Gentle Bioanalysis of Proteins

Transcription

1 Gentle Bioanalysis of Proteins APPLICA 2017 Patrick Endres, Judith Vajda, Egbert Müller GmbH September 7 th, 2017

2 Demand on chromatographic analysis Fast Inexpensive (Column and buffer) Robust (Easy method development) (Non-denaturing) à Is RPC always the best option? I think not! 2

3 Small Molecules large Molecules Convential API s Biologics Sizing factor aspirin 180,16 g/mol IgG > g/mol *EvaluatePharma World Preview 2014, Outlook to

4 Biological Products are complex! Amino acid sequence Secondary structure Alpha, beta Tertiary structure Protein folding Quaternary structure Spherical arrangement of different domains Plus: glycosylation, deamidation, hydroxylation etc. 4

5 Monoclonal Antibodies >60 approved mabs 5 of the top 10 drugs are mabs #1: Humira with 16 billion US $ (2016) > 600 candidates in clinical trials λ or κ light chain Characterization: Antigen binding Glycosylation site Fab Size Charge variants Glycosylation Peptides after enzymatic digest Biological activity Fc Synthetic modifications *Zahlen 2017, FDA & Wikipedia 5

6 Chromatography Toolbox Characteristic Size Charge Hydrophobicity Titer LC method SEC (gel filtration) IEX RPC/HIC Analytical Protein A 6

7 MAb Heterogeneity Aggregation Fragmentation Incorrect disulfide bonds N-terminal cyclization (Glu/Gln àpglu) Deamidation (AsnàAsp) Isomerization (AspàisoAsp) Glyco pattern Met oxidation Artificial modifications (PEG, isotopes) C-terminal Lys truncation Characterization by Size Exclusion Chromatography 7

8 Size Exclusion Chromatography Separation of molecules according to the hydrodynamic radius Larger molecules have no or limited pore access Non-adsorptive Column Calibration TSKgel SuperSW mab HR 1000 MW [kda] , retention time [min] 8

9 Speed or the applied flow rate Separation in SEC relies on pore diffusion High flow rates decrease separation performance mab aggregate separation on TSKgel UP-SW cm L 0,06 0,05 0,04 y = 0,0565x + 0,0154 R² = 0,98805 H [mm] 0,03 0,02 0, ,2 0,4 0,6 0,8 superficial velocity [mm/s] àthere is always a trade-off between performance and time 9

10 SEC-UHPLC UHPLC systems: Smaller system dead volume Optimized detector flow cells Can be operated at pressures >1000 bars UHPLC columns: Particle size, ID, superficial velocity, backpressure Two dimensions: 4.6 mm ID x 15 cm à fast analysis and high throughput. 4.6 mm ID x 30 cm à maximum performance. Guard columns for direct coupling reduce dead volume Increase of efficiency: time-wise or performance-wise 10

11 Charge Variants Various changes at amino acids and a varying sialinic acid content can lead to charge variants of a mab. Aggregation Fragmentation Incorrect disulfide bonds N-terminal cyclization (Glu/Gln àpglu) Deamidation (AsnàAsp) Isomerization (AspàisoAsp) Glyco pattern Met oxidation Artificial modifications (PEG, isotopes) C-terminal Lys truncation 11

12 Specifications of IEX analytical Columns Hydrophilic polymerbeads (7 µm) are alkaline resistant Non-porous particles have fast mass transfer properties Innovative surface design à comparatively high capacity for a nonporous resin Fast, high-resolution power for the analysis of proteins and peptides TSKgel CM-STAT Carboxymethyl ligand (WCX) TSKgel SP-STAT - Sulfopropyl ligand (SCX)

13 Example: C-terminal Lysine mau K K K digested with carboxypeptidase B Column: TSKgel CM-STAT (4.6 x 100 mm) Eluent A: 20 mmol/l MES (ph 6.0) Eluent B: 20 mmol/l MES mol/l NaCl (ph 6.0) Gradient: 0 min 10 %B 15 min 30 %B 15.1 min 100 %B 18 min 100 %B 18.1 min 10 %B Flow rate: 1.0 ml/min Detection: UV 280 nm Temp.: 25 Inj. vol.: 20 µl Conc. : 0.5 g/l Samples: Therapeutic antibody, treated and untreated with carboxypeptidase B min before digestion Procedure: To a 35 µl of therapeutic antibody (10 g/l), 1 µl of carboxypeptidase B (Sigma C9584, 140 U/mg protein, 5 g/ L in PBS) was added and incubated for 3 hr at 37. After adding 664 µl of 20 mmol/l MES (ph 6.0) to dilute the antibody concentration of 0.5 g/l, 20 µl of the diluted sample was injected. Varying number of positive charges at a mab (lysine) can be resolved by CEX 13

14 Why are UHPLC systems beneficial for IEX? mab separation (TSKgel SP-STAT 4.6 x 100 mm) µm UHPLC system HPLC system Comparison of Resolution and plate numbers 14

15 Benefits of a UHPLC System - IEX 1 ml/min, A: 10 mm phosphate, ph 7.0, B: A + 1 M NaCl, gradient: 0-50 % B in 25 min, 5 µl Injection volume. Peaks are sharper: higher resolution and plate numbers Due to smaller dead volume: Elution occurs earlier à Increase Gradient delay volume to keep the same integration method 15

16 Artificial Modifications Artifical modifications can be introduced for diagnostic use, to prolong half-life or to increase potency of a therapy Aggregation Fragmentation Incorrect disulfide bonds N-terminal cyclization (Glu/Gln àpglu) Deamidation (AsnàAsp) Isomerization (AspàisoAsp) Glyco pattern Met oxidation Artificial modifications (PEG, ADCs) C-terminal Lys truncation 16

17 Modern MAb formats Antibody - Drug - Conjugates Lysine Conjugation Cysteine Conjugation Site directed conjugation *Genentech, World ADC Summit US

18 Hydrophobic Interaction Chromatography Analogue to reversed phase in biochromatography Ligands comparable to RPC, often shorter alkyl chains and lower ligand density Mild conditions Hydrophobic interactions induced by high salt concentrations Elution in a decreasing salt gradient TSKgel Butyl-NPR: Non-porous base material C4 ligand 18

19 ADC Drug/Antibody Ratio (DAR) mabs (e.g. Herceptin) are usually characterized by SEC BUT: conjugates are often too small to resolve ADC from mab but are hydrophobic (Drug Size: ~750 Da) RPC (small molecules) - HIC (proteins) Herceptin ADC DAR=2 DAR=0 DAR=4 DAR=6 DAR=8 Column: TSKgel Butyl-NPR (2.5 µm, 4.6 x 100 mm) Eluent: A) 25 mmol/l phosphate (ph 7.0),1.5 mol/l ammonium sulfate B) 25 mmol/l phosphate (ph 7.0) / 2-propanol = 8 / 2 Gradient: % B (20 minutes) Flow rate: 0.5 ml/min Detection:: 280 nm Injection: 10 µl Sample: Herceptin; 0.24 g/l, ADC 2.2 g/l 19

20 Things to consider Low pressure or high pressure gradient? Flowrate is not constant with high pressure gradient due to excess volume 20

21 Temperature 40 C 40 C T 40 C y 50 C 30 C T 40 C y 21

22 Temperature 40 C 40 C 40 C Band broadening due to laminar flow profile Temperature Laminar flow profile Combined effect 50 C 30 C 40 C Sharper peak due to combined effect of laminar flow profile and viscosity 22

23 Analytical Protein A Chromatography Most of the monoclonal antibody biotherapeutics on the market today are based on IgG 1. Interest in IgG 2 and IgG 4 is rapidly growing. These samples must be screened for mab titer; affinity protein A columns are often employed for this purpose. With many samples to be screened for different purposes, a reliable and high throughput column is needed for this workflow. 23

24 High Flow Rates for High Throughput Analysis 20 µl of CHO cell supernatant spiked with polyclonal antibody (0.5 mg/ml) TSKgel Protein A-5PW column shows similar recovery of IgG up to 4.0 ml/min. Less than 1 minute analysis was available at 4.0 ml/min with similar peak profile. 24

25 Dynamic Range and Linearity Sample: Purified polyclonal IgG TSKgel Protein A-5PW column shows a wide dynamic range from g/l (2-200 µg) with good linearity (R 2 > 0.999) for IgG. 25

26 Durability study using CHO crude Feedstock containing IgG 1 Cleaning protocol reversed flow at 0.5 ml/min for 20 CV: a. 0.1 mol/l NaOH b. DI Water c. 1 mol/l acetic acid normal flow for 20 CV: a. DI water b. 0.5 mol/l sodium phosphate, ph CV: 20mm sodium phosphate ph 7.4 The column can be used with high flow rate while still maintaining peak area consistency with RSD of 1.7% The column was cleaned after 1230 injections using a stepwise cleaning protocol 26

27 Summary Points to consider for method transfer from HPLC to UHPLC of biomolecules mab aggregate analysis can be accomplished in 4 min! UHPLC systems are beneficial for separation efficiency Charge variants can rapidly be analyzed with non-porous stationary phases connected to UHPLC systems Antibody-drug-conjugates (ADC) can be analyzed with HIC Titer determenation with analytical Protein A Chromatography

28 Acknowledgements Dr. Judith Vajda (All Data besides Protein A) Keegan Gyke (Protein A Data) PD Dr. Egbert Müller 28

29 Questions? Phone: Mail: Im Leuschnerpark Griesheim Germany