Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody Dávid Suba a,∗ , Zoltán Urbányi a , András Salgó b a b
Chemical Works of Gedeon Richter Plc, Gyömroi ˝ út 19.–21., 1103 Budapest, Hungary Budapest University of Technology and Economics, Muegyetem rakpart 3., 1111 Budapest, Hungary ˝
a r t i c l e
i n f o
Article history: Received 12 February 2015 Received in revised form 21 April 2015 Accepted 24 April 2015 Available online 6 May 2015 Keywords: Capillary electrophoresis Isoelectric focusing Monoclonal antibody Method development Validation
a b s t r a c t Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some “out-of-the-box” applications commercially available which provide easy and rapid isoelectric focusing solutions for investigating monoclonal antibody drug proteins. However use of these kits in routine testings requires high costs. A capillary isoelectric focusing method was developed and validated for identification testing of monoclonal antibody drug products with isoelectric point between 7.0 and 9.0. A method was developed providing good pH gradient for internal calibration (R2 > 0.99) and good resolution between all of the isoform peaks (R = 2), minimizing the time and complexity of sample preparation (no urea or salt used). The method is highly reproducible and it is suitable for validation and method transfer to any QC laboratories. Another advantage of the method is that it operates with commercially available chemicals which can be purchased from any suppliers. The interaction with capillary walls (avoid precipitation and adsorption as far as possible) was minimized and synthetic isoelectric small molecular markers were used instead of peptide or protein based markers. The developed method was validated according to the recent ICH guideline (Q2(R1)). Relative standard deviation results were below 0.2% for isoelectric points and below 4% according to the normalized migration times. The method is robust to buffer components with different lot numbers and neutral capillaries with different type of inner coatings. The fluoro-carbon coated column was chosen because of costs-effectivity aspects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The emerging market of biosimilar monoclonal antibodies (MABs) generated need for more and more accurate determination of similarity of biosimilar candidates to the originator’s products [1]. Characterization of a monoclonal antibody is a very difficult task in every analytical aspect, due to the various post-translational modifications such as glycosylation, phosphorylation or lysine clipping. During the whole process and storage the protein drug molecules may suffer from many physico-chemical stresses, resulting in the formation of charge variants like oxidation of tryptophan or deamidation of asparagine [2]. All of these modifications (both biological and chemical) contribute to the full charge profile of
∗ Corresponding author. Tel.: +36 15057457. E-mail addresses: [email protected], suba [email protected] (D. Suba). http://dx.doi.org/10.1016/j.jpba.2015.04.037 0731-7085/© 2015 Elsevier B.V. All rights reserved.
the protein. Some of these charge variants may have possible effect on biological activity of the molecule. Glycosylation profile of monoclonal antibody highly influences the biological activity of the protein [3,4]. For example, there are several evidences of the negative effect of the acidic charge variants on the antigen-binding capability of monoclonal antibody [5]. Monitoring charge variants and product related impurities during storage is essential from the point of view of safety and activity [6], consequently it is one of the most important analytical aspect of biosimilar development. The most advanced techniques such as liquid chromatography coupled to tandem mass spectrometry [7,8] are powerful tools for characterization of biological drug products, but are difficult to implement into the routine analysis and regulated quality control. Capillary isoelectric focusing (cIEF) is a powerful tool to characterize charge profile of the monoclonal antibodies, because different charge variants can be distinguished according to their isoelectric points using internal calibration [9]. As cIEF provides
54
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
very high reproducibility and specificity, this is one of the most popular tools for monoclonal antibody analysis. There are some different approaches of isoelectric focusing in capillaries [10–12,19]. The basis of the methods is the same: the separation according to the pI takes place in pH gradient formed by carrier ampholytes in silica capillary (coated or uncoated). The separations can be performed in two steps consecutive of the focusing and the separation or in one step only [12]. According to the conventional approach the peaks can be detected in “continuous” mode through a capillary window by UV detector, or by on-capillary-detected solutions called imaged [13,14] capillary isoelectric focusing. These later techniques operate just with the focusing step and no mobilization is required providing fast separations. Other solution needs special instrumentation [15,16], but cannot be implemented into routine analysis. In this study we intended to adjust a high resolution and highly reproducible capillary isoelectric focusing application for identification testing of monoclonal antibody with isoelectric points between 7.4 and 8.0. Moreover our aim was to make this method capable of analysing different MABs within pI range 7–9. 2. Materials and methods 2.1. Materials Hydroxypropyl methylcellulose (HPMC), iminodiacetic acid (IDA), arginine (ARG), Pharmalytes (pH 5–8 and pH 3–10), phosphoric acid, sodium hydroxide, acetic acid, Tris buffer, carboxypeptidase-B (CPB) enzyme and UV–visible synthetic cIEF markers were purchased from Sigma–Aldrich (St Louis, MO, USA). Commercially available monoclonal antibodies were purchased from Hoffmann La Roche (Basel, Switzerland) and Janssen Biotechnology Inc. (Horsham, PA, USA). 2.2. Instrumentation All measurements were performed on Beckman Coulter (Hercules, CA, USA) PA800 and PA800 Plus capillary electrophoresis systems. 50 m inner-diameter fluorocarbon coated capillaries (Sil-FC) were purchased from Agilent Technologies (Santa Clara, CA, USA) and 50 m inner-diameter polyacrylamide (PAAM) coated capillaries were purchased from Beckman Coulter and Sepax Technologies (Newark, DE, USA). The polyimide coating was burnt on both ends of the capillaries by Microsolv CE Window Maker apparatus (Microsolv, USA Monmouth). 2.3. Buffers and solutions 40 mM phosphoric acid solution was used as anode buffer, 80 mM sodium hydroxide solution was used as cathode buffer and 100 mM acetic acid was used as mobilizer and rinse solution. 1% (m/v) HPMC solution was prepared as separation gel buffer, 500 mM arginine solution as anodic stabilizer and 20 mM iminodiacetic acid as cathodic stabilizer according to Mack et al. [17]. The formulated MAB solution was diluted with capillary electrophoresis (CE) grade purified water to 0.5 mg/ml concentration to minimize any interference with salts. 2.4. Sample solution The investigated sample solution contained 46% (v/v) HPMC solution, 41% (v/v) 0.5 mg/ml MAB solution, 3.2% (v/v) mixture of Pharmalyte 3–10 and 5–8 (1:5), 5.5% (v/v) arginine solution, 0.9% (v/v) iminodiacetic acid solution and 0.9% (v/v) of each pI markers. In the case of blank and system suitability solution 41% (v/v) purified water was used instead of the 0.5 mg/ml MAB solution. The
same sample solution setup was applied for all of the samples used for specificity measurements. 2.5. General setup The basic setup, which was not modified during method development, was the following: the amount of the cathodic and anodic stabilizers (arginine and iminodiacetic acid), the percentage of HPMC in the solution and the amount of pI markers in each case were the same described in Section 2. The injection was performed during 60 s with 20 psi hydrodinamical pressure, the focusing step for 10 min and the mobilization step for 20 min were used. The chemical mobilization was done by changing catholyte buffer to 100 mM acetic acid. 20 ◦ C capillary temperature was used according to Cao et al. [9] and the samples were stored at 10 ◦ C. 2.6. Marker selection Accurate pI determination is based on the pI markers with well-defined isoelectric points. However, most of the recent cIEF methods for therapeutic proteins used peptide-based isoelectric markers [9,17]. Synthetic pI markers were chosen in our experiments, due to their wide pI range, good UV absorbance, easy accessibility from the market and minimal interaction with the capillary walls. During development three synthetic markers were chosen with different pI below and over the pI of the analyte indicating the goodness of the pH gradient by the R2 coefficient of the migration time–pI function. As the analyte charge distribution is between 7.4 and 8.0 markers were selected from Sigma with pI 7.0, 8.4 and 9.0. 2.7. Anolyte, catholyte and chemical mobilization buffer 40 mM phosphoric acid was used as anolyte, 80 mM sodiumhydroxide as catholyte and 100 mM acetic acid as chemical mobilizer. No difference was detected between the different concentrations (data not shown). Fresh buffers were used after every third injections to avoid contamination and dilution of electrolytes. 3. Results and discussion 3.1. Method development Our aim was to achieve the maximum resolution combined with as short migration time as possible. The setups with shorter migration times were chosen. In this study a commercially available human IgG1 type monoclonal antibody with five main isoforms was chosen as a model protein for the investigations. As indicator of the optimal Pharmalyte ratio the goodness of migration time–isoelectric point plot was chosen. The regression coefficient of the plot (R2 ) was investigated and 0.99 as minimum value was determined (data not shown). Runs with R2 value lower than minimum were rejected. According to Refs. [9,17,18] the following critical parameters were chosen for method development: focusing and mobilization voltage, amount of salt and urea in the sample. In every case 10 min focusing voltage was used according to Cao et al. [9] to achieve short separation. Equal resolutions between all the peaks were aimed during development. Resolutions between peaks were determined by Eq. (1): R=
tm(N+1) − tm(N) pI(N) − pI(N+1)
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
55
Fig. 1. Electropherograms obtained by using different Pharmalyte ratios of Pharmalyte 3–10 and 5–8. (a) Pharmalyte 3–10; (b) Pharmalyte 5–8; (c) Pharmalyte 3–10:5–8:4:2. A, B and C are the synthetic pI markers 9.0, 8.4 and 7.0. 1–5 are the sample peaks belonging to the different isoforms of the investigated monoclonal antibody MAB A. Focusing for 10 min at 30 kV, mobilization for 20 min at 30 kV with 100 mM acetic acid. * indicates peak splicing.
where tm is the migration time (min) of the sample peak, pI is the isoelectric point of the sample peak and N is the peak order. The migration time differences between the neighbouring peaks were divided with their pI difference. This resolution numbers (R)
simply show the best separation performance of the method for each peak pairs. The proper conditioning of the separation capillary is the key of the reproducibility. Different cleaning strategies for PAAM coated
56
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
Table 1 Summary of results obtained during development for the different parameter setups. Parameter adjusted
1 2 3 4 5 6 7
Pharmalyte 3–10 Pharmalyte 5–8 Focusing voltage 20 kV Focusing voltage 25 kV 25 mM Tris 4 M Urea Final setup
R (resolution)
R2
Migration time (min)
Peak 1 and 2
Peak 2 and 3
Peak 3 and 4
Peak 4 and 5
Marker 1
Marker 2
Marker 3
2.0 2.8 1.8 2.3 1.5 2.4 2.0
1.9 2.9 1.8 2.3 1.6 2.4 2.0
1.9 2.9 1.8 2.3 1.6 2.4 2.0
2.0 2.9 1.8 2.3 1.5 1.4 2.0
17.70 17.68 16.47 17.68 18.18 24.34 17.38
19.51 18.85 18.33 19.65 19.14 25.93 18.68
21.65 22.68 20.88 22.28 21.28 29.20 21.33
0.97 0.99 0.98 0.98 0.99 0.99 0.99
Fig. 2. Effect of focusing voltage on the isoelectric profile and the goodness of the pH gradient. (a) 20 kV focusing voltage; (b) 25 kV focusing voltage; (c) 30 kV focusing voltage. A, B and C are the synthetic pI markers 9.0, 8.4 and 7.0. 1–5 are the sample peaks belonging to the different isoforms of the investigated monoclonal antibody MAB A. Focusing for 10 min and mobilization for 20 min at 30 kV with 100 mM acetic acid. * indicates peak splicing.
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
57
Fig. 3. Effect of salt on the electrophoretic profiles. (a) 25 mM Tris; (b) 4 M urea; (c) no Tris or urea was added. A, B and C are the synthetic pI markers 9.0, 8.4 and 7.0. 1–5 are the sample peaks belonging to the different isoforms of the investigated monoclonal antibody MAB A. Focusing for 10 min at 30 kV and mobilization for 20 min at 30 kV with 100 mM acetic acid. * indicates peak splicing.
capillaries were described recently [22]. We used acetic acid and purified water only to clean the FC-coated capillary. All the performance characteristics tests were performed without any extra conditioning program described below. Pre-run and post-run cleaning programs of measuring methods were built and performed during every run. The following cleaning sequence was used: prerun conditioning with purified water rinse at 50 psi for 3 min followed by a 3 min rinse with 100 mM acetic acid at 50 psi and a 6 min long purified water rinse at 70 psi, and post-run conditioning with three-steps purified water rinse at 20, 50 and 70 psi for 3, 3 and 6 min sequentially. 3.2. Pharmalyte ratio Amphoteric electrolytes form the pH gradient in the capillary, so they determine the resolution and selectivity of the method. In every case Pharmalytes were used in 3 v/v% total of the sample solution. To reach the maximum resolution the optimal Pharmalyte ratio was investigated. Pharmalytes with pH from 5 to 8 and with pH from 3 to 10 were tested. Initially the Pharmalyte 3–10 was investigated (Fig. 1) and acceptable peak shapes but poor resolution (Table 1 line 1) and gradient (R2 = 0.97) were observed. In the case of Pharmalyte 5–8 the resolution and the goodness of the pH gradient (R2 = 0.99) were acceptable, but there was a peak-slicing at peak 3 (Fig. 1b, Table 1 line 2). Finally the mixture of Pharmalyte 3–10
and 5–8 in the ratio of 4:2 proved to be optimal. Good resolution (Table 1 line 7), and gradient were obtained (R2 = 0.99) (Fig. 1c).
3.3. Effect of focusing voltage Prior to determine the effect of focusing voltage, 20, 25 and 30 kV were investigated. Results are shown in Fig. 2(a–c). Poor pH gradient (R2 = 0.98), resolution (Table 1 line 3) and peak slicing-up of peak 3 were obtained during mobilization at 20 kV focusing. Focusing with 25 kV and at 30 kV produced similar isoelectric profile. The 25 kV focusing setup provided better resolution (Table 1 line 4), but the best pH gradient (R2 = 0.99) belonged to the 30 kV focusing voltage.
3.4. Effect of mobilization voltage The effect of mobilization voltage on the resolution and migration time was investigated. Our aim was to minimize migration time as far as possible. 20, 25 and 30 kV mobilization voltages were applied. Almost the same profile and pH gradient were obtained in each case (data not shown). The set up with 30 kV mobilization voltage produced the shortest migration time, consequently this was chosen for the final method setup.
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D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
Fig. 4. (a) Graphical interpretation of intermediate precision data: isoelectric points of the five isoforms of MAB A. (b) Graphical interpretation of intermediate precision data: normalized migration times of the five isoforms of MAB A.
3.5. Salt and urea content of the sample
4. Validation procedure
Mack et al. [17] describe the positive effect of salt and urea concentration on the resolution. 50 mM Tris solution was used to dilute the sample to 0.5 mg/ml, the final salt concentration was approximately 25 mM (Fig. 3a). The salt resulted in good pH gradient (R2 = 0.99), but produced instability: slicing in Peak 3 and poor resolution between all of the peaks (Table 1 line 5). Urea is widely used for isoelectric focusing to keep protein in solution [19]. For testing the effect of urea a solution of 8 M urea was used to dilute sample to 0.5 mg/ml concentration. The urea concentration was about 4 M in the final solution. Fig. 3b shows that the goodness of the pH gradient is acceptable, but the peaks migrate late and the resolution is better except between peaks 4 and 5 (Table 1 line 6), than those in the case of using Tris in the sample mixture (Fig. 3a). The best setup was in case of using no salt or urea in the solution (Table 1 line 7).
The aim of an analytical validation is to prove that the applied analytical method is suitable for the purpose of use (e.g. identification tests). According to the Guideline [18] specificity should be investigated during validation of an identification method. Additionally the intermediate precision of the developed method was investigated.
3.6. The optimised method In former methods [9,17] 25 kV focusing voltage was applied; in our study 30 kV focusing voltage was found to be optimal. The same 30 kV mobilization voltage was applied in our final method, but no urea or salt was added to the sample mixture. The other parameters are the same described as general setup in the beginning of the method development session. This optimised method proved to be ready for validation. In this method the resolutions between all of the peaks were equal: short migration time and good pH gradient were obtained (Table 1 line 7).
4.1. Method performances 4.1.1. System suitability Investigating system suitability is inevitable in every case for each single run. System suitability was determined by the R2 value of the migration time–isoelectric point function in the case of the sample and reference solutions.
4.1.2. Intermediate precision Intermediate precision tests were performed to investigate the effects of random errors on the precision of the developed analytical method. This procedure was carried out on two different instruments with two different operators on three different days. We applied six independent sample preparations with single injections on each day. All of the 18 runs were evaluated, the mean pI values of the five main peaks were determined and the relative standard deviations were calculated. Graphical interpretation of the standard deviation of the isoelectric points belonging to Peaks 1–5 can be seen in Fig. 4. The relative standard deviation for all of the peaks was below 0.2%, the highest one (0.173) belongs to Peak 3. Variability of migration times of the pI markers was also investigated. Good standard deviations (
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody Dávid Suba a,∗ , Zoltán Urbányi a , András Salgó b a b
Chemical Works of Gedeon Richter Plc, Gyömroi ˝ út 19.–21., 1103 Budapest, Hungary Budapest University of Technology and Economics, Muegyetem rakpart 3., 1111 Budapest, Hungary ˝
a r t i c l e
i n f o
Article history: Received 12 February 2015 Received in revised form 21 April 2015 Accepted 24 April 2015 Available online 6 May 2015 Keywords: Capillary electrophoresis Isoelectric focusing Monoclonal antibody Method development Validation
a b s t r a c t Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some “out-of-the-box” applications commercially available which provide easy and rapid isoelectric focusing solutions for investigating monoclonal antibody drug proteins. However use of these kits in routine testings requires high costs. A capillary isoelectric focusing method was developed and validated for identification testing of monoclonal antibody drug products with isoelectric point between 7.0 and 9.0. A method was developed providing good pH gradient for internal calibration (R2 > 0.99) and good resolution between all of the isoform peaks (R = 2), minimizing the time and complexity of sample preparation (no urea or salt used). The method is highly reproducible and it is suitable for validation and method transfer to any QC laboratories. Another advantage of the method is that it operates with commercially available chemicals which can be purchased from any suppliers. The interaction with capillary walls (avoid precipitation and adsorption as far as possible) was minimized and synthetic isoelectric small molecular markers were used instead of peptide or protein based markers. The developed method was validated according to the recent ICH guideline (Q2(R1)). Relative standard deviation results were below 0.2% for isoelectric points and below 4% according to the normalized migration times. The method is robust to buffer components with different lot numbers and neutral capillaries with different type of inner coatings. The fluoro-carbon coated column was chosen because of costs-effectivity aspects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The emerging market of biosimilar monoclonal antibodies (MABs) generated need for more and more accurate determination of similarity of biosimilar candidates to the originator’s products [1]. Characterization of a monoclonal antibody is a very difficult task in every analytical aspect, due to the various post-translational modifications such as glycosylation, phosphorylation or lysine clipping. During the whole process and storage the protein drug molecules may suffer from many physico-chemical stresses, resulting in the formation of charge variants like oxidation of tryptophan or deamidation of asparagine [2]. All of these modifications (both biological and chemical) contribute to the full charge profile of
∗ Corresponding author. Tel.: +36 15057457. E-mail addresses: [email protected], suba [email protected] (D. Suba). http://dx.doi.org/10.1016/j.jpba.2015.04.037 0731-7085/© 2015 Elsevier B.V. All rights reserved.
the protein. Some of these charge variants may have possible effect on biological activity of the molecule. Glycosylation profile of monoclonal antibody highly influences the biological activity of the protein [3,4]. For example, there are several evidences of the negative effect of the acidic charge variants on the antigen-binding capability of monoclonal antibody [5]. Monitoring charge variants and product related impurities during storage is essential from the point of view of safety and activity [6], consequently it is one of the most important analytical aspect of biosimilar development. The most advanced techniques such as liquid chromatography coupled to tandem mass spectrometry [7,8] are powerful tools for characterization of biological drug products, but are difficult to implement into the routine analysis and regulated quality control. Capillary isoelectric focusing (cIEF) is a powerful tool to characterize charge profile of the monoclonal antibodies, because different charge variants can be distinguished according to their isoelectric points using internal calibration [9]. As cIEF provides
54
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
very high reproducibility and specificity, this is one of the most popular tools for monoclonal antibody analysis. There are some different approaches of isoelectric focusing in capillaries [10–12,19]. The basis of the methods is the same: the separation according to the pI takes place in pH gradient formed by carrier ampholytes in silica capillary (coated or uncoated). The separations can be performed in two steps consecutive of the focusing and the separation or in one step only [12]. According to the conventional approach the peaks can be detected in “continuous” mode through a capillary window by UV detector, or by on-capillary-detected solutions called imaged [13,14] capillary isoelectric focusing. These later techniques operate just with the focusing step and no mobilization is required providing fast separations. Other solution needs special instrumentation [15,16], but cannot be implemented into routine analysis. In this study we intended to adjust a high resolution and highly reproducible capillary isoelectric focusing application for identification testing of monoclonal antibody with isoelectric points between 7.4 and 8.0. Moreover our aim was to make this method capable of analysing different MABs within pI range 7–9. 2. Materials and methods 2.1. Materials Hydroxypropyl methylcellulose (HPMC), iminodiacetic acid (IDA), arginine (ARG), Pharmalytes (pH 5–8 and pH 3–10), phosphoric acid, sodium hydroxide, acetic acid, Tris buffer, carboxypeptidase-B (CPB) enzyme and UV–visible synthetic cIEF markers were purchased from Sigma–Aldrich (St Louis, MO, USA). Commercially available monoclonal antibodies were purchased from Hoffmann La Roche (Basel, Switzerland) and Janssen Biotechnology Inc. (Horsham, PA, USA). 2.2. Instrumentation All measurements were performed on Beckman Coulter (Hercules, CA, USA) PA800 and PA800 Plus capillary electrophoresis systems. 50 m inner-diameter fluorocarbon coated capillaries (Sil-FC) were purchased from Agilent Technologies (Santa Clara, CA, USA) and 50 m inner-diameter polyacrylamide (PAAM) coated capillaries were purchased from Beckman Coulter and Sepax Technologies (Newark, DE, USA). The polyimide coating was burnt on both ends of the capillaries by Microsolv CE Window Maker apparatus (Microsolv, USA Monmouth). 2.3. Buffers and solutions 40 mM phosphoric acid solution was used as anode buffer, 80 mM sodium hydroxide solution was used as cathode buffer and 100 mM acetic acid was used as mobilizer and rinse solution. 1% (m/v) HPMC solution was prepared as separation gel buffer, 500 mM arginine solution as anodic stabilizer and 20 mM iminodiacetic acid as cathodic stabilizer according to Mack et al. [17]. The formulated MAB solution was diluted with capillary electrophoresis (CE) grade purified water to 0.5 mg/ml concentration to minimize any interference with salts. 2.4. Sample solution The investigated sample solution contained 46% (v/v) HPMC solution, 41% (v/v) 0.5 mg/ml MAB solution, 3.2% (v/v) mixture of Pharmalyte 3–10 and 5–8 (1:5), 5.5% (v/v) arginine solution, 0.9% (v/v) iminodiacetic acid solution and 0.9% (v/v) of each pI markers. In the case of blank and system suitability solution 41% (v/v) purified water was used instead of the 0.5 mg/ml MAB solution. The
same sample solution setup was applied for all of the samples used for specificity measurements. 2.5. General setup The basic setup, which was not modified during method development, was the following: the amount of the cathodic and anodic stabilizers (arginine and iminodiacetic acid), the percentage of HPMC in the solution and the amount of pI markers in each case were the same described in Section 2. The injection was performed during 60 s with 20 psi hydrodinamical pressure, the focusing step for 10 min and the mobilization step for 20 min were used. The chemical mobilization was done by changing catholyte buffer to 100 mM acetic acid. 20 ◦ C capillary temperature was used according to Cao et al. [9] and the samples were stored at 10 ◦ C. 2.6. Marker selection Accurate pI determination is based on the pI markers with well-defined isoelectric points. However, most of the recent cIEF methods for therapeutic proteins used peptide-based isoelectric markers [9,17]. Synthetic pI markers were chosen in our experiments, due to their wide pI range, good UV absorbance, easy accessibility from the market and minimal interaction with the capillary walls. During development three synthetic markers were chosen with different pI below and over the pI of the analyte indicating the goodness of the pH gradient by the R2 coefficient of the migration time–pI function. As the analyte charge distribution is between 7.4 and 8.0 markers were selected from Sigma with pI 7.0, 8.4 and 9.0. 2.7. Anolyte, catholyte and chemical mobilization buffer 40 mM phosphoric acid was used as anolyte, 80 mM sodiumhydroxide as catholyte and 100 mM acetic acid as chemical mobilizer. No difference was detected between the different concentrations (data not shown). Fresh buffers were used after every third injections to avoid contamination and dilution of electrolytes. 3. Results and discussion 3.1. Method development Our aim was to achieve the maximum resolution combined with as short migration time as possible. The setups with shorter migration times were chosen. In this study a commercially available human IgG1 type monoclonal antibody with five main isoforms was chosen as a model protein for the investigations. As indicator of the optimal Pharmalyte ratio the goodness of migration time–isoelectric point plot was chosen. The regression coefficient of the plot (R2 ) was investigated and 0.99 as minimum value was determined (data not shown). Runs with R2 value lower than minimum were rejected. According to Refs. [9,17,18] the following critical parameters were chosen for method development: focusing and mobilization voltage, amount of salt and urea in the sample. In every case 10 min focusing voltage was used according to Cao et al. [9] to achieve short separation. Equal resolutions between all the peaks were aimed during development. Resolutions between peaks were determined by Eq. (1): R=
tm(N+1) − tm(N) pI(N) − pI(N+1)
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
55
Fig. 1. Electropherograms obtained by using different Pharmalyte ratios of Pharmalyte 3–10 and 5–8. (a) Pharmalyte 3–10; (b) Pharmalyte 5–8; (c) Pharmalyte 3–10:5–8:4:2. A, B and C are the synthetic pI markers 9.0, 8.4 and 7.0. 1–5 are the sample peaks belonging to the different isoforms of the investigated monoclonal antibody MAB A. Focusing for 10 min at 30 kV, mobilization for 20 min at 30 kV with 100 mM acetic acid. * indicates peak splicing.
where tm is the migration time (min) of the sample peak, pI is the isoelectric point of the sample peak and N is the peak order. The migration time differences between the neighbouring peaks were divided with their pI difference. This resolution numbers (R)
simply show the best separation performance of the method for each peak pairs. The proper conditioning of the separation capillary is the key of the reproducibility. Different cleaning strategies for PAAM coated
56
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
Table 1 Summary of results obtained during development for the different parameter setups. Parameter adjusted
1 2 3 4 5 6 7
Pharmalyte 3–10 Pharmalyte 5–8 Focusing voltage 20 kV Focusing voltage 25 kV 25 mM Tris 4 M Urea Final setup
R (resolution)
R2
Migration time (min)
Peak 1 and 2
Peak 2 and 3
Peak 3 and 4
Peak 4 and 5
Marker 1
Marker 2
Marker 3
2.0 2.8 1.8 2.3 1.5 2.4 2.0
1.9 2.9 1.8 2.3 1.6 2.4 2.0
1.9 2.9 1.8 2.3 1.6 2.4 2.0
2.0 2.9 1.8 2.3 1.5 1.4 2.0
17.70 17.68 16.47 17.68 18.18 24.34 17.38
19.51 18.85 18.33 19.65 19.14 25.93 18.68
21.65 22.68 20.88 22.28 21.28 29.20 21.33
0.97 0.99 0.98 0.98 0.99 0.99 0.99
Fig. 2. Effect of focusing voltage on the isoelectric profile and the goodness of the pH gradient. (a) 20 kV focusing voltage; (b) 25 kV focusing voltage; (c) 30 kV focusing voltage. A, B and C are the synthetic pI markers 9.0, 8.4 and 7.0. 1–5 are the sample peaks belonging to the different isoforms of the investigated monoclonal antibody MAB A. Focusing for 10 min and mobilization for 20 min at 30 kV with 100 mM acetic acid. * indicates peak splicing.
D. Suba et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 53–61
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Fig. 3. Effect of salt on the electrophoretic profiles. (a) 25 mM Tris; (b) 4 M urea; (c) no Tris or urea was added. A, B and C are the synthetic pI markers 9.0, 8.4 and 7.0. 1–5 are the sample peaks belonging to the different isoforms of the investigated monoclonal antibody MAB A. Focusing for 10 min at 30 kV and mobilization for 20 min at 30 kV with 100 mM acetic acid. * indicates peak splicing.
capillaries were described recently [22]. We used acetic acid and purified water only to clean the FC-coated capillary. All the performance characteristics tests were performed without any extra conditioning program described below. Pre-run and post-run cleaning programs of measuring methods were built and performed during every run. The following cleaning sequence was used: prerun conditioning with purified water rinse at 50 psi for 3 min followed by a 3 min rinse with 100 mM acetic acid at 50 psi and a 6 min long purified water rinse at 70 psi, and post-run conditioning with three-steps purified water rinse at 20, 50 and 70 psi for 3, 3 and 6 min sequentially. 3.2. Pharmalyte ratio Amphoteric electrolytes form the pH gradient in the capillary, so they determine the resolution and selectivity of the method. In every case Pharmalytes were used in 3 v/v% total of the sample solution. To reach the maximum resolution the optimal Pharmalyte ratio was investigated. Pharmalytes with pH from 5 to 8 and with pH from 3 to 10 were tested. Initially the Pharmalyte 3–10 was investigated (Fig. 1) and acceptable peak shapes but poor resolution (Table 1 line 1) and gradient (R2 = 0.97) were observed. In the case of Pharmalyte 5–8 the resolution and the goodness of the pH gradient (R2 = 0.99) were acceptable, but there was a peak-slicing at peak 3 (Fig. 1b, Table 1 line 2). Finally the mixture of Pharmalyte 3–10
and 5–8 in the ratio of 4:2 proved to be optimal. Good resolution (Table 1 line 7), and gradient were obtained (R2 = 0.99) (Fig. 1c).
3.3. Effect of focusing voltage Prior to determine the effect of focusing voltage, 20, 25 and 30 kV were investigated. Results are shown in Fig. 2(a–c). Poor pH gradient (R2 = 0.98), resolution (Table 1 line 3) and peak slicing-up of peak 3 were obtained during mobilization at 20 kV focusing. Focusing with 25 kV and at 30 kV produced similar isoelectric profile. The 25 kV focusing setup provided better resolution (Table 1 line 4), but the best pH gradient (R2 = 0.99) belonged to the 30 kV focusing voltage.
3.4. Effect of mobilization voltage The effect of mobilization voltage on the resolution and migration time was investigated. Our aim was to minimize migration time as far as possible. 20, 25 and 30 kV mobilization voltages were applied. Almost the same profile and pH gradient were obtained in each case (data not shown). The set up with 30 kV mobilization voltage produced the shortest migration time, consequently this was chosen for the final method setup.
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Fig. 4. (a) Graphical interpretation of intermediate precision data: isoelectric points of the five isoforms of MAB A. (b) Graphical interpretation of intermediate precision data: normalized migration times of the five isoforms of MAB A.
3.5. Salt and urea content of the sample
4. Validation procedure
Mack et al. [17] describe the positive effect of salt and urea concentration on the resolution. 50 mM Tris solution was used to dilute the sample to 0.5 mg/ml, the final salt concentration was approximately 25 mM (Fig. 3a). The salt resulted in good pH gradient (R2 = 0.99), but produced instability: slicing in Peak 3 and poor resolution between all of the peaks (Table 1 line 5). Urea is widely used for isoelectric focusing to keep protein in solution [19]. For testing the effect of urea a solution of 8 M urea was used to dilute sample to 0.5 mg/ml concentration. The urea concentration was about 4 M in the final solution. Fig. 3b shows that the goodness of the pH gradient is acceptable, but the peaks migrate late and the resolution is better except between peaks 4 and 5 (Table 1 line 6), than those in the case of using Tris in the sample mixture (Fig. 3a). The best setup was in case of using no salt or urea in the solution (Table 1 line 7).
The aim of an analytical validation is to prove that the applied analytical method is suitable for the purpose of use (e.g. identification tests). According to the Guideline [18] specificity should be investigated during validation of an identification method. Additionally the intermediate precision of the developed method was investigated.
3.6. The optimised method In former methods [9,17] 25 kV focusing voltage was applied; in our study 30 kV focusing voltage was found to be optimal. The same 30 kV mobilization voltage was applied in our final method, but no urea or salt was added to the sample mixture. The other parameters are the same described as general setup in the beginning of the method development session. This optimised method proved to be ready for validation. In this method the resolutions between all of the peaks were equal: short migration time and good pH gradient were obtained (Table 1 line 7).
4.1. Method performances 4.1.1. System suitability Investigating system suitability is inevitable in every case for each single run. System suitability was determined by the R2 value of the migration time–isoelectric point function in the case of the sample and reference solutions.
4.1.2. Intermediate precision Intermediate precision tests were performed to investigate the effects of random errors on the precision of the developed analytical method. This procedure was carried out on two different instruments with two different operators on three different days. We applied six independent sample preparations with single injections on each day. All of the 18 runs were evaluated, the mean pI values of the five main peaks were determined and the relative standard deviations were calculated. Graphical interpretation of the standard deviation of the isoelectric points belonging to Peaks 1–5 can be seen in Fig. 4. The relative standard deviation for all of the peaks was below 0.2%, the highest one (0.173) belongs to Peak 3. Variability of migration times of the pI markers was also investigated. Good standard deviations (
