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Optimization Strategies to Determine Charged Protein Variants in a Therapeutic Protein Using Capillary Isoelectric Jon S

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Optimization Strategies to Determine Charged Protein Variants in a Therapeutic Protein Using Capillary Isoelectric Jon S Vikas Dhingra, Ph.D. Kelly Hoberg B.S. Optimization Strategies to Determine Charged Protein Variants in a Therapeutic Protein using Capillary Isoelectric Jon S. Kauffman, Ph.D. Biopharmaceutical Services Introduction METHODOLOGY PRESSURE RESULTS METHODOLOGY CHEMICAL METHODOLOGY COMPARISON l Isoelectric focusing is a powerful charge-based 0.025 0.025 Repeatability using Pressure Mobilization l A cIEF profile was obtained using a 45 cm neutral coated capillary with a UV analytical technique used in the separation of A cIEF profile was obtained using a 45 cm neutral coated capillary with a UV 0.020 0.020 mAb detector set at 280 nm on a ProteomeLab CE system. Sample proteins were Repeatability using Chemical Mobilization Parameter Pressure Method Specification Chemical Method Specification Comparison proteins based on their isoelectric points. It is widely detector set at 280 nm on a ProteomeLab CE system. Sample proteins were 3.5-9.3 pI markers 0.015 0.015 allowed to reach their pI’s in a two-dimensional separation scheme which allowed to reach their pI’s in a two-dimensional separation scheme which utilized Anolyte 91 mM Phosphoric Acid in cIEF Gel 200 mM Phosphoric Acid in water Reduces migration time shifts within runs utilized in the biopharmaceutical industry for the utilized 200 mM Phosphoric Acid as the anolyte and 300 mM Sodium Hydroxide 0.06 0.06 91 mM Phosphoric Acid as the anolyte and 20 mM Sodium Hydroxide as the 0.06 0.06 Catholyte 20 mM Sodium Hydroxide in water 300 mM Sodium Hydroxide in water Reduces migration time shifts within runs AU mAb AU 1 2 3 4 0.010 0.010 as the catholyte with an applied voltage of 25 KV for focusing. Mobilization was characterization and identification of protein-based catholyte with an applied voltage of 15 KV for focusing. Mobilization was 0.05 0.05 Sample To the ampholyte gel solution (200 µL of To the ampholyte gel solution (200 µL of Sample preparation similar. performed under 30 KV in either 20 mM Formic Acid or 175 mM Acetic Acid 0.05 0.05 performed under 21 KV with 0.5 psi pressure to mobilize the proteins from the Preparation cIEF gel + 4 µL of Ampholyte 4-7), add cIEF gel + 4 µL of Ampholyte 4-7), add biotherapeutics, as well as in stability studies. 0.005 0.005 10 µL of monoclonal Ab solution 10 µL of monoclonal Ab solution chemical mobilization solutions to mobilize the proteins from the capillary for 0.04 0.04 capillary for subsequent analysis. 0.04 0.04 subsequent analysis. Focusing Condition 15 KV focusing (500 V/cm) for 6 minutes, 25 KV focusing in Anolyte and Catholyte 0.000 0.000 0.17 minute ramping time in Anolyte and solutions for 6 minutes, 0.17 minute l 0.03 0.03 0.03 0.03 Catholyte ramping time AU AU In IEF proteins are separated in a pH gradient AU AU Mobilization 21 KV mobilization (700 V/cm) for 34 30 KV mobilization in Chemical Shorter mobilization time for shorter Parameter Specification 6 8 10 12 14 16 18 20 0.02 0.02 generated by carrier ampholytes 0.02 0.02 Minutes Parameter Specification minutes with 0.5 psi forward pressure, Mobilization solutions (either 20 mM analysis; Reduces broadening of band 0.17 minute ramping time in Anolyte and Formic Acid or 175 mM Acetic Acid) for 20 and laminar flow; Increases capillary life. 0.01 0.01 Catholyte minutes with 0.1 psi forward pressure, Electrophoresis System Beckman Coulter ProteomeLab PA 800 Capillary Electrophoresis Electrophoresis System Beckman Coulter ProteomeLab PA 800 Capillary Electrophoresis 0.01 0.01 - Broad and narrow range 0.17 minute ramping time System System Neutral Capillary 50 µm × 45 cm, 2” (Beckman Coulter) Neutral Capillary 50 µm × 45 cm, 2” (Beckman Coulter) 0.00 0.00 0.00 0.00 Capillary Rinse capillary with 10 mM Phosphoric Rinse capillary in water for 2 minutes at Pressure requires more washing time as - Gel based Gel profile of mAb cIEF profile of mAb Conditioning Acid for 2 minutes at 30 psi followed by a 50 psi, followed by rinsing in Chemical compared to chemical. Chemical pI Marker cIEF 3-10 Protein Standards (Beckman Coulter) pI Marker cIEF 3-10 Protein Standards (Beckman Coulter) 15 minute water rinse at 30 psi. Mobilization Solution for 2 minutes at 50 conditioning good for entire continual Capillary Temperature 20°C 0 5 10 15 20 25 0 5 10 15 20 25 Conditioning performed daily before psi, then finally a cIEF gel rinse for 5 analysis. Capillary Temperature 20°C Minutes Minutes analysis. minutes at 50 psi. - Capillary based l l Sample Temperature 10°C Sample Temperature 10°C Slab gel electrophoresis Automation Conditioning performed as needed. l UV Parameters Wavelength – 280 nm; Data rate – 2 Hz; Filter – High Resolution Six separate mAb preparations were analyzed to Capillary isoelectric focusing (cIEF) is a technique UV Parameters Wavelength – 280 nm; Data rate – 2 Hz; Filter – High Resolution l Serva violet staining l High throughput Peak width – More than 25 Capillary Cleaning No cleaning methodology. Rinse capillary with Cleaning Solution (6 Reduces aggregation and increases Peak width – More than 25 RSD Migration time: 0.52% determine consistency M Urea, 2 M Thiourea in water) for 3 capillary performance. Focusing Step 25 KV focusing in Anolyte and Catholyte solutions for 6 minutes, minutes at 50 psi, followed by a 2 minute that separates amphoteric molecules along a 0.17 minute ramping time Focusing Step 15 KV focusing (500 V/cm) for 6 minutes, 0.17 minute ramping l Labor / time intensive l Time labor – ~30-60 min RSD pI: 0.43% A consistent profile was observed water rinse at 50 psi. time Mobilizing Step 30 KV mobilization in Chemical Mobilization solutions for 20 Cleaning performed as needed. capillary according to their pI values, resulting in the RSD Migration time: 0.83% minutes with 0.1 psi forward pressure, 0.17 minute ramping time Easy to achieve reproducible peak patterns Mobilizing Step 21 KV mobilization (700 V/cm) for 34 minutes with 0.5 psi forward l Semi-quantitative l Green technology formation of a stabilized pH gradient. pressure, 0.17 minute ramping time RSD pI: 0.39% Focusing time can be optimized in a single run l cIEF offers many advantages over conventional gel- based techniques including automation, increased accuracy, and improved sensitivity, efficiency, and l Equipment inexpensive l Sample preparation Desalting of mAb on with Pressure Mobilization e lt in gDsa : Factors imputing to cIEF cIEF functionalities: resolution of protein separation. l Sample preparation l Simple, less time consuming Possible Problems 1. S p in p r o t e in de s a l ti n g sp in co lumn a t 1 , 5 0 0 g RSD Migration time: 0.14% Effect of Varying Chemical Mobilization Solutions separation fo r 1 minu t e to elute bu ffe r . l Determine pI of proteins in solution l l RSD pI: 0.09% Chemical Mobilization with l Evaluation of capillary IEF technique with various l Gel preparation High throughput 2. Ad d 50 0 μ L of Mon o c l o n a l Ab to co l u mn . Pressure Mobilization with 0.5 PSI l Pressure mobilization 175 mM Acetic acid Ampholytes - pH range / l Differentiate between homologous l Sample loading technique l Short capillary preparation time 3. Sp in at 1, 5 0 0 g fo r 2 minu t e s . Elu a n t is th e optimization methodologies using the Beckman PA - Broadening of bands de s a lt e d Mon o c lon a l Ab . concentration - should cover pI’s of proteins 0.025 0.025 l pH gradient is established via carrier l pH gradient is established via carrier 0.06 0.06 0.06 0.06 sample and give satisfactory 800 ProteomeLab system was performed. Their l - Laminar flow issues 0.06 0.06 Monitor process consistency ampholytes ampholytes 0.05 0.05 0.05 0.05 resolution following results were obtained from each profile. 0.020 0.020 l l Protein pI marker Low Pressure rinse 0.5 PSI l Positioning of anode and cathode can l Linear pH gradient between the 0.05 0.05 0.04 0.04 0.04 0.04 l Protein - Concentration - should be Monitor process stability affect band migration cathode and anode 0.015 0.015 AU 0.03 0.03 AU l Protein precipitation & aggregation 0.04 0.04 AU 0.03 0.03 AU high enough to detect minor species, l Obtain signature profiles l l Pore size Noisy baselines 0.02 0.02 0.02 0.02 but should not saturate the detector AU AU AU 0.03 0.03 AU -Huge issue 0.010 0.010 l Protein characterization l Densitometer quantitation and staining 0.01 0.01 0.01 0.01 l Sample - Composition -Can generate artifactual spikes on electropherogram 0.02 0.02 l Dynamic process characteristics can affect analysis 0.005 0.005 0.00 0.00 0.00 0.00 l -Block capillary and fluctuate/reduce current to produce 0.01 0.01 Focusing Voltage/Time - too short 0 5 10 15 20 25 0.000 0.000 0 5 10 15 20 25 will lead to non-reproducible peaks - Minutes variable migration time 0.00 0.00 Minutes too long will lead to aggregation -Favored by high protein concentration in the zone 0 5 10 15 20 25 0 5 10 15 20 25 30 35 Minutes Minutes l Chemical Mobilizer: 175 mM Acetic Acid Chemical Mobilizer: 20 mM Formic Acid Selection of pI markers - should -Interaction with Capillary bracket sample peaks l Gradient decay over time Additional isoforms of antibody observed Schematic diagram of cIEF ProteomeLab PA 800 CE cIEF has a bi-directional flow Possible Solutions Effect of Urea Wash in Pressure Mobilization l Mobilization Robustness: Change in Focusing Conditions with Pressure Mobilization Conclusion - Chemical Pre Urea Wash Post Urea Wash Comparison Overlay 0.025 0.025 0.025 0.025 - Capillary degradation l The IEF profile from cIEF method was comparable to the profile from gel IEF, which suggests that the 0.020 0.020 0.020 0.020 15 KV Focusing 10 KV Focusing 18 KV Focusing 0.020 0.020 l 0.025 0.0250.025 0.025 0.025 0.025 Monoclonal Ab 9.22 Protein pI marker Monoclonal Ab 9.19 0.015 0.015 cIEF method is accurate and may replace the gel IEF.
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