Higher Order mab Aggregate Analysis using New Innovative SEC Technology

Transcription

1 Higher Order mab Aggregate Analysis using New Innovative SEC Technology Ronald E. Majors, Ph.D. LCGC No. America West Chester, PA USA WCBP 2016 Washington, DC Linda Lloyd, Ph.D. Agilent Technologies Church Stretton United Kingdom 1

2 Top 200 pharmaceutical products by U.S. retail sales ( )* (those in RED represent a biopharmaceutical product) *Charts courtesy of the Njardarson Group, Univ. of Arizona, M. Brichacek, N. McGrath, E. Rogers, J.Morton, L.Batory, R. Bauer, J.A.Wurst, and J.T. Njardarson. URL: World Preview Outlook to 2020, EvaluatePharma (2014), In 2020, the top 100 prescription drugs based on biologicals will account for 52% of the sales in the U.S.

3 BioPharma trends: implications for liquid phase separations Traditional Pharma shifting to biopharmaceuticals Need to speed up the analysis Accurate characterization Analysis of native biomolecules in biocompatible environments Connect LC to higher order LC/MS Innovative columns/methods needed for biomolecules Some biologics moving off patent Require unique new technologies Workflow driven Move to UHPLC and smaller particles Superficially porous particle technology Require selectivity, resolution and reproducibility Minimize degradation caused by analysis Non-denaturing eluents aqueous (Not RP) LC techniques with MS friendly eluents Low concentration of volatile salts Require complete solution with documentation Defined workflow solutions Need products, workflows suitable for biosimilars Rapid analysis for reduced development time

4 BioPharmaceutical Product Critical Quality Attribute (CQA) HIC/RP HIC/CEX SEC SEC CEX SEC Glycan column CEX Glycan column CEX Glycan column HIC/RP Peptide mapping/rp CEX Reference: Amgen poster

5 Aggregation Proteins including mabs are relatively unstable Do not always adopt native conformation Aggregates may form due to improper production, storage or handling Aggregates can be: -Tangled clusters of denatured mabs - Order structures of native mabs Can have important consequences for the safety and efficacy of biopharmaceuticals - Will reduce the process yield - Will increase the cost of production - May cause patient immunogenicity

6 Factors that impact aggregation Manufacturing Processes Steps and Products Fermentation Purification Formulation Storage Shipping Administration Stress Conditions Heat Freeze-thaw Cross-linking Protein concentration Formulation change ph, salt Chemical modification Mechanical stress/ surface

7 Classical techniques for aggregate determination WHO has set limit for aggregates as <5%

8 Size Exclusion Chromatography (SEC) Size in solution is related to retention time -Smaller molecules spend longer in the pores and elute later. -Larger molecules spend less time in the pores and elute sooner.

9 Molecular weight determination Apparent molecular weight is determined by running samples of known molecular weight and plotting a calibration curve Unknown is β-lactoglobulin MW of 18.4 kd AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7µm β-lactoglobulin dimer, apparent MW of 37 kd

10 Molecular weight determination However - separation is based on hydrodynamic volume in solution and not molecular weight shape dependency Light Scattering methods provide absolute molecular weights and increased sensitivity for aggregates

11 What does that mean for SEC columns? SEC columns must deliver accurate, precise quantitation for mabs and mab conjugates High resolution for more accurate quantitation Faster analysis speeds for delivery to deadlines Must be stable for long periods of time and high number of injections Should cause no change to sample integrity Sensitivity for quantitation at low levels - 1 to 5% Methods must be easily transferred to other locations, including QA/QC

12 Pain points for current SEC columns Limited resolution over molecular size range of interest - incomplete resolution makes automated quantitation more difficult - will compromise reproducibility SEC method impacts sample integrity - non-specific interactions drive mobile phase selection to achieve size separation and will contribute to poor peak shape, recovery and carry over - will compromise accuracy; also, shear forces may be caused by small porosity frits and particles Long analysis times large particle sizes and long columns run at low linear velocity - will compromise lifetime if run faster Limited sensitivity - larger column IDs impact limits of detection and reduces the accuracy of the quantitation for low level aggregation SEC columns are not very robust and have limited pressure stability

13 So, what is a good SEC column? Need high pore volume and optimum pore size No interaction between particle surface and samples Mobile phases that do not impact the sample composition

14 Introducing AdvanceBio SEC a new generation of column technology A totally new way to make the base silica particle (fully porous) allowing better control of particle diameter, pore diameter and pore volume High degree of particle stability allowing for a robust column able to withstand up to 1500 injections, higher flow rates (speed) and no particulate bleed for LS detector Novel bonding chemistry minimizes non-specific interactions allowing improved peak shape, excellent recoveries, high degree of inertness and better quantitation (data accuracy), no or low salt in mobile phase and use of pure aqueous mobile phases (no organic). A 2.7µm particle to allow good resolution, moderate pressure (can use 400 bar systems) and use of 2µm frits (ruggedness)

15 Introducing AdvanceBio SEC a new generation in column technology Initial pore size offering: 130 Å and 300Å Column dimensions: 4.6mm and 7.8mm i.d s. 150mm and 300mm lengths Guard columns: same i.d s but 50mm length Recommended for proteins and peptides with emphasis on monoclonal antibodies analysis and quantification including higher order mab aggregates and degradation fragments* Suitability for different sample types - modified proteins including ADCs and pegylated proteins* *with same mobile phase

16 Column performance report & batch test: AdvanceBio SEC 130Å

17 Typical operating conditions Recommended starting conditions Parameter Mobile phases Conditions ph Aqueous buffers 150 mm phosphate buffer, ph 7.0 Aqueous organic mixes Operating temp Operating pressure Flow rate Protein resolving ranges o C (recommended) 80 o C (maximum) <200 bar per column (recommended) 400 bar (maximum) ml/min for 7.8 mm ID ml/min for 4.6 mm ID kd for 130Å 5-1,250 kd for 300Å No compromise in operating conditions

18 Features of AdvanceBio SEC and resulting benefits Feature Advantages Benefits New high pore volume silica Increased resolution Accuracy of quantitation 300Å Pore size 130Å Pore size Unique hydrophilic polymeric bonded phase 2.7µm particle Higher mab aggregates are not excluded Resolution of mab fragments, small proteins, peptides Narrow symmetrical peaks High resolution with lower back pressure Quantitation of dimer and higher aggregates in single run More information about a wider range of samples Gain accuracy and reproducibility of results Performance benefits with HPLC and UHPLC systems 300 mm column length High resolution Increased accuracy of quantitation 150 mm column length Shorter analysis times Increased sample throughput 7.8 mm ID Robust separations Reproducibility 4.6 mm ID Increased sensitivity Able to quantify low level aggregation

19 To increase speed of analysis Sample: IgG I9640 Reduce the column length from 250 to 150 mm Increase Flow: 0.5mL/min, 1.0mL/min, 1.5mL/min Normalized and aligned 1.5 ml/min 0.5 ml/min 1.0 ml/min Run time reduced from 12 to 4 minutes 3X sample throughput Resolution maintained

20 Reproducibility protein standards: four production batches Oval Myo Protein Standards BioRad Gel Filtration test mix Batch RS Oval/Myo No batch or column qualification is required - Replace and go

21 Reproducibility mab aggregate samples: four production batches Mono Dim Target application Batch RS Dim/Mono Multiple batches available for customer validation

22 Sample integrity: recovery Five replicate injections of BSA 0.1mg/mL, 1µL equivalent to 0.1 µg of BSA on column BSA peak area is consistent even with 0.1 µg on-column loads Injection Monomer Peak Area No protein recovery issues observed Sample integrity maintained Accurate data

23 Sample integrity: run-to-run 1 2 Injection 1 Target application: mab monomer and aggregate(s) 1. Dimer 2. Monomer Injection 1 Rs monomer/ dimer Monomer 73.9% 74.3% Dimer 12.2% 11.5% Injection Injection 1500 Reproducible quantitation from injection 1 to injection reduces rework as column ages

24 Separations of different sample types using same columns and conditions mau mau C A Biotherapuetic mabs, innovators, biosimilars and ADC Rituximab Innovator min Cetuximab, Innovator min mau mau B Rituximab Biosimilar min D Herceptin Innovator min Samples Retention Time Mean (min) RSD Peak Area Mean (mau/min) RSD Rituximab Innovator Rituximab Biosimilar Cetuximab Innovator mau nm, Intact ADC 220nm, Heat/pH stressed ADC Intact ADC Herceptin Innovator ADC (T-DM1) Degraded ADC Aggregated ADC min The same column [AdvanceBio SEC-3, 300Å, 7.8 x 300 mm, 2.7 μm] and mobile phase: [(PBS) 50 mm sodium phosphate, 150 mm sodium chloride, ph 7.4] for mabs and ADCs - Eliminates stocking multiple column types - Eliminates multiple methods

25 Different types of samples: complex conjugated proteins PEGylated Proteins - increase bioavailability, increase serum half-life, decrease immunogenicity Pegfilgrastin (PEG GCSF)

26 Summary: AdvanceBio SEC delivers Accuracy of quantitation: Unique chemistry: improved peak shapes with high efficiency for increased resolution and provides better sensitivity for low level aggregate/fragment analysis. Unique chemistry: reduced non-specific interactions over a wide range of ph and salt concentrations to maintain sample integrity and provide high data accuracy Narrow particle size distribution and high pore volume: increased column efficiency and resolution Increase resolution: run two columns of the same pore size in series increases resolution Extended resolving range: run two columns of different pore size in series and enable analysis of multiple attributes in one run Decreased analysis time: run columns at higher flow rates; increases sample throughput Method robustness: Particle integrity: reduces soluble and insoluble column bleed for mass spec and light scattering detectors Control of manufacturing process: ensures batch-to-batch and column-to-column reproducibility Particle size and 2µm frit: reduces the risk of column clogging and early failure; operates at lower pressure. Increased pressure stability: reduces risk of column collapse with high viscosity eluents or during solvent transfers QA testing: application specific protein test mix

27 Acknowledgements o Thanks to co-author Linda Lloyd for helping to put together the presentation. o Thanks to Agilent R/D and Applications chemists in Church Stretton, UK and Wilmington, DE, USA for developing an innovative product and for generating the technical data.