Understanding the Effects of Common Mobile Phase Additives on the Performance of Size Exclusion Chromatography

Transcription

1 Understanding the Effects of Common Mobile Phase Additives on the Performance of Size Exclusion Chromatography 1

2 Presentation Importance of aggregate analysis in BioPharma manufacturing Analytical techniques used for aggregate analysis Agilent s aggregate analysis solution Routine workflow Advanced workflow Impact of mobile phase on protein characterization Summary 2

3 Importance of Analyzing Aggregates in Biopharma Researches need to make precise measurement of the extent of aggregation for their drug compound. Below are some of the challenges they are faced with: Typically need to demonstrate <1% aggregation (some studies use 5%) with precision and accuracy. What solution can I use that will offer me the analytical sensitivity and reproducibility I need? There isn t one analytical technique that is perfect to measure the wide MW range of protein aggregates. Which techniques should I be using? Maintaining sample integrity Traditional SEC columns and mobile phase can impact the sample integrity and result in poor peak shape and inaccurate data. Is there a solution I can use that will not introduce artifacts? 3

4 Approximate range of detectable protein sizes (diameter) of various analytical methods Aggregate Analytical Techniques SEC, SDS-PAGE Static & Dynamic Light Scattering AUC & FFF-MALS Sub-visible and Visible particles Microscope Flow Imaging Microscope Counter Visual Inspection Monomers & Oligomers Hans-Christian M Protein Aggregation : Pathways, Induction Factors and Analysis. Journal of Pharm. Science. Vol 98, No.9, Sep

5 Frequently Used Methods for the Analysis of Protein Aggregation Hans-Christian M Protein Aggregation : Pathways, Induction Factors and Analysis. Journal of Pharm. Science. Vol 98, No.9, Sep

6 Protein aggregation Aggregates may be a combination of two (i.e. dimer) or more (multimer) protein molecules Proteins aggregate when exposed to stress conditions This can include, but is not restricted to, upstream and downstream processes changes in concentration changes in ph changes in ionic strength changes in temperature exposure to surfaces or shear forces For accurate quantitation of the level of aggregation it is essential that the SEC analysis does not impact the sample integrity 6

7 Agilent s LC Aggregate Solution Size exclusion columns AdvanceBio SEC columns Standards AdvanceBio SEC Protein Standards Routine workflow 1260 Infinity II Bio-inert system with UV/DAD Advanced workflow With 1260 Infinity Multi-Detector Bio-SEC Solution 7

8 What if..? Would working with inert column buffers and chemistries benefit your analysis? You can start developing optimized, efficient separations while minimizing negative aggregation artifacts. Agilent AdvanceBio SEC column Are you concerned about your proteins and aggregates potentially having non-specific surface binding and interaction with your chromatography metal surfaces? Agilent s 1260 II Bio-Inert UHPLC system Do you need to report absolute, accurate, and reproducible molecular weights for your proteins, aggregates and biologics. Do you need to have full confidence in your reports for routine analysis and compliance submission? Agilent 1260 Infinity Multi-Detector Bio-SEC Solution 8

9 Size Exclusion Columns Agilent Bio-LC Columns Size Exclusion Restricted access to pore structure for larger molecules 4.6 x 150 mm or 7.8 x 150 mm for higher throughput, faster separations AdvanceBio SEC Bio SEC-3 Bio SEC-5 ProSEC 300S Zorbax GF250 & GF x 300 mm or 7.8 x 300 mm for higher resolution, increased sensitivity 9

10 What s different about Agilent s AdvanceBio SEC Columns? Columns must deliver accurate, precise quantitation for mabs and next generation biologics High resolution for more accurate quantitation Faster analysis speeds for delivery to deadlines Stable baselines with light scattering detection No change to sample integrity Analytical sensitivity for quantitation at low levels - 1 to 5% Methods must be easily transferred to other locations AdvanceBio SEC columns improve lab productivity by providing robust, reliable methods that eliminate sample re-analysis and increased sample throughput. 10

11 Standards Agilent AdvanceBio SEC Protein Standard, 300Å Agilent AdvanceBio SEC Protein Standard, 130Å Protein/Peptide MW Protein/Peptide MW 1. Thyroglobulin 670, Ovalbumin 45, γ-globulin 150, Myoglobin 17, Ovalbumin 45, Aprotinin 6, Myoglobin 17,000 4 Neurotensin 1, Angiotensin II 1, Angiotensin II 1,000 P No P No Norm. Norm Standards for column calibration system suitability/performance verification checks min min 11

12 Detecting and Quantifying mab Aggregation 4 # Time Area Area% Aggregates Fragments

13 1260 Infinity II Bio-inert LC System Aggregate Analysis Higher Analytical Sensitivity Aggregates Peak Detection by MSD Sensitive Aggregates by MDS Advance detector Quantitation by UV DAD Routine detector AdvanceBio SEC Higher Aggregates Peaks by MDS 13

14 Agilent 1260 Infinity II Bio-inert Quaternary LC The New Standard in Bioanalysis 100% Bio-inert Precious sample does not touch metal surfaces ph range 1-13 (shortterm 14) 2 M salt, 8 M urea No stainless steel/iron in mobile phase flow path Special bio-inert capillary technology New Bio-inert Multisampler for ultralow carry-over UHPLC capability 600 bar Ease of Use and Robustness Corrosion resistant Active seal wash Quaternary buffer mixing Bio-HPLC columns for biotherapeutic characterization The choice for both, bioanalytical and biopurification up to 10 ml/min 14

15 Impact of Mobile Phase on SEC Trastuzumab 5mg/mL 150mM Sodium phosphate, ph 7 Phosphate Buffered Saline (PBS) The 150 mm sodium phosphate mobile phase gives imporved peak shape and resolution compared to the phosphate buffered saline but is this the optimum? 15

16 Buffer Advisor and How It Helps You BufferAdvisor is an independent utility software Provides the foundation for automated mixing of acid, base, water and salt to prepare on-instrument mobile phases Can help calculate the selected buffer concentration and ph with high accuracy Can suggest the most suitable stock solutions and even provides recipes Includes more than 50 different buffers and more than 20 were experimentally evaluated Has additional option to add user defined buffers 16

17 Buffer Advisor Simplifies Size Exclusion Method Development Ease of use Automates preparation of mobile phases from stock solutions Eliminates the need to prepare multiple mobile phases for a method development/robustness study Provides recipes for buffer preparation Increase of productivity Simplifies ph scouting studies Simplifies salt concentration/mobile additive studies Increase of Robustness and run to run repeatibility Compensates for ph shifts in salt gradients Calculates optimized gradient steps 17

18 Buffer Advisor Composition for Robustness Investigation Used 20 different mobile phase compositions to determine impact of : buffer mm salt to 50 mm ph to 7.5 Expt % A % B % C % D Init. ph Buffer mm NaCl mm IS 18

19 Robustness Differing buffer composition at different ph values mau ph ph 6.5 * 200 * 150 mau 100 * Ovalbumin, Vitamin B12 * * min min mau mau ph 7.0 ph 7.5 * 200 * 150 * * min min 19

20 Robustness Impact of phosphate concentration and ph on resolution Rs IgA/IgG Rs Ovalbumin/Myoglobin ph 6.0 ph ph 6.0 ph 6.5 ph 7.0 ph ph ph Phosphate mm Phosphate mm 20

21 The Advantage of Buffer Advisor Manual method One bottle for each mobile phase vs BufferAdvisor method Four bottles only Buffer Advisor helps you achieve more in less time 21

22 Bio-inert 1260 and MDS Solution Size exclusion chromatography (SEC) is the standard method used to determine and quantify monomers, dimers, aggregates, and potential degradants. The Agilent 1260 Infinity Multi-Detector Bio-SEC Solution is optimized for reproducible advanced analysis of protein-based pharmaceuticals. Light scattering detection is essential to be able to report absolute, accurate, and reproducible molecular weights. The light scattering detector shows much greater analytical sensitivity to the aggregation present. Agilent is the only company able to supply all the components required for the characterization of biomolecules, instruments, columns, detectors, and dedicated Bio-SEC software. 22

23 Require Columns with Low Particulate Bleed Traditional Diol Chemistry Column (15 Baseline) AdvanceBio SEC 300Å (15 Baseline) Improved baseline stability with the AdvanceBio SEC column 23

24 Increase Aggregate Sensitivity with LS detection Advance aggregate workflow adds critical information 24

25 Agilent 1260 Infinity Bio-Inert SEC Multi-Detector Solution (MDS) Accurate Molecular Weight Determination The 1260 Infinity Multi-Detector Bio-SEC is a solution for sizing and aggregation studies of proteins and other large biomolecules using Size Exclusion Chromatography The Agilent 1260 Infinity Bio-inert LC System combines true bio-inert sample flow path with UHPLC power range. The sample never touches metal surfaces since there is no stainless steel in the mobile phase flow path. The combined Static and Dynamic Light Scattering Detector simultaneously determines the absolute Molecular Weight (Mw) as well as the size of the molecule (Rh). Agilent Bio-SEC Software is a dedicated Chromatography Data System for an easy, yet powerful, workflow based solution 25

26 Agilent 1260 Infinity Bio-SEC Solution A Monoclonal Antibody (mab) Linear response to all species UV (280nm) Higher order aggregates Dimer Monomer Fragments LS (90 ) More sensitive to aggregates Rh (nm) Size in solution The dynamic light scattering response has been used to determine the hydrodynamic radius, Rh, and is represented by the purple spots 26

27 MDS Bio SEC Software 27

28 Simple to Use Data Analysis Software - Routine Analysis Wizard How Agilent s Bio-SEC Data Analysis works - in 5 clicks? Define Baseline Data fit limits can be All viewed parameters and adjusted are if drawn required from Results include Define peak(s) Molar Mass, the instrument size, automatically and Concentration saved in the and % recovery sample file 28

29 Does surfactant impact size exclusion chromatography? Surfactants may be added to protein formulations to stabilize a protein in solution or to denature a protein as in SDS-PAGE Size exclusion chromatography is normally performed under non-denaturing conditions but additives such as surfactants may be added to the mobile phase In this study size exclusion chromatography together with light scattering detection is used to investigate the impact that sodium dodecyl sulphate (SDS) on SEC data, protein retention time, apparent molecular weight and hydrodynamic radius 29

30 Sodium Dodecyl Sulphate (SDS) O O S O O - n 62 Na + Micelles form when ~ 62 SDS molecules associate into a spherical structure with the hydrophobic core at the centre and the change end facing outward To denature proteins SDS is used above its critical micelle concentration (CMC) - typically 2% SDS Micelle 30

31 Bovine Serum Albumin Native heart shaped conformation Process of protein denaturation SDS below CMC protein becomes elongated (in theory) SDS above CMC protein become decorated with micelles (in theory; beads on a string ) 31

32 SEC Chromatograms of BSA with Increasing SDS (UV, 280nm) 0.0% SDS in PBS 0.2% SDS in PBS 0.4% SDS in PBS 1.4% SDS in PBS Using UV detection the level of SDS does not impact the overall shape of the chromatogram with monomer, dimer and aggregates present However the as the concentration of SDS increases so the peaks are shifted to the left 32

33 Retention Time and Total Peak Area % SDS in Mobile Phase Retention Time (min) Higher Order Aggregates Dimer Monomer Total Peak Area 0.0% % % % % % % % Retention Time (min) % 0.4% 0.9% 1.4% % SDS in Mobile Phase Higher Order Aggregates Dimer Monomer At low concentrations of SDS retention times are reduce indicating a increase in protein size At concentrations above 0.4% SDS there is no further reduction in retention time (increase in size) 33

34 Use of Light Scattering Detector to Determine Molecular Weight UV 280 nm LS Higher order aggregates 2. Dimer 3. Monomer 1 34

35 Use of Light Scattering Detector to Determine Molecular Weight SDS w/w% Molecular Weight Higher Order Aggregates Dimer Monomer Number of Monomer Units Higher Order Aggregate s Dimer Monomer As the concentration of SDS increases the molecular weight increases but then stabilizes at an SDS concentration of 0.6% The number of monomer units is consistent with monomer, dimer and trimer 35

36 Mass Gain for BSA with Increasing SDS Concentration SDS w/w% Mass Gain SDS : BSA (g / g) Higher Order Aggregates Dimer Monomer Higher Order Aggregates Dimer Monomer Plateau 36

37 Comparison of UV 280 nm Signal of BSA in 0.0% SDS and 1.4% SDS 0.0% SDS in PBS 1.4% SDS in PBS Less resolution Low molecular weight peaks 37

38 Overview of LS 90 Signal of BSA in 0.0% SDS and 1.4% SDS LS 90 ; 0.0% SDS in PBS LS 90 ; 1.4% SDS in PBS Increased peak area is seen at higher SDS concentrations LS detector is mass sensitive and not concentration dependent as UV Vertical offset is attributed to background due to the presence of SDS micelles in the mobile phase Vertical offset 38

39 Overview of LS 90 in 0.0% SDS and 1.4% SDS with Rh added Vertical offset is attributed to background due to the presence of SDS micelles in the mobile phase 39

40 Does surfactant impact size exclusion chromatography? Advance detector 1260 Infinity II Bio-inert LC System Agilent 1260 Infinity Multi-Detector AdvanceBio SEC columns Using the Agilent Advance workflow for SEC it is possible to answer the question - YES 40

41 Conclusion Bio-inert 1260 and MDS Solution Agilent offers a routine solution to determine and quantify monomers, dimers, aggregates, and potential degradants based around UV DAD detection The Agilent 1260 Infinity Multi-Detector Bio-SEC Solution is optimized for reproducible advanced analysis of protein-based pharmaceuticals Light scattering detection is essential to be able to report absolute, accurate, and reproducible molecular weights. While providing greater analytical sensitivity for aggregation analysis Agilent is a one-stop company. We are able to supply all the components required for the characterization of biomolecules, instruments, columns, standards, UV, DAD, LS detectors, and dedicated Bio-SEC software. 41

42 Thank you 42