm PTFE filter (Jinan, Shandong, China) prior to analysis and preparation.

HPLC determination
Mobile phases of the HPLC were phosphate solution (A) and acetonitrile (B) [4]. The maximum absorption wavelength was determined at 214 nm by full range scan of linaclotide on UV spectrometry. The eluting gradient was shown in Table 1. The volume of injection was 20 μL. The flow rate was 1.0 mL/min. Optimised analytical conditions were as follows: the analytical column was SinoChrom ODS-BP (5 μm, 250 mm × 4.6 mm); mobile phase A was 30 mmol·L -1 phosphate (pH-2.8) and mobile phase B was acetonitrile; column temperature was set to 40°C.

Optimisation of solvents
Five different proportions of acetonitrile as solvent to dissolve crude linaclotide were compared in the solubility and the peak shape [5].

Optimisation of pH
In order to investigate the effect of pH on linaclotide analysis, phosphate was applied to adjust the pH of mobile phase A from 2.0 to 3.2.

Optimisation of phosphate concentration
Phosphate concentration of mobile phase A was optimised in the range of 10~50 mmol·L -1 to investigate the effect of buffer concentration on linaclotide analysis [6].

Optimisation of oven temperature
Oven temperature was compared as an effect on resolution in the range of 17~40°C controlled by column oven.

Linearity and Limit of Detection (LOD)/Limit of Quantification (LOQ)
The calibration curve was plotted in the range of 0.1-1.6 mg/mL of linaclotide and fitted with least squares linear regression. The LOD and LOQ were estimated considering the ratio of back-ground noise and analytical signal. Linaclotide LOD and LOQ were determined based on the calibration curve and calculated according to Eq. (1) and Eq. (2).
LOD=3σ/S Eq. (1) LOQ=10σ/S Eq. (2) Where σ represents the standard deviation of the response, S represents the slope of the calibration curve.

Precision
Precision was evaluated in terms of repeatability. Repeatability (intra-assay) was verified for one concentration of linaclotide with 6 replicates.

Preparation of linaclotide
In chromatographic preparation of linaclotide, analytical method was modified due to the modification of flow rate, the volume of sample, and the diameter of column [7]. The preparative chromatographic conditions were as follows: the column was SinoChrom ODS-BP (250 × 30 mm, 10 μm); mobile phase A was water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid; the elution gradient was 7%→25% B% from 0 to 32 min and was 25% from 32 to 50 min; room temperature was applied; the flow rate was 30 mL·min -1 ; wavelength of detection was at 214 nm.

Optimisation of the HPLC column
Three types of chromatographic columns were compared to analyse crude linaclotide. The analytical results were shown in Table  2. The SinoChrom ODS-BP was selected for the further analysis due to its best resolution results with impurities before and after linaclotide.

Optimisation of solvents
Results (Table 3) showed that with increasing portion of acetonitrile in mobile phase, the chromatographic peaks of linaclotide were deformed and open. Optimised portion was set to water: acetonitrile=2:1 (v/v).

Optimisation of pH
Results were shown in Figure 2. The resolution of the impurity before linaclotide and linaclotide (R1) was gradually increased with the increase of pH. The resolution of linaclotide and the impurity after linaclotide (R2) was increased till pH 2.8, and then decreased. The optimised pH was set to 2.8 adjusted by phosphate buffer.

Optimisation of phosphate concentration
Results were shown in Figure 3. The R1 gradually decreased with the increase of phosphate concentration. The R2 increased with the increase of phosphate concentration. 30 mmol·L-1 was chosen as the optimised concentration of phosphate solution as mobile phase A.

Optimisation of oven temperature
Results was shown in Figure 4. With the increase of oven temperature, R1 and R2 increased. The oven temperature was optimised to 40°C. The optimised chromatography diagram was shown in Figure 5.

Linearity and LOD/LOQ
The linear equation of the calibration curve is: y=231.9, x=4.310, r=0.9995, where y represents the peak area and x represents the concentration of linaclotide. Limit of detection (LOD) and limit of quantification (LOQ) were 0.5 µg/mL and 1.0 µg/mL with the signal-

Precision
The RSD of intraday results were less than 1%. Results were listed in Table 4.

Stability
Peak area and retention time were compared. Results suggested that linaclotide solution was stable in 24 hours, shown in Table 5. RSD was less than 1%.

Application in preparative chromatography
Preparative chromatography diagram was shown in Figure 6, in accordance with linaclotide was collected between the vertical lines. Analysis of freeze-dried linaclotidewas shown in Figure 7. Batch preparation results were shown in Table 6. The purity of linaclotide after preparation was over 98%.

Conclusion
Analytical RP-HPLC method was developed for analysis of linaclotide. Optimisation of solvent and chromatographic conditions including pH of mobile phase, phosphate concentration and oven temperature was studied. Method validation was carried out in linearity, LOD, LOQ, precision and stability. Linaclotide was successfully prepared on preparative HPLC with this method. Modified method for linaclotide preparation on a preparative liquid chromatography was    Table 6: HPLC preparation results of three batches of crude linaclotide.