Get there, faster with ice3

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1 Get there, faster with ice3

2 Method development in hours, not weeks.

3 The ice3 Tomorrow s challenges are always looming, so why not catapult ahead? ice3 lets you move beyond the limits of traditional protein analysis. Quick and simple method development gets you to product approval sooner, or as we like to say: FDA, PDQ.

4 Streamlines ice3 fast-tracks your development timelines. 10-minute start to finish runs let you optimize method conditions in an afternoon. As an added bonus, you can use the same method for multiple molecules no need for product-specific methods. You can now standardize platform methods across product development and QC, so you ll save time and costs and get more consistent data and information in the process. MINUTES/SAMPLE ice Traditional cief IEX Chromatography

5 Simplifies Techniques like IEF gels, ion exchange and traditional capillary IEF have great benefits, but each has its own set of challenges too. ice3 combines the best of these three worlds, giving you high resolution, quantitation and automation without the hassles. No variability, no manual processes, no long productspecific method development, and no mobilization steps that double analysis times. Sample Loading Separate by Charge Quantitate

6 Resolves Separation of mab Isoforms Proteins are complex, no way around it. They behave differently, come in a variety of shapes and sizes, and some are linked to other molecules. ice3 easily separates proteins in the most challenging samples. In fact, that s what it does best. Absorbance pi marker pi pi marker 7.65

7 USA USA % 80% 60% 40% 20% 0% 22 min 18 min 14 min 10 min 8 min 6 min 4 min 22 min 18 min 14 min 10 min 8 min 6 min 4 min mab-2 mab-3 pi Value /0 100/6 100/12 100/30 100/60 120/0 120/30 Total Peak ± ± ± ± ± ±0.801 pi Value of Main Peak 8.54± ± ± ± ± ±0.02 Peak Area 67.1± ± ± ± ± ±0.008 Assures Over 100 publications, presentations, and posters can t be wrong. Customers across sites, countries and continents rely on ice systems to solve their toughest challenges and meet regulatory requirements. Here s the real proof: A 12-lab study at 11 biopharms verified ice technology as robust and reliable for charge heterogeneity analysis of monoclonal antibodies Zoran Sosic Damian Houde Andy Blum Tyler Carlage Yelena Lyubarskaya BiogenIdec, Cambridge, MA, Received March 7, 2008 Revised June 26, 2008 Accepted June 26, Introduction Research Article Application of imaging capillary IEF for characterization and quantitative analysis of recombinant protein charge heterogeneity Monitoring, quantitative analysis and characterization of the charge heterogeneity of recombinant proteins is an important part of biopharmaceutical product development. Various modifications in a protein structure, such as deamidation, amino acid substitution/deletion, differential glycosylation, glycation, etc., can constitute the sources of charge heterogeneity [1]. In biopharmaceutical development slab gel IEF and/or IEC have traditionally been used to monitor protein charge heterogeneity for batch-to-batch process consistency, product quality assessment and providing information on protein stability and purity. IEC is known to require a significant method development time In this work several aspects of imaging capillary IEF (icief) application for charge heterogeneity analysis of recombinant proteins and monoclonal antibodies have been discussed. Advantages of the method as compared with traditional approaches for determination of biomolecule charge heterogeneity, such as gel and IEC, have been demonstrated. Correlation of icief-detected protein isoforms with the charge heterogeneity determined by IEC has been shown for a representative recombinant monoclonal antibody. Identification of charged variants collected from IEC has been performed by ESI-MS. Qualification of an icief method for use in quality control environment for quantitative analysis of recombinant protein charge heterogeneity and monitoring protein stability has also been discussed. The intermediate precision for determination of pi of main or main acidic species was r0.2% RSD. Relative % peak areas for acidic, main and basic species were reproducible within 1.9, 0.9 and 16.6% RSD, respectively. Based on the assay performance evaluation, icief assay has been shown to allow for fast method development, short analysis time and high sample throughput. Some aspects of the method specificity for use as an identity test in biopharmaceutical development have been discussed. Keywords: Biopharmaceutical process development / Charge heterogeneity / Imaging capillary IEF / Recombinant monoclonal antibody / Recombinant proteins DOI /elps Correspondence: Dr. Zoran Sosic, Analytical Development, Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA 02142, zoran.sosic@biogenidec.com Fax: Abbreviations: CIEX, cation ion exchange chromatography; ESI-q-TOF MS, electrospray ionization quadrupole time-offlight mass spectrometry; icief, imaging capillary IEF; rmab, recombinant mab Electrophoresis 2008, 29, and it often does not provide adequate separation for isoform quantitation. The use of shallow salt gradient in IEC can achieve better peak separation; however, such chromatographic conditions are not robust enough and prone to be affected by minute changes (buffer ph, column lot). Traditional gel IEF suffers from low throughput and often does not provide accurate quantitative information. The introduction of capillary IEF (cief) by Hjerten and Zhu [2] offered significant advantages over slab gel IEF as it enabled faster, automated and quantitative analysis. Compared with IEC, cief demonstrates superior resolution, with an advantage of shorter method development time, a higher throughput, and minimal solvent consumption. In a typical cief analysis, protein isoforms are first focused along the capillary column according to their isoelectric point and then mobilized toward on-column detector located at one end of the capillary [3 5]. The mobilization step often requires optimization as poor migration time and peak area reproducibility due to distortion of ph gradient and non-uniform mobilization speed have been reported [6]. A recent introduction of imaging capillary IEF (icief) overcomes the issues with single point detection as it eliminates the need for a mobilization step [7]. In this approach the whole separation capillary column is stripped off the polyimide coating and is used as Effect on Peak Area & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim % main peak area Absorbance total peak area (x10000) Absorbance 100/0 Electrophoresis 2009, 30, 1 9 CE and CEC 7 100/0 100/6 100/12 100/30 100/60 120/0 120/30 Transfer time/ transfer delay time (Sec) Effect on % Main Peak Area 100/ Figure 6. The effect of focusing time and voltage on ice separation. 100/12 100/30 100/60 120/0 120/30 Transfer time/ transfer delay time (Sec) pi value of main peak ing time range for mab-2 appeared to be 4 8 min. On the other hand, the focusing range for mab-3 was much wider; complete focusing was achieved from 6 up to 18 min. In summary, 1 min initial focusing at 1500 V followed by 5 min at 3000 V was found to be optimal for all mabs tested with good reproducibility. Therefore, this condition is recommended as the generic condition in ice separation for mabs Sample transfer time and delay time In the current instrument and software setting for ice separation using Convergent Bioscience s ice280 Analyzer, transfer time and transfer delay time are two parameters that facilitate the delivery of analytes to the focus zone of the capillary. To find out how sample transfer time and transfer delay time affect ice analysis, studies were carried out to determine the optimal conditions for these parameters. Transfer time and transfer delay time were tested with Alcott autosampler at an injection protein concentration of 0.3 mg/ml. Prepared samples were analyzed with seven different transfer time/transfer delay time combinations at 100/0, 100/6, 100/12, 100/30, 100/60, 120/0 and 120/30 s. All other parameters were set according to the optimal ice conditions described earlier Journal of Pharmaceutical and Biomedical Analysis 43 (2007) Transfer Time / Transfer Delay Time (Second) Figure 7. The effect of sample transfer time/transfer delay time on ice separation. Effect on pi Value of Main Peak 100/0 100/6 100/12 100/30 100/60 120/0 120/30 Transfer time/ transfer delay time (Sec) Area (x10000) Percent Main & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Development of a High Throughput Protein A Well-Plate Purification Method for Monoclonal Antibodies Jennifer Hopp, Ross Pritchett, Maria Darlucio, Junfen Ma, and Judy H. Chou Oceanside Process Research and Development, Genentech Inc., One Antibody Way, Oceanside, CA DOI /btpr.247 Published online July 27, 2009 in Wiley InterScience ( We have developed a new high throughput method for the purification of monoclonal antibodies from harvested cell culture fluid for analytical characterization. This method uses Protein A resin in a 96 well-plate format with protein loading sufficient to perform multiple analyses per well. Resin and buffer conditions were optimized to obtain aggregate and charge variant comparability with three preparative Protein A purified monoclonal antibodies. We are able to successfully demonstrate comparability for aggregate within 0.25% based upon size-exclusion chromatography. Acidic species were found to be within 2% from the preparative purified control based upon cation-exchange chromatography, 5% based upon capillary zone electrophoresis, and 3% based upon imaged capillary isoelectric focusing. Glycan distribution was analyzed and was within 1% of the preparative purified controls. A tryptic digest was performed and all peaks in the preparative purified control were found in the first elution from the well-plate format. VC 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: , 2009 Keywords: protein A, high throughput, monoclonal antibody purification Introduction Scale-down purification techniques can shorten timelines analysis of size, charge, and glycan distribution to column for process development activities such as cell line selection purification. The platform for this purification method is a and purification optimization. Protein A purification is one 96 well-plate format. Protein A resins, elution buffers, and of the most widely used chromatographic techniques for the production of monoclonal antibodies 1,2 neutralization buffers were optimized to produce representative protein in sufficient quantity for multiple downstream but it is under-represented in the literature with regards to development of scaledown purification methods. Advances in high throughput pu- analyses. The recovered protein has comparable aggregate content, glycan distribution, and charge heterogeneity compared to protein obtained on a preparative scale. This purifirification methods focus on purification optimization for cation exchange chromatography screening in 96 well-plate 3 11 or miniaturized column formats. 12 cation method also meets the material requirements for These techniques offer multiple analytical assays per well including size-exclusion rapid methods for purification optimization with minimal chromatography (SEC), ion exchange chromatography (IEC), protein requirements. capillary zone electrophoresis (CZE), capillary electrophoresis glycan (CE-glycan), imaged capillary isoelectric focusing Protein A purification is often utilized at the time of cell line selection to obtain an early read of product quality. (icief), and peptide mapping. This method can be fully High throughput Protein A methods can be used at this critical path activity to quickly obtain purified antibodies with ture fluid. automated for rapid parallel processing of harvested cell cul- minimal cell culture volume. This enables the evaluation of product quality during early cell line development prior to extensive expansion and cell culture optimization. High throughput Protein A methods have been developed for sample preparation, 13,14 but without demonstrating optimization Reagents for product quality comparability to process scale Protein A MabSelect and MabSelect SuRe affinity chromatography chromatography. This is a critical aspect that must be evaluated to ensure analytical data from the scale down method Poros A resin was from Applied Biosystems (Foster City, resin was obtained from GE Healthcare (Uppsala, Sweden). is representative of the preparative scale. CA) and ProSep A resin was obtained from Millipore (Billerica, MA). In this study, we describe the development of a Protein A well-plate purification method for purifying monoclonal antibodies from harvested cell culture fluid (HCCF). The focus recombinant humanized monoclonal IgG1 was obtained from Chinese hamster ovary (CHO) cell culture fluid containing Genentech (Oceanside, CA). HCCF from three different antibodies were used in this work and denoted as mab-1, mab- 2, and mab-3. The titers of antibody in the HCCF ranged Evaluation of the ice280 Analyzer as a potential high-throughput tool for formulation development Ning Li a,, Kendall Kessler a, Laura Bass b, David Zeng a a Department of Pharmaceutical Research and Development, Pfizer Global Biologics, St. Louis Laboratory, Pfizer Inc., St. Louis, MO 63017, USA b Department of Analytical Research and Development, Pfizer Global Biologics, St. Louis Laboratory, Pfizer Inc., St. Louis, MO 63017, USA Received 27 July 2006; received in revised form 8 September 2006; accepted 9 September 2006 Available online 12 October 2006 Abstract The ice280 Analyzer (ice280) was evaluated for its potential application as a high-throughput tool to determine pi and separate charge related species using glycosylated, non-glycosylated and pegylated protein therapeutics as models. Resolution was achieved for glycosylated and nonglycosylated molecules, but remained a challenge for pegylated proteins. The sources of charge variants were determined to be the presence of C-terminal lysine residues, sialic acid content, and deamidation. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of g/ml of IgG with linear regression coefficients of 0.984, 0.998, and for acidic, main and basic species, respectively. Limit of detection and limit of quantitation were determined to be 3 and 11 g/ml. The R.S.D. for intra- and inter-day precision as well as reproducibility was determined to be 0.2% or less for all pi values and 1.4% or less for acidic and main peak area distribution; the R.S.D. for basic peak area distribution was 5.7% or less. Robustness testing was performed by deliberately deviating ±50% of pharmalyte concentration away from the desired condition. This deviation revealed a pi shift of only 0.06 units and resulted in no significant impact on area percent distribution. Utilization of ice280 Analyzer eliminated the mobilization step associated with traditional capillary isoelectric focusing analysis and increased analytical throughput at least 2-fold Elsevier B.V. All rights reserved. Keywords: Imaged capillary isoelectric focusing; ice280 Analyzer; Deamidation; Protein; Pegylation 1. Introduction Slab gel isoelectric focusing (IEF) technique has been routinely used to determine the isoelectric point (pi) of proteins and to monitor their purity, stability and microheterogeneity [1 7]. Nevertheless, the technique is labor-intensive and semi-quantitative [1,8]. Recently, isoelectric focusing performed in a capillary format (cief) has demonstrated and offers many advantages over conventional slab gel IEF. These include improved resolution [1], better quantitation, and automation capability [1]. Several years ago, an imaged cief instrument (ice280 Analyzer) was introduced to the market by Convergent Bioscience. Prior to the introduction of this instrument, most cief analyses were carried out using traditional CE instruments with a two step approach: (1) protein focusing after being Corresponding author. Tel.: ; fax: address: ning.x.li@pfizer.com (N. Li) /$ see front matter 2006 Elsevier B.V. All rights reserved. doi: /j.jpba Correspondence concerning this article should be addressed to J. Hopp at hopp.jennifer@gene.com. of this work was to develop a high throughput purification method resulting in comparable product quality based on Materials and Methods VC 2009 American Institute of Chemical Engineers 1427 introduced into the capillary and (2) post-focusing mobilization of protein passing through a capillary detector window located at one end of the capillary [7,9,10 12]. There were problems associated with the mobilization step. The ph gradient established during the focusing step could be distorted [10 12]. This often resulted in band broadening, reduced resolution, and poor reproducibility [10 12]. Imaged cief completely eliminated the mobilization step by taking the whole capillary absorption image using a charge-coupled device camera. The detection system consists of a whole column optical absorption imaging detector operated at 280nm. The light source of the absorption detector is a deuterium (D2) lamp. During the focusing, the light beam from the lamp is focused onto the separation capillary by a bundle of optical fibers and a cylindrical lens. The final whole capillary UV absorption image is captured by a camera with an imaging lens and a charge-coupled device sensor [11]. This work was intended to evaluate the ice280 Analyzer as a potential high-throughput tool for formulation development. 1 Robustness of icief methodology for the analysis of monoclonal antibodies: An interlaboratory study, O Salas-Solano, et al., Journal of Separation Science, Nov 2012; 35(22):

8 Specifications ice3 Instrument PrinCE Next Autosampler Description Specification Description Specification Sample Volume/Run Sample Delivery μl Alcott 720 Autosampler or PrinCE Next Microinjector Tray Capacity Sample Cooling 4 40 C, ± 1 C Buffer Tray: 50 (11 mm) vials Sample Tray: 50 (11 mm) vials or one 96-well microplate Typical Run Time minutes Dimensions 47 cm H x 33 cm W x 66 cm D Detection UV absorption at 280 nm Weight 28 kg (61 lbs) Focusing Voltage 600 V/cm Power V AC, 50/60 Hz Dimensions Weight Power 66 cm H x 28 cm W x 31 cm D 20 kg (45 lbs) V AC, 50/60 Hz Alcott 720 Autosampler Description Tray Capacity Specification Sample Cooling 4 40 C, ± 1 C 48/4 Tray: 48 (11 mm) vials plus 4 (10 ml) vials 96/4 Tray: 96-well microplate plus 4 (10 ml) vials Dimensions Weight Power 33 cm H x 32 cm W x 55 cm D 16 kg (35 lbs) V AC, 50/60 Hz Toll-free: (888) Tel: (408) Fax: (408) info@proteinsimple.com proteinsimple.com / Rev D ProteinSimple. ice, ProteinSimple and the ProteinSimple logo are trademarks and/or registered trademarks of ProteinSimple.