A method for purifying telopeptides

Abstract

This invention belongs to the field of peptide purification technology and discloses a purification method for telpolide, aiming to solve the problems of easy column damage and low purification efficiency in existing technologies. The method includes: dissolving crude telpolide in ammonia; performing a first liquid chromatography purification, with mobile phase A being an aqueous solution containing volatile salts at pH 6.0-7.0; performing a second liquid chromatography purification, with mobile phase A being an aqueous solution containing volatile acids at pH 1.5-2.5; and finally, concentrating, adjusting pH, allowing crystallization, and lyophilizing to obtain the final product. This invention uses volatile salts to avoid damage to the chromatographic column caused by phosphates, and the purity of the obtained telpolide can reach up to 99.6%, with a total recovery rate of over 75%. This method is suitable for the efficient purification and preparation of telpolide raw materials.

Description

Technical Field

[0001] This invention belongs to the field of peptide purification technology, specifically relating to a highly efficient purification method for telpoeptide suitable for industrial production. Background Technology

[0002] Tirzepatide is a 39-amino acid long-chain modified polypeptide with C20 fatty acid diacid side chain modification and a molecular weight of approximately 4813.45 Da. Tirzepatide is a dual agonist of the gastric inhibitory polypeptide glucagon-like peptide-1 receptor. It is the world's first and currently only dual agonist of GIP (glucose-dependent insulinotropic peptide) / GLP-1 (glucagon-like peptide-1) receptors. By simultaneously activating two incretin receptors, it produces a synergistic effect and shows significant advantages in glycemic control and weight management. Treatment of type 2 diabetes: Based on the SURPASS series of phase III clinical trials, tirzepatide (15 mg) can reduce glycated hemoglobin (HbA1c) by an average of 2.3%, which is significantly better than smegglutide (1 mg, reduction of 1.86%) and insulin degludec. Obesity treatment: Studies show that telpotetide (15mg) can reduce the average weight of non-diabetic obese patients by 22.5% (approximately 24 kg), far exceeding the weight loss effect of smegglutide (12.4%). OSA treatment: Approved in June 2025 for moderate to severe obstructive sleep apnea (OSA) in obese adults. Therefore, the demand for telpotetide's active pharmaceutical ingredient is huge, and improving its production process to increase efficiency or purity is currently a hot research topic.

[0003] Existing technologies, such as the Chinese invention patent with publication number CN118702801A, use a phosphate / triethylamine system for the first purification pass. Although the peak shape is symmetrical, the phosphate buffer system easily leads to column clogging, and subsequent complex cleaning steps are required, which is not conducive to continuous production. Another Chinese invention patent with publication number CN116693629A involves the chemical preparation of functionalized silica gel packing material, which is complex and introduces additional chemical modification steps, increasing the risk of impurities. Existing technologies typically require multi-step reversed-phase preparation, which is complex and time-consuming. Desalting processes often rely on gel chromatography or dialysis, resulting in high solvent consumption, long processing time, low yield, high column wear, easy peptide loss during ultrafiltration and salt conversion, difficulty in separating impurities during extraction, and significant equipment investment. Therefore, based on the above-mentioned problems with existing technologies, there is an urgent need to find a purification method with higher product purity, shorter process cycle, higher overall recovery rate, and less equipment wear. Summary of the Invention

[0004] To address the challenges of long purification cycles, high equipment wear and tear, and low product recovery rates in existing technologies for telpoide, this invention provides a purification method for telpoide. This method yields products with high purity, short process cycles, and high recovery rates. The purification process does not clog columns and eliminates the need for complex steps to prepare functionalized silica fillers, thus reducing the risk of impurities.

[0005] A method for purifying telpoeptide, characterized by comprising the following steps: The crude telpolide was dissolved in an ammonia solution to obtain a crude telpolide solution. The crude solution of telpolide was purified by reversed-phase high-performance liquid chromatography. Phase A consisted of water and volatile salts, and phase B consisted of organic solvents. The pH of phase A was 6.0-7.0. The telpoide fraction with a purity of over 95% obtained from the first purification was subjected to a second purification using reversed-phase high-performance liquid chromatography. Phase A consisted of water and volatile acids, while phase B consisted of organic solvents. The pH of phase A was 1.5–2.5. Take the telpoeptide fraction with a purity of over 99% obtained from the second purification, adjust the volume percentage of organic solvent in the telpoeptide fraction to be less than 15% and the pH value to 3-4, let it stand to crystallize, centrifuge, add sodium hydroxide solution to dissolve the obtained solid, freeze dry it to obtain pure telpoeptide.

[0006] Preferably, the ammonia solution is an ammonia solution with a volume percentage of 0.5% to 1.0%.

[0007] Preferably, in the first purified phase A, Volatile salts include one or more of trimethylammonium acetate, ammonium acetate, or ammonium bicarbonate; and / or The concentration of volatile salts is 4.0~8.0 g / L.

[0008] Trimethylammonium acetate can precisely stabilize the pH of the system at 6.0–7.0. Under this mild environment, the acetate anion formed by its dissociation may enhance the retention behavior of the peptide on the reversed-phase chromatography column through electrostatic interaction with the positively charged telpoeptide molecule. However, what is particularly noteworthy is that this salt may not only perform well in conventional retention control, but also produce beneficial changes in the microscopic separation mechanism. It is speculated that it can weaken the non-specific secondary interaction between the peptide and the stationary phase to a certain extent, thereby improving the symmetry of the main peak and effectively suppressing tailing. This peak shape modification effect is likely not an inherent property of ammonium salts or acetates, but is attributed to the unique hydrophobic trimethylammonium cation structure of trimethylammonium acetate selectively shielding the silanol groups on the surface of the stationary phase under this specific pH window. Its improvement is better than that of ammonium acetate or ammonium bicarbonate at the same concentration.

[0009] This ammonium salt system operates under neutral conditions at pH 6.0–7.0, suggesting that it is chemically mild and non-corrosive to hydrophobic packing materials such as C8, thus extending the column's lifespan. In contrast, phosphate systems, due to decreased solubility within this pH range, are prone to crystallization at the column inlet, leading to column pressure spikes and reduced separation performance. This invention reduces the risk of such clogging, thereby improving process stability and batch-to-batch reproducibility. Furthermore, all three ammonium salts exhibit excellent volatility, allowing them to be removed during subsequent rotary evaporation and freeze-drying processes, avoiding the risk of phosphate residues. This eliminates the need for costly desalination steps such as gel chromatography or ultrafiltration, potentially simplifying the post-processing workflow. Moreover, the waste liquid does not contain recalcitrant phosphates, meeting the environmental requirements of green pharmaceutical manufacturing.

[0010] Preferably, in the second purified phase A, Volatile acids include one or more of 2-chlorobutyric acid, trifluoroacetic acid, or formic acid; and / or The volatile acid content is 0.1% to 0.5% by mass.

[0011] This invention utilizes 2-chlorobutyric acid, trifluoroacetic acid, or formic acid as mobile phase additives under low pH conditions (1.5~2.5). All three acids provide sufficient protons to effectively inhibit the dissociation of silanol groups, thereby weakening secondary retention effects and improving the symmetry of the main peak. Their anions can also act as ion-pairing reagents, participating in separation through hydrophobic adsorption and enhancing the retention behavior of peptides on a C8 column. Specifically, 2-chlorobutyric acid exists primarily as an undissociated hydrophobic molecule at low pH, capable of adsorbing onto the C8 stationary phase surface to form a dynamic "pseudo-stationary phase" or "solvent layer," further regulating the separation environment. Notably, the presence of trifluoroacetic acid and formic acid synergistically regulates the ionic strength and solvent polarity of the mobile phase, helping to stabilize the molecular adsorption state of 2-chlorobutyric acid and optimizing the overall distribution behavior of the acidic additives, thus improving separation selectivity and peak quality. Experiments show that when the three acids are used in combination, the purity can reach 99.6%, and the recovery rate reaches 85.7%. In addition, all three acids have excellent volatility and are easily and completely removed during subsequent rotary evaporation concentration and freeze drying processes, eliminating the need for an additional desalting step and simplifying the post-processing procedures.

[0012] Preferably, the first purified phase B is acetonitrile and isopropanol, with the concentration of acetonitrile and isopropanol increasing from 40% to 50% by volume; and / or The second purified phase B is acetonitrile, with the acetonitrile concentration increasing from 42% to 52% by volume. More preferably, the first purified phase B organic solvent is acetonitrile and isopropanol in a volume ratio of 4:1 to 2:1.

[0013] Preferably, 2% to 7% of the crude telpopeptide seed crystals are added to the fraction before it settles and crystallizes.

[0014] Preferably, the amount of sodium hydroxide added is 1.0% to 1.5% of the total mass of telpoeptide.

[0015] In the crystallization step, the efficiency of isoelectric point crystallization and crystal quality are improved by controlling the acetonitrile ratio and adding seed crystals in a synergistic manner: controlling the volume percentage of acetonitrile in the crystallization system to below 15% can moderately reduce the solubility of peptides in the peptide solution, further reducing the solubility of telpoide molecules near the isoelectric point, thereby effectively improving the crystallization yield, while avoiding oil precipitation or co-precipitation impurities caused by excessive acetonitrile ratio; the synergistic effect of the two combined is that the seed crystals solve the problem of uncontrollable nucleation, and the acetonitrile regulates the solubility, transforming the crystallization process from spontaneous random nucleation to controlled directional growth, achieving both high yield and high purity. At the same time, the regular large-particle crystals exhibit excellent operational performance in subsequent centrifugation and lyophilization reconstitution, and the lyophilized cake is loose and uniform, and reconstitutes rapidly, effectively reducing the filtration difficulty in industrial production.

[0016] Beneficial effects This invention abandons the phosphate / triethylamine buffer system commonly used in traditional separation and purification processes. Although phosphate systems can retain the function of silanol packing materials to some extent, they are prone to salting out and crystallizing within the column due to changes in the solvent environment. This not only severely impairs column efficiency but also leaves residual salts that are difficult to remove completely through conventional cleaning, shortening the column's lifespan with long-term use. To address this issue, this invention selects trimethylammonium acetate, ammonium acetate, or ammonium bicarbonate as alternative buffer salts, and uses them in combination with a mixed solvent of acetonitrile and isopropanol. This combination fully leverages the ease of removal of these volatile salts in subsequent processing and, through the fine adjustment of solvent polarity by isopropanol, effectively improves the separation selectivity for long-chain hydrophobic peptides such as telpoide. Experiments show that, compared to a single acetonitrile system, this mixed solvent system improves the target peak resolution while being gentler on the column material, reducing the risk of stationary phase damage and thus helping to extend the column's lifespan. In the gradient elution strategy, this invention introduces 2-chlorobutyric acid, trifluoroacetic acid, or formic acid as acidic modifiers in the second elution stage. This enhances the removal of various process-related impurities in telpoeptide, especially difficult-to-remove impurities whose retention behavior is very similar to the main peak. Notably, the acid and alkali additives used in both elution processes are volatile compounds, which facilitates subsequent processing—most solvents and salts can be efficiently removed primarily through rotary evaporation, eliminating the need for expensive and membrane-clogging ultrafiltration systems. This reduces equipment investment and maintenance costs while simplifying the operation. The overall yield of the process described in this invention can reach over 75%, an improvement of 5-10% compared to traditional methods. This invention proposes first controlling the acetonitrile content through rotary evaporation, then utilizing isoelectric point precipitation, low-temperature crystallization, and also incorporating telpoeptide crystals to assist crystallization. This not only removes salts but also directly concentrates and precipitates the peptide fragments from a large volume solution, resulting in fast and high-quality crystal precipitation. This significantly reduces the volume and energy consumption of subsequent freeze-drying, improving production efficiency. Attached Figure Description

[0017] The applicant will further describe the invention in detail with reference to the accompanying drawings.

[0018] Figure 1 This shows the HPLC chromatogram of the finished product, telpolide. Figure 2 This shows the mass spectrum of telpoeptide. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] Example 1: Dissolution and first-step purification of crude telpolide The crude telpoeptide with a purity of 84.5% was dissolved in an ammonia aqueous solution (ammonia volume percentage 1.0%), and sonicated until completely dissolved to obtain a 40 g / L crude telpoeptide solution. The crude telpolide solution was subjected to gradient elution by RP-HPLC for 60 min. A DAC-200 column with a height of 250 mm was used, the flow rate was 900 mL / min, and the detection wavelength was 215 / 280 nm. The telpolide fraction was collected from the appearance of the main peak to its cessation. The fraction with a purity of ≥95% was separated for further purification. The gradient elution used an aqueous solution with a pH of 6.0–7.0 as the mobile phase A, containing one or more of trimethylammonium acetate, ammonium acetate, and ammonium bicarbonate. The CAS number of trimethylammonium acetate was 6850-27-7. An acetonitrile:isopropanol ratio of 4:1 (v:v) was used as the mobile phase B, with the acetonitrile concentration increasing from 40% to 50% by volume. Octylsilane-bonded silica gel was used as the stationary phase. The yield and purity were calculated. The DAC-200 column, after 300 uses, was evaluated for its peak shape symmetry, elution time variation, and column pressure variation. The results are shown in Table 1.

[0022] Table 1 - Detection results of purification of different volatile salts in Phase A of the first purification process.

[0023] The results of experiments 1 to 6 show that different mobile phase additive compositions have a certain impact on the purification effect and column condition of telpolide. Among them, Experiment 1, which uses a buffer system combining trimethylammonium acetate, ammonium acetate, and ammonium bicarbonate, exhibits the best overall performance among all test groups: its target analyte recovery rate reaches 90.2%, purity is increased to 97.5%, and the maximum proportion of single impurity peaks is only 0.20%, with all indicators superior to other comparative schemes. Regarding single ammonium salt systems, Experiments 2 (trimethylammonium acetate), 3 (ammonium acetate), and 4 (ammonium bicarbonate) all achieved relatively ideal purification effects, with purity ranging from 97.1% to 97.2% and recovery rates ranging from 89.12% to 89.86%. However, their recovery rates and purity were slightly inferior to the scheme using the three combined, indicating that the separation ability of a single ammonium salt is limited and it is difficult to fully utilize the complementary advantages of each salt component in terms of ionic strength and selectivity. Experiment 5 used a two-component combination of ammonium acetate and ammonium bicarbonate. Its recovery rate (90.00%) and purity (97.4%) were improved compared to the single-salt system, but still not as good as the effect of the three-salt combination in Experiment 1. This further illustrates that the introduction of trimethylammonium acetate has a beneficial effect on optimizing separation selectivity and improving purification efficiency. In contrast, when phosphoric acid was used as the mobile phase additive in Experiment 6, all indicators were the lowest among all experimental groups, with a recovery rate of only 88.12%, a purity of 96.5%, and a maximum single impurity rate as high as 0.5%. At the same time, the phosphate system caused significant irreversible damage to the chromatographic column. In the column lifespan comparison experiment, the phosphate system's column pressure rose above 20 MPa after approximately 150 injections, and the chromatographic peak shape began to show tailing, indicating irreversible changes in the stationary phase, severely affecting separation reproducibility and preparation efficiency. In contrast, the ammonium salt system used in this invention maintained a column pressure of only 17 MPa after 300 injections, with symmetrical peak shape and no significant change in peak time, indicating that the column performance remained good. This difference clearly demonstrates that ammonium salt systems not only provide excellent separation results but are also gentler on chromatographic packing materials, effectively extending column life and reducing consumable costs and equipment maintenance frequency in industrial production. Therefore, the volatile buffer salt system using trimethylammonium acetate, ammonium acetate, and ammonium bicarbonate offers advantages in separation performance, column protection, and process stability, making it suitable as the preferred mobile phase for the first step of purification and separation.

[0024] Example 2: The telpoeptide fraction with a purity of over 95% collected after the first purification was subjected to gradient elution again using water as mobile phase A. The volatile acids in phase A included one or more of 2-chlorobutyric acid, trifluoroacetic acid, and formic acid. The CAS number for 2-chlorobutyric acid is 4170-24-5. Formic acid or 2-chlorobutyric acid was not used alone in the experiment because their pH could not be adjusted to 1.5–2.5 independently. Acetonitrile was used as mobile phase B to separate telpoeptide with a purity of over 99%. The acetonitrile concentration increased from 42% to 52% by volume. Octylsilane-bonded silica gel was used as the stationary phase. The yield and purity were calculated, and the results are shown in Table 2.

[0025] Table 2 - Purification Detection Results of Different Volatile Acids in Phase A of the Second Purification Process

[0026] Based on the results of experiments 7 to 9, the effects of using 2-chlorobutyric acid alone or in combination with dichlorobutyric acid and trifluoroacetic acid on the maximum single impurity rate were not significantly different for each system. However, the separation effect was most ideal when all three acids were used in combination, with a recovery rate of 85.7% and a purity increase to 99.6%. Furthermore, mass spectrometry analysis showed that the total impurity content of the deleted peptides Gly4, Gly30, and their combinations (with or without Gly4 or Gly30) was controlled below 0.1%, and the total impurity content of the deleted peptides Ser11, Ile12, and Ser11-Ile12 was also below 0.1%, indicating that the synergistic effect of the three acids positively contributes to impurity removal. In addition, it is noteworthy that during the second-step separation and purification process, the maximum single impurity rate in all groups decreased compared to the first purification step, indicating that the multi-step purification strategy helps to further improve product purity and provides a feasible approach for efficiently obtaining high-purity target peptides.

[0027] Example 3: The telpoeptide fraction with a purity of over 99% was concentrated by rotary evaporation until the acetonitrile volume percentage in the fraction was less than 15%. Crystallization was then carried out under the conditions of 2–8℃, pH 3–4, and a seed crystal addition of 2%–7% of the crude telpoeptide mass. After centrifugation, the resulting solid was dissolved in 1.25% sodium hydroxide solution and freeze-dried to obtain pure telpoeptide. The yield and purity were calculated, and the results are shown in Table 3. The obtained telpoeptide was analyzed by liquid chromatography using a C18 column (4.6 × 250 mm, 5 μm). Gradient elution was performed with 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B. The flow rate was 1.0 mL / min, the column temperature was 25–30℃, the detection wavelength was 220 nm, the injection volume was 5 μL, and the run time was approximately 55 min. Under these conditions, the retention time of the telpoeptide main peak was approximately 40.9 min, and the separation between the main peak and adjacent impurity peaks was good. The obtained telpoeptide (Experiment 10) was analyzed by liquid chromatography. Figure 1The chromatographic peak analysis data of this sample are shown in Table 4. The peak shape is symmetrical and without tailing, indicating good detection performance. The obtained telpolide was also subjected to mass spectrometry, using ESI positive ion mode for detection, with telpolide [M+4H]. 4+ The precursor ion was m / z 1204.3~1204.4, and the quantitative product ion was m / z 396.2. The ion spray voltage was 5500V, the source temperature was 400℃, and the internal standard was smegglutinin (MRM: m / z 1029.4). Chromatographic separation was performed using an ACQUITY Premier Peptide CSH C18 column (2.1×50~150 mm, 1.7μm), with gradient elution of 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile (0~0.25min: 35% B → 0.25~2.0min: 50% B → 2.0~3.0min: 75% B → 3.0~3.1min: 90% B → 3.1~5.0min: 35% B equilibration). The flow rate was 0.3~0.4mL / min, the column temperature was 50~55℃, and the total run time was 5min. Detection results are shown in [link to detection details]. Figure 2 Mass spectrometry showed that the molecular ion peak was consistent with the theoretical molecular weight of telpoeptide.

[0028] Table 3 - Comparison of crystallization of telpopeptide under different conditions

[0029] Table 4 - Chromatographic peak analysis data of telpolide

[0030] Experiments 10 to 16 systematically investigated the effects of acetonitrile volume and seed crystal mass on product yield and purity. A comparison of Experiments 10, 11, and 12 showed that an excessively high acetonitrile ratio reduced the yield to some extent; the yield was 95.4% when the acetonitrile ratio reached 20%, a significant decrease. Further comparisons of Experiments 13-15 revealed that seed crystal mass had a certain impact on both yield and purity, with the optimal yield and purity achieved at a seed crystal mass of 4%. Experiment 16, using an ultrafiltration membrane instead of rotary evaporation, only yielded 61.7%. Considering all factors, the conditions corresponding to Experiment 10 were the optimal scheme, achieving a yield of 98.7% and a purity of 99.6%, and producing crystals with regular shape and large particle size, which is beneficial for subsequent filtration, drying, and other operations. In this embodiment, using the same batch of crude telpoeptide as the starting material, the product of three consecutive purification steps (recovery rate 90.2%), purification steps (recovery rate 85.7%), and crystallization (recovery rate 98.5%) resulted in a total recovery rate of 90.2% × 85.7% × 98.7% ≈ 76.14% (three consecutive steps). The total recovery rate for each step was calculated as the ratio of the mass of the target peptide input to the mass of the target peptide recovered (target peptide mass = total peptide mass × HPLC purity).

[0031] In summary, the entire purification process can be categorized as a synergistic combination of two liquid chromatography purification steps and subsequent crystallization control. In the first liquid chromatography purification, a volatile buffer salt system composed of trimethylammonium acetate, ammonium acetate, and ammonium bicarbonate is used. This effectively adjusts the mobile phase pH, maintaining a suitable separation environment, and extends the column life while ensuring initial purification efficiency. The second liquid chromatography purification introduces the combined use of 2-chlorobutyric acid, trifluoroacetic acid, and formic acid to further purify telpolide, reducing the content of each impurity to below 0.1%, thus improving product purity. Subsequently, by precisely controlling the acetonitrile concentration and the amount of telpolide seed crystals added, the target product is effectively induced to precipitate rapidly, forming regular, large-particle crystals, which facilitates subsequent separation and drying operations. The overall process design is reasonable, the functions of each step are clearly defined, and the synergistic effect achieves a balance between high yield, high purity, and good operability.

Claims

1. A method for purifying telpolide, characterized in that... Includes the following steps: The crude telpolide was dissolved in an ammonia solution to obtain a crude telpolide solution. The crude solution of telpolide was purified by reversed-phase high-performance liquid chromatography. Phase A consisted of water and volatile salts, and phase B consisted of organic solvents. The pH of phase A was 6.0-7.

0. The telpoide fraction with a purity of over 95% obtained from the first purification was subjected to a second purification using reversed-phase high-performance liquid chromatography. Phase A consisted of water and volatile acids, while phase B consisted of organic solvents. The pH of phase A was 1.5–2.

5. Take the telpoeptide fraction with a purity of over 99% obtained from the second purification, adjust the volume percentage of organic solvent in the telpoeptide fraction to be less than 15% and the pH value to 3-4, let it stand to crystallize, centrifuge, add sodium hydroxide solution to dissolve the obtained solid, freeze dry it to obtain pure telpoeptide.

2. The purification method for telpolide according to claim 1, characterized in that, The ammonia solution is an ammonia solution with a volume percentage of 0.5% to 1.0%.

3. The purification method for telpolide according to claim 1, characterized in that, In the first purified phase A, Volatile salts include one or more of trimethylammonium acetate, ammonium acetate, or ammonium bicarbonate; and / or The concentration of volatile salts is 4.0~8.0 g / L.

4. The purification method for telpolide according to claim 1, characterized in that, In the second purified phase A, Volatile acids include one or more of 2-chlorobutyric acid, trifluoroacetic acid, or formic acid; and / or The volatile acid content is 0.1% to 0.5% by mass.

5. The purification method for telpolide according to claim 1, characterized in that, The first purified phase B is acetonitrile and isopropanol, with the concentration of acetonitrile and isopropanol increasing from 40% to 50% by volume; and / or The second purified phase B is acetonitrile, and the acetonitrile concentration increases from 42% to 52% by volume percentage.

6. The purification method for telpolide according to claim 5, characterized in that, The first purified phase B organic solvent is acetonitrile and isopropanol, with a volume ratio of 4:1 to 2:

1.

7. The purification method for telpolide according to claim 1, characterized in that, After standing and crystallizing, add 2% to 7% of the crude telpopeptide seed crystals to the distillate.

8. The purification method for telpolide according to claim 1, characterized in that, The amount of sodium hydroxide added is 1.0% to 1.5% of the total mass of telpoeptide.

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