Facile spectrophotometric assay of molar equivalents of N-hydroxysuccinimide esters of monomethoxyl poly-(ethylene glycol) derivatives
- Ang Gao†1,
- Xiaolan Yang†1,
- Chun Zhang1,
- Gaobo Long1,
- Jun Pu1,
- Yonghua Yuan1,
- Hongbo Liu1,
- Yuanli Li1 and
- Fei Liao1Email author
© Gao et al; licensee Chemistry Central Ltd. 2012
Received: 17 August 2012
Accepted: 25 October 2012
Published: 23 November 2012
A new method is developed to quantify molar equivalents of N-hydroxysuccinimide (NHS) esters of derivatives of monomethoxyl poly-(ethylene glycol) (mPEG) in their preparations with NHS acetate ester as the reference.
NHS ester of succinic monoester or carbonate of mPEG of 5,000 Da was synthesized and reacted with excessive ethanolamine in dimethylformamide at 25°C for 15 min. Residual ethanolamine was subsequently quantified by absorbance at 420 nm after reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS) at pH 9.2 for 15 min at 55°C followed by cooling with tap water. Reaction products of ethanolamine and NHS esters of mPEG caused no interference with TNBS assay of residual ethanolamine. Reaction between ethanolamine and NHS acetate ester follows 1:1 stoichiometry. By the new method, molar equivalents of NHS esters of carbonate and succinic monoester of mPEG in their preparations were about 90% and 60% of their theoretical values, respectively. During storage at 37°C in humid air, the new method detected spontaneous hydrolyses of the two NHS esters of mPEG more sensitively than the classical spectrophotometric method based on absorbance at 260 nm of NHS released by reaction with ammonia in aqueous solution.
The new method is favorable to quantify molar equivalents of NHS esters of mPEG derivatives and thus control quality of their preparations.
KeywordsN-hydroxysuccinimide esters Monomethyl ether of poly-(ethylene glycol) Ethanolamine 2,4,6-trinitrobenzenesulfonic acid Molar equivalent
Monomethyl ether of poly-(ethylene glycol) (mPEG) is a pivotal biomaterial for formulating therapeutic proteins and similar biomolecules [1–6]. The modification of biomolecules with mPEGs is denoted PEGylation, and mPEGs are usually activated for selective PEGylation of amino groups of biomolecules under mild conditions [1, 3, 6, 7]. N-hydroxysuccinimide (NHS) esters of succinic monoester of mPEGs (NHS-SC-mPEG), and of carbonate of mPEGs (NHS-CB-mPEG), are classical active forms for PEGylating accessible amino groups of biomolecules. For PEGylation, molar ratios of NHS esters of mPEGs to amino groups of biomolecules are primary determinants . NHS esters of mPEGs are either synthesized in laboratories or commercial products; NHS esters of mPEGs and mPEGs are large polymers difficult to purify. More importantly, NHS esters of mPEGs easily undergo spontaneous hydrolyses during storage to yield mPEGs. The use of preparations of NHS esters of mPEGs with low purity for PEGylation of biomolecules brings more unwanted substances in PEGylated products to challenge the subsequent purification process. Hence, molar equivalents of NHS esters of mPEGs rather than mPEG lengths in commercial or laboratory preparations are their critical characteristics , and facile methods are needed for accurate analysis of molar equivalents of NHS esters of mPEGs in such preparations.
Chromatographic methods can validate NHS esters of mPEGs and quantify their molar equivalents [9, 10], but are laborious and require a reference compound for each NHS ester of mPEG that is usually unavailable. Ionized NHS has an absorbance peak at 260 nm; molar equivalents of NHS esters of mPEGs can be quantified based on absorbance of NHS released by reactions of NHS esters of mPEGs with ammonia and/or hydroxide ion in alkaline aqueous solutions [9, 11]. However, NHS esters of mPEGs undergo so fast reactions with ammonia and/or hydroxide ion that pre-existed NHS from spontaneous hydrolysis can not be accurately measured. Additionally, NHS is unstable in alkaline aqueous solutions and should be quantified within 20 min since reaction initiation. The glycyl-glycine test is commonly used to quantify molar equivalents of NHS esters of mPEGs [1, 8, 12, 13]. However, it employs borate buffer at pH 8.0 for the reaction between glycylglycine and NHS esters; some NHS esters like that of mPEG-O-CH2-COOH undergo rapid hydrolyses at pH 8.0 and this glycyl-glycine test can not reliably quantify their molar equivalents. Hence, for controlling quality of preparations of NHS esters of mPEGs, new facile methods are still needed for accurate analysis of molar equivalents of NHS esters of mPEGs in such preparations.
In organic solvents free of water, NHS esters react rapidly and irreversibly with alkyl primary amines to yield amide derivatives and NHS, but basically undergo no spontaneous decomposition [10, 14–17]. When an alkyl primary amine in an organic solvent can be facilely and reliably quantified in the presence of NHS and amide derivatives of mPEGs, the reactions between NHS esters of mPEGs and the alkyl primary amine in the organic solvent can be employed to quantify molar equivalents of NHS esters in preparations based on established stoichiometry of the reactions. The conjugate of 2,4,6-trinitrobenzenesulfonic acid (TNBS) with an alkyl primary amine has a strong absorbance peak around 420 nm and can be utilized for facile assay of the alkyl primary amine [18–20]. Herein, we reported a new facile method for quantifying molar equivalents of NHS esters of mPEG of 5000 Dalton (mPEG5k) in their preparations via reactions with ethanolamine and subsequent spectrophotometric assay of residual ethanolamine with TNBS.
Results and discussion
Selection of a suitable alkyl primary amine and a proper organic solvent
The designed method to quantify molar equivalents of NHS esters of derivatives of mPEGs in preparations requires a suitable alkyl primary amine, a proper organic solvent, an optimized concentration of the alkyl primary amine in the organic solvent and stoichiometry for the reaction between the primary amine and NHS esters of mPEGs. In appearance, any alkyl primary amine, any organic solvent compatible with NHS esters of mPEGs may be applicable. However, the consecutive reaction with TNBS to quantify the residual alkyl primary amine requires alkaline aqueous solutions, in which most alkyl primary amines and some organic solvents are incompatible. This complicated situation requires delicate selection of an organic solvent and an alkyl primary amine. Glycylglycine surely has unfavorable solubility in common organic solvents. Methylamine, ethylamine and propylamine are easily evaporated under room temperature. NHS esters have negligible reactivity with alcohol . Hence, ethanolamine, glycine, ethylenediamine, n-butylamine with reasonable solubility in water were tested as candidate amines, while tetrahydrofurane (THF) and dimethylformamide (DMF) of unlimited solubility in water were compared as candidate organic solvents.
Those alkyl primary amines produced similar absorbance spectra after reaction with TNBS at 25°C for 120 min in alkaline aqueous solutions (Figure 1). To quantify an alkyl primary amine with TNBS, the absorbance at 420 nm was measured, but the conjugates of tested amines and TNBS had two absorption peaks, one around 420 nm while another around 350 nm. The conjugates of ethanolamine and n-butylamine with TNBS all had absorbance at 420 nm no less than that around 350 nm while that of glycine had slightly stronger absorbance at 350 nm. The situation with ethylenediamine is complicated. The conjugate of excessive ethylenediamine with TNBS had stronger absorbance at 420 nm than that at 350 nm, but ethylenediamine can not be quantified in this case. With TNBS in molar excess, both amino groups on ethylenediamine were conjugated with TNBS but the conjugate had stronger absorbance around 350 nm than that at 420 nm. These results made ethylenediamine unfavorable to quantify molar equivalents of NHS esters of mPEGs by the new method. On the other hand, in alkaline aqueous solution at 37°C, ethanolamine, glycine, ethylenediamine and n-butylamine displayed reaction rates with TNBS in a descent order (Figure 2). Solubility in water of ethanolamine or ethylenediamine ranks the first, that of glycine acts as the second while that of n-butylamine is the lowest. Solubility in THF and DMF of ethanolamine, ethylenediamine and n-butylamine is comparable, but that of glycine is too low for this new method to quantify molar equivalents of NHS esters of mPEGs. Hence, ethanolamine may be a suitable alkyl primary amine for the new method.
The reaction mixture of commercial DMF with TNBS produced negligible absorbance at 420 nm. The conjugate of TNBS with any of the tested primary amines has millimolar absorptivity over 10 (mmol/L·cm)-1 (Figure 2) [18–20]. Assuming the measurable absorbance below 1.500, the highest concentration of a suitable alkyl primary amine in DMF should be below 1.7 mmol/L if the reaction mixture is diluted by 21-fold for reaction with TNBS in an alkaline aqueous solution (the addition of just 50 μL DMF solution of ethanolamine and an NHS ester of mPEG as the sample to a total of 0.95 mL borate buffer plus 50 μL 0.4% aqueous solution of TNBS to quantify residual ethanolamine). For measuring absorbance below 1.200 at 420 nm with common spectrophotometers, final 0.83 mmol/L ethanolamine is employed in DMF to react with NHS esters of mPEGs for the new method.
Optimization of reaction conditions of ethanolamine with TNBS
Taken together, the optimized conditions for the new method are preset as follows. The reaction between 0.83 mmol/L ethanolamine in excess and a tested NHS ester of mPEG in DMF is allowed to continue for 15 min at 25°C for complete consumption of the NHS ester (Figure 3). The reaction between residual ethanolamine and TNBS in borate buffer at pH 9.2 continues for 15 min at 55°C followed by cooling with tap water to measure absorbance at 420 nm. These optimized conditions were used throughout, unless otherwise stated.
Preliminary applications of the new method
By TLC analysis, no free NHS was detectable in the two NHS esters of mPEG5k. By the new method, there was a linear decrease in absorbance at 420 nm to quantities of either of the two NHS esters of mPEGs in DMF for reactions with ethanolamine. Based on the response slopes and 1:1 reaction stoichiometry, NHS-CB-mPEG5k and NHS-SC-mPEG5k in their preparations were about 90% and 60% of their theoretical values calculated from their average molecular weights, respectively. By the classical spectrophotometric method based on absorbance at 260 nm of NHS released upon the reaction with ammonia, consistent molar equivalents of these two NHS esters of mPEG5k in their preparations were obtained, correspondingly (data not given). NHS-CB-mPEG5k in its preparation had a molar equivalent close to its theoretical value, supporting its high purity and the reaction stoichiometry of 1:1 between NHS-CB-mPEG5k and ethanolamine. It is a putative that NHS-SC-mPEG has reactivity with ethanolamine comparable to NHS-CB-mPEG5k [21, 22]. The reaction between NHS-SC-mPEG and ethanolamine should be completed under the optimized reaction conditions; the lower molar equivalent of NHS-SC-mPEG in its preparation thus indicated its lower purity. Hence, both the new method and the classical spectrophotometric method are effective to check homogeneity of NHS esters of mPEGs in the absence of pre-existed NHS.
In conclusion, a facile method is developed to quantify molar equivalents of NHS esters of mPEGs in laboratory or commercial preparations via their reactions with ethanolamine in dimethylformamide and subsequent spectrophotometric assay of residual ethanolamine with 2,4,6-trinitrobenzenesulfonic acid. The new method consumes just about 35 min for each analysis, displays resistance to pre-existed NHS, is universally applicable to common active esters as long as they are stable in DMF, and requires just one easily-accessible reference compound for different NHS esters. The glycylglycine test of active esters requires over 60 min for each analysis and is unreliable to active esters susceptible to hydrolysis [1, 8, 13]. The classical spectrophotometric method based on NHS absorbance at 260 nm is susceptible to the interference of pre-existed NHS originated from partial decomposition/hydrolysis of NHS esters. Taken together, the new method for controlling quality of commercial or laboratory preparations of active esters of mPEG derivatives and optimizing PEGylation process of therapeutic proteins is advantageous over other conventional methods.
Materials and methods
Chemicals and instruments
5% aqueous solution of TNBS and mPEG5k were from Sigma-Aldrich. NHS, triphosgene, glycine, n-butylamine, dicyclohexanylcarbodiimide (DCC), ethanolamine, ethylenediamine hydrochloride, and organic solvents were domestic reagents of analytical grade and used as received. Shimadzu UV 2550 and Mapada UV 1600 PC spectrophotometers were used.
Syntheses of NHS active esters
Reactions of NHS esters with primary amines and quantification with TNBS
Each NHS ester was reacted with an indicated alkyl primary amine in THF or DMF at room temperature for a stated period [11, 14–17]. Then, 50 μL reaction mixture was withdrawn and mixed with 0.95 mL sodium borate buffer (200 mmol/L at pH 9.2); to this alkaline solution in 1.0 mL, a total of 50 μL TNBS aqueous solution (0.4%) was added and mixed by vortex. The mixture was kept at room temperature (25°C) or an indicated temperature for a stated period. Finally, absorbance of reaction mixture with TNBS at 420 nm (after cooling to room temperature) was measured using reagent blank to correct background absorbance [18–20]. NHS-AC was used as the reference owing to its high purity.
Quantification of NHS esters based on NHS absorbance
An aliquot of THF solution of each NHS ester was transferred into a small bottle, dried in vacuum at 25°C and was then sealed. The effects of two conditions on storage stability were tested. Firstly, samples were kept at 37°C in sealed bottles. Secondly, samples were kept at 37°C in opened bottles in a closed cell culture incubator with saturated humidity. A bottle of powder sample was withdrawn at indicated time for analysis.
Data were determined at least in triplicate and represented as mean ± standard deviation (SD). Coefficients of variation (CV) were below 10% unless otherwise stated. Student’s t-test was used for comparison.
Monomethoxyl poly-(ethylene glycol)
NHS acetate ester
NHS esters of succinic monoester of mPEGs
NHS esters of carbonate of mPEGs
This project was supported by Natural Science Foundation Project of CQ (CSTC2011BA5039), National Natural Science Foundation of China (no. 30672009), Program for New Century Excellent Talent in University (NCET-09-0926), and “863”-High-Technology Program of China (No. 2011AA02A108). Dr. Yonghua Yuan presently works in Children’s Hospital, Chongqing Medical University.
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