www.electrophoresis-journal.com
Page 1
Electrophoresis
Analytical QbD: Development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates Mili Pathak, Debayon Dutta, Prof. Anurag Rathore* Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India
*
Corresponding author: AnuragRathore Department of Chemical Engineering Indian Institute of Technology HauzKhas, New Delhi, 110016, India Phone: +91-9650770650 Email: [email protected] Website: www.biotechcmz.com
Keywords:mAb aggregate, native polyacrylamide gel electrophoresis (N-PAGE), Quality by Design (QbD), size exclusion chromatography (SEC).
Received: 25-Nov-2013; Revised: 04-Mar-2014; Accepted: 07-Mar-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400055. This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 2
Electrophoresis
Abstract This paper proposes a Quality by Design (QbD) based development of a novel Native polyacrylamide gel electrophoresis (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies (mAbs). Comparability to the present gold standard of size exclusion chromatography (SEC) has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making NPAGE relevant to those academicians and other small companies involved in early stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Further, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and the analyte resulting in a higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A Quality by Design (QbD) based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments (DOE). The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 3
Electrophoresis
1. Introduction Monoclonal antibodies (mAbs) have been successfully demonstrated to target extracellular proteins with high specificity for treatment of a variety of autoimmune diseases, such as cancer and multiple sclerosis. [1, 2] Since their introduction in the early 1990’s, the demand for monoclonal antibodies has increased rapidly[3] and they presently comprise more than 40% of the marketed biotech therapeutics. [4] Typical processing scheme for production of mAbs involves product expression in mammalian cells followed by clarification of the harvested cell culture broth. The purification process comprises of Protein A affinity chromatography for product capture and removal of host cell related impurities, low pH inactivation for virus removal followed by a combination of cation exchange chromatography (CEX), anion exchange chromatography (AEX) and/or hydrophobic interaction chromatography (HIC) for removal of a gamut of product
related,
process
related
and
host
cell
related
impurities
[5].
Ultrafiltration/diafiltration (UF/DF) steps are intermittently used for product concentration and/or buffer exchange. The final steps include nanofiltration for viral clearance followed by UF/DF into the final formulation buffer [5, 6].During processing, the process intermediates are analyzed for the various host cell related, process related and product related impurities. This is performed so as to ensure adequate process control and process monitoring is in place [7-12]. Aggregates are one of the important factors responsible for increased risk of immune response. The unknown nature of the formed aggregates as well as the wide size range of (six orders of magnitude, from a few nm to a few mm in diameter) poses challenge in analyzing them. None of the currently available techniques is able to cover this entire size range (KDa to mm) and as a result a combination of several techniques is used for this analysis. Techniques that may be included in this analytical package are SEC, PAGE, asymmetrical flow field flow fractionation (AF4)), electron microscopy, This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 4
Electrophoresis
atomic force microscopy, SAXS/SANS (Scattering of X-ray/neutron beam), “Native” mass spectrometry, macro-ion mobility spectrometry, analytical ultra centrifugation [13]. Each technique has its own strengths and weaknesses. Moreover, the available techniques differ with respect to the underlying physical principle and, consequently, differ with respect to the results and type of information obtained. Of these, while SEC is the most commonly used tool due to its excellent robustness, it suffers with certain disadvantages including sample dilution, limited resolution, limited particle size range and possibility of secondary interactions between the stationary phase and the analyte resulting in higher elution time and therefore underestimation of the analyte size [14]. Also, SEC requires expensive instrumentation and consumables [14, 15]. SDS-PAGE is also an often used quality control method to differentiate disulfide mediated and non-disulfide mediated covalent aggregates but is unable to quantify the total aggregates as covalent aggregates have a tendency to break down in the presence of chaotropes such as SDS. Native PAGE on the other hand is easy to perform and can analyze both covalent and non-covalent aggregates. However, as the electrophoretic mobility depends both on size and charge, quantification is a difficult task. Quality by design (QbD) has been described by the ICH Q8 guideline as “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management” [16-18]. Design of Experiment (DOE) is a key element in QbD application towards process development [19]. In this approach, all relevant variables are altered simultaneously through a set of planned experiments followed by statistical analysis of the results. DOE approach has been shown to deliver significant amount of information through a minimum number of experiments [20]. While numerous examples exist in the literature
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 5
Electrophoresis
demonstrating QbD based process development [21-23], applications towards analytical development are relatively recent [24-27]. N-PAGE is based on the premise that any alteration in the charge, conformation or binding events of the protein occurring during mAb processing would change their relative migration [28-33]. As a result it can be used for stability studies of mAbs and other proteins [34] and also for demonstrating product comparability. It can also be used as an orthogonal tool to SEC for mAb analysis [35]. Thus the focus of this paper is to develop and validate a novel, robust Native PAGE protocol for quantification aggregates of mAbs with pI >8 using QbD approach. Performance of the final protocol has been compared to the traditional SEC method. The manuscript demonstrates how the principles of QbD can be applied for development and optimization of analytical methods.
2. Materials and methods 2.1 Materials and equipment Acrylamide, N,N-methylenebisacrylamide, tris, N,N,N′,N′-Tetramethylethylenediamine and ammonium persulfate (APS) were obtained from Sigma–Aldrich Co., St. Louis, MO, USA. Silver nitrate, EDTA, sodium carbonate anhydrous, methanol and glacial acetic
acid
used
for
silver
staining
were
obtained
from
Merck
KGaA,
Darmstadt,Germany. Neutralized pool of monoclonal antibody was used for experimentation. The mAb was donated to us from a major Indian biotech manufacturer. The Mini-PROTEAN Tetra cell electrophoresis unit was purchased from Bio-Rad, Hercules, California, USA, and was used for gelcasting and performing electrophoresis. GS800 calibrated densitometer with Quantity One® software was purchased from BioRad and used for gel scanning and analysis.
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 6
Electrophoresis
2.2 Methodology A QbD based methodology was adopted for development and optimization of the NPAGE based analytical method for measurement of aggregates [17, 18]. First, key performance attributes were defined for the method. For this study, we defined these as the number of bands (to quantitate if complete separation is being achieved), error in % aggregate (to quantitate deviation from SEC) and RMSD for % aggregate (to quantitate accuracy fo the method). Based on our understanding of development of N-PAGE method, we performed a few scouting studies at the start to prioritize the parameters that have significant effect on the quality fo the separation. Next, we optimized the conditions of the running gel followed by optimization of gel percentage. Both of these studies employed DOE so as to get the required information from as few experiments as possible. Finally, the optimized method was compared to the SEC and the data analyzed statistically to demonstrate that the method successfully meets the expectations. 2.2.1 Scouting studies Method attributes that lead to separation of biomolecules (from literature and trial experiments)
include
running
buffer
pH,
running
buffer
molarity,
aminocaproicacid ratio, gel buffer molarity, gel buffer pH, loading dye, concentration of sample, voltage and percentage of resolving gel. All these parameters were studied with the ranges mentioned in Table1. Effect of each parameter was examined and parameters that were found to be significant were examined further.
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 7
Electrophoresis
2.2.2 Optimization of running gel conditions (study 1) In this study, the important parameters from initial screening that were forced to impact protein stability were examined and the data analyzed by JMP® 8 (SAS Institute Inc., Cary, NC) statistical software. 2.2.3 Optimization of gel percentage (study 2) In this study, a two level, three parameter, full-factorial DOE with two center points was performed (to optimize the gel percentage) and the resulting data was analyzed using JMP® 8 (SAS Institute Inc., Cary, NC) statistical software. 2.2.4 Statistical analysis for robustness and comparability Samples with different aggregate content were analyzed by SEC and the N-PAGE protocol in triplicates to check the robustness of the proposed method as well as to compare the performance of the two methods. The Student’s t-test and RMSD value were used to examine the comparability of the measurements using the Student’s t distribution test. If the calculated t value was less than the t value at p = 0.05, the data was deemed to be comparable.
3. Results and Discussion 3.1 Scouting studies In a typical N-PAGE separation, the buffer provides a conducting medium for the separation, maintains the pH and the ionic strength of the sample similar to that of the surrounding gel. For proteins with pI
Page 1
Electrophoresis
Analytical QbD: Development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates Mili Pathak, Debayon Dutta, Prof. Anurag Rathore* Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India
*
Corresponding author: AnuragRathore Department of Chemical Engineering Indian Institute of Technology HauzKhas, New Delhi, 110016, India Phone: +91-9650770650 Email: [email protected] Website: www.biotechcmz.com
Keywords:mAb aggregate, native polyacrylamide gel electrophoresis (N-PAGE), Quality by Design (QbD), size exclusion chromatography (SEC).
Received: 25-Nov-2013; Revised: 04-Mar-2014; Accepted: 07-Mar-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400055. This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 2
Electrophoresis
Abstract This paper proposes a Quality by Design (QbD) based development of a novel Native polyacrylamide gel electrophoresis (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies (mAbs). Comparability to the present gold standard of size exclusion chromatography (SEC) has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making NPAGE relevant to those academicians and other small companies involved in early stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Further, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and the analyte resulting in a higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A Quality by Design (QbD) based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments (DOE). The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 3
Electrophoresis
1. Introduction Monoclonal antibodies (mAbs) have been successfully demonstrated to target extracellular proteins with high specificity for treatment of a variety of autoimmune diseases, such as cancer and multiple sclerosis. [1, 2] Since their introduction in the early 1990’s, the demand for monoclonal antibodies has increased rapidly[3] and they presently comprise more than 40% of the marketed biotech therapeutics. [4] Typical processing scheme for production of mAbs involves product expression in mammalian cells followed by clarification of the harvested cell culture broth. The purification process comprises of Protein A affinity chromatography for product capture and removal of host cell related impurities, low pH inactivation for virus removal followed by a combination of cation exchange chromatography (CEX), anion exchange chromatography (AEX) and/or hydrophobic interaction chromatography (HIC) for removal of a gamut of product
related,
process
related
and
host
cell
related
impurities
[5].
Ultrafiltration/diafiltration (UF/DF) steps are intermittently used for product concentration and/or buffer exchange. The final steps include nanofiltration for viral clearance followed by UF/DF into the final formulation buffer [5, 6].During processing, the process intermediates are analyzed for the various host cell related, process related and product related impurities. This is performed so as to ensure adequate process control and process monitoring is in place [7-12]. Aggregates are one of the important factors responsible for increased risk of immune response. The unknown nature of the formed aggregates as well as the wide size range of (six orders of magnitude, from a few nm to a few mm in diameter) poses challenge in analyzing them. None of the currently available techniques is able to cover this entire size range (KDa to mm) and as a result a combination of several techniques is used for this analysis. Techniques that may be included in this analytical package are SEC, PAGE, asymmetrical flow field flow fractionation (AF4)), electron microscopy, This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 4
Electrophoresis
atomic force microscopy, SAXS/SANS (Scattering of X-ray/neutron beam), “Native” mass spectrometry, macro-ion mobility spectrometry, analytical ultra centrifugation [13]. Each technique has its own strengths and weaknesses. Moreover, the available techniques differ with respect to the underlying physical principle and, consequently, differ with respect to the results and type of information obtained. Of these, while SEC is the most commonly used tool due to its excellent robustness, it suffers with certain disadvantages including sample dilution, limited resolution, limited particle size range and possibility of secondary interactions between the stationary phase and the analyte resulting in higher elution time and therefore underestimation of the analyte size [14]. Also, SEC requires expensive instrumentation and consumables [14, 15]. SDS-PAGE is also an often used quality control method to differentiate disulfide mediated and non-disulfide mediated covalent aggregates but is unable to quantify the total aggregates as covalent aggregates have a tendency to break down in the presence of chaotropes such as SDS. Native PAGE on the other hand is easy to perform and can analyze both covalent and non-covalent aggregates. However, as the electrophoretic mobility depends both on size and charge, quantification is a difficult task. Quality by design (QbD) has been described by the ICH Q8 guideline as “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management” [16-18]. Design of Experiment (DOE) is a key element in QbD application towards process development [19]. In this approach, all relevant variables are altered simultaneously through a set of planned experiments followed by statistical analysis of the results. DOE approach has been shown to deliver significant amount of information through a minimum number of experiments [20]. While numerous examples exist in the literature
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 5
Electrophoresis
demonstrating QbD based process development [21-23], applications towards analytical development are relatively recent [24-27]. N-PAGE is based on the premise that any alteration in the charge, conformation or binding events of the protein occurring during mAb processing would change their relative migration [28-33]. As a result it can be used for stability studies of mAbs and other proteins [34] and also for demonstrating product comparability. It can also be used as an orthogonal tool to SEC for mAb analysis [35]. Thus the focus of this paper is to develop and validate a novel, robust Native PAGE protocol for quantification aggregates of mAbs with pI >8 using QbD approach. Performance of the final protocol has been compared to the traditional SEC method. The manuscript demonstrates how the principles of QbD can be applied for development and optimization of analytical methods.
2. Materials and methods 2.1 Materials and equipment Acrylamide, N,N-methylenebisacrylamide, tris, N,N,N′,N′-Tetramethylethylenediamine and ammonium persulfate (APS) were obtained from Sigma–Aldrich Co., St. Louis, MO, USA. Silver nitrate, EDTA, sodium carbonate anhydrous, methanol and glacial acetic
acid
used
for
silver
staining
were
obtained
from
Merck
KGaA,
Darmstadt,Germany. Neutralized pool of monoclonal antibody was used for experimentation. The mAb was donated to us from a major Indian biotech manufacturer. The Mini-PROTEAN Tetra cell electrophoresis unit was purchased from Bio-Rad, Hercules, California, USA, and was used for gelcasting and performing electrophoresis. GS800 calibrated densitometer with Quantity One® software was purchased from BioRad and used for gel scanning and analysis.
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 6
Electrophoresis
2.2 Methodology A QbD based methodology was adopted for development and optimization of the NPAGE based analytical method for measurement of aggregates [17, 18]. First, key performance attributes were defined for the method. For this study, we defined these as the number of bands (to quantitate if complete separation is being achieved), error in % aggregate (to quantitate deviation from SEC) and RMSD for % aggregate (to quantitate accuracy fo the method). Based on our understanding of development of N-PAGE method, we performed a few scouting studies at the start to prioritize the parameters that have significant effect on the quality fo the separation. Next, we optimized the conditions of the running gel followed by optimization of gel percentage. Both of these studies employed DOE so as to get the required information from as few experiments as possible. Finally, the optimized method was compared to the SEC and the data analyzed statistically to demonstrate that the method successfully meets the expectations. 2.2.1 Scouting studies Method attributes that lead to separation of biomolecules (from literature and trial experiments)
include
running
buffer
pH,
running
buffer
molarity,
aminocaproicacid ratio, gel buffer molarity, gel buffer pH, loading dye, concentration of sample, voltage and percentage of resolving gel. All these parameters were studied with the ranges mentioned in Table1. Effect of each parameter was examined and parameters that were found to be significant were examined further.
This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 7
Electrophoresis
2.2.2 Optimization of running gel conditions (study 1) In this study, the important parameters from initial screening that were forced to impact protein stability were examined and the data analyzed by JMP® 8 (SAS Institute Inc., Cary, NC) statistical software. 2.2.3 Optimization of gel percentage (study 2) In this study, a two level, three parameter, full-factorial DOE with two center points was performed (to optimize the gel percentage) and the resulting data was analyzed using JMP® 8 (SAS Institute Inc., Cary, NC) statistical software. 2.2.4 Statistical analysis for robustness and comparability Samples with different aggregate content were analyzed by SEC and the N-PAGE protocol in triplicates to check the robustness of the proposed method as well as to compare the performance of the two methods. The Student’s t-test and RMSD value were used to examine the comparability of the measurements using the Student’s t distribution test. If the calculated t value was less than the t value at p = 0.05, the data was deemed to be comparable.
3. Results and Discussion 3.1 Scouting studies In a typical N-PAGE separation, the buffer provides a conducting medium for the separation, maintains the pH and the ionic strength of the sample similar to that of the surrounding gel. For proteins with pI
