Developing Quantitative UPLC Assays with UV

Transcription

1 Developing Quantitative UPLC Assays with UV Detection for Antibodies & Other Proteins Steve Taylor 2011 Waters Corporation 1

2 Outline UPLC technology for RP protein separations Method development parameters for RP protein separations Quantitation of proteins and their variants Conclusions 2011 Waters Corporation 2

3 ACQUITY UPLC H-Class Bio UPLC designed for biomolecules 15,000 psi maximum pressure capability Quaternary solvent blending Flow-through needle sample manager Stainless steel free flow path AutoBlend Plus Technology Automates blending of acid modifiers and multiple organic solvents Automates formation of ph buffers Excellent gradient reproducibility Minimized dispersion 2011 Waters Corporation 3

4 Columns for RP UPLC of Proteins ACQUITY UPLC BEH300 C4 300Å pore size C4 ligand Minimal secondary interactions Long column life at elevated temperature Minimal carryover and maximum recovery 2011 Waters Corporation 4

5 Outline UPLC technology for RP protein separations Method development parameters for RP protein separations Quantitation of proteins and their variants Conclusions 2011 Waters Corporation 5

6 Influencing Protein Separations Operating Factors Mobile phase modifier Temperature Column Length Organic solvent Gradient slope Flow rate 2011 Waters Corporation 6

7 Effects of Mobile Phase Modifier Trifluoroacetic acid (TFA) is commonly preferred for good chromatography with proteins and peptides Formic acid (FA) often used for increased signal in MS For quantitative LC assays with UV detection TFA is the preferred modifier 2011 Waters Corporation 7

8 Effect of Acid Concentration Protein Mix % TFA 0.15 A B C D E F A. Ribonuclease B. Cytochrome c C. BSA D. Myoglobin E. Enolase F. Phosphorylase b % TFA % 02% TFA Change selectivity by changing modifier concentration Waters Corporation 8

9 Protein Separation in RP LC Effects of Increasing Temperature Benefits Usually reduces retention Often improves protein peak shape Often improves recovery Often improves resolution by changing selectivity Often makes peaks narrower, but not universally Risks May precipitate proteins May promote chemical modification May increase denaturation 2011 Waters Corporation 9

10 Recovery with Increasing Temperature Intact Murine IgG, BEH300 C % TFA, ACN separation C C C With the monoclonal IgG, elevated temperature is absolutely required for reasonable chromatography Waters Corporation 10

11 Example of Increasing Temperature 0.02% TFA separation A. Ribonuclease B. Cytochrome c C. BSA D. Myoglobin E. Enolase F. Phosphorylase b A B C D E 40ºC 0.08 F Elution order reversal ºC 0.16 A U Waters Corporation 11

12 Effect of Column Length on Separation x 50mm x 150mm Waters Corporation 12

13 Effect of Solvent on Separation at 40ºC 0.1% TFA ACN MeOH EtOH :3 IPA:ACN 5 1 IPA Ribonuclease 2. Cytochrome c 3. BSA 4. Myoglobin 5. Enolase 6. Phosphorylase b Waters Corporation 13

14 Effect of Solvent on Separation at 80ºC Mixture of IgG Monoclonal Antibodies; 0.1% TFA ACN H C M RsH,C = MeOH Rs H,C = coelution H, C EtOH H C M Rs H,C = : IPA:ACN H C M 1.20 IPA H C M ACQUITY UPLC BEH300 C4, 1.7µm, RsH,C = x 50 mm A: 0.1% TFA in waters B: 0.1% TFA in organic solvent 0.2mL/min 20% B to 71.4% B over 29.6 mins, 80 C Detection: 220nm H Humanized; C Chimeric; M Murine Rs H,C = Waters Corporation 14

15 Observed Backpressures (0.2mL/min, 1.7 µm, BEH300 C4 2.1x150mm column) 1.5%/ Column Volume Gradient Slope Mobile Phase B 100% ACN 7:3 IPA: ACN 100% IPA 100% MeOH Temp (ºC) Initial PSI Highest PSI ~ % B at Highest PSI % EtOH values are approximate 2011 Waters Corporation 15

16 ACQUITY UPLC H-Class Bio AutoBlend Plus 95% 0% 45% 50% Water Acetonitrile Water Acetonitrile 0% 5% 0% 5% Isopropanol 1%TFA Isopropanol 1%TFA 100% Water 50% Water 0% Acetonitrile 50% Acetonitrile 0.05% TFA 0.05% TFA Automatically blends up to four solvents formation of desired % TFA or other modifiers (e.g. 0.1% vs 0.05% TFA) formation of new organic solvent mixture (e.g. ACN vs 7:3 IPA:ACN) Routine assays become more rugged (less human error) Exploring different acid modifier percentages and different blends of organic solvents is simplified 2011 Waters Corporation 16

17 Effect of Gradient Slope on Separation of a Monclonal Antibody Mix; 0.1% TFA, ACN, 80ºC %/CV R H,C = %/CV R H,C = %/CV R H,C = Waters Corporation 17

18 Gradient Slope Effects Myoglobin / Enolase Rs Enolase Height () Res solution Pe eak Height () Gradient slope (%B/CV) 2011 Waters Corporation 18

19 Effect of Flow Rate; ACN Separation 2.1mm i.d. column µl/min µl/min Waters Corporation 19

20 Outline UPLC technology for RP protein separations Method development parameters for RP protein separations Quantitation of proteins and their variants Conclusions 2011 Waters Corporation 20

21 Wavelength, sensitivity & detector saturation Humanized IgG4 quantitation at 220nm 2.40 ACQUITY UPLC BEH300 C4 2.1x50mm 0.1% TFA, IPA 20-37%B 0.2mL/min 3.3 µl injection 1%/CV gradient slope TUV at 220 nm µg 0.05 µg 10 µg 7.5 µg 5 µg 1 µg 0.5 µg Detector saturation at 220nm limits dynamic range to a 50-fold range of concentrations Waters Corporation 21

22 280nm for wider dynamic range applications Humanized IgG4 Quantitation nm ACQUITY UPLC BEH300 C4 2.1x50mm µg on column nm Peak height reduced Baseline drift reduced 10x U A Waters Corporation 22

23 Linear Dynamic Range at 280 nm Amount on Area Column (µg) Avg. Area % RSD Humanized IgG μg on column y = 55003x R² = Area Humanized IgG Triplicate injections µg on column 2011 Waters Corporation 23

24 Impurity Quantitation (murine IgG spiked into humanized IgG) 50 µg API on Column % % % % % % Waters Corporation 24

25 Quantitating Impurities in Presence of Large Amount of Protein Impurity API Nominal % of API Measured % of API Area % RSD Area % RSD %RSD values based on 5 replicates 2011 Waters Corporation 25

26 Carryover Carryover and memory effects appearance of constituents of one sample in the next gradient analysis Must determine if the source of carryover is system or column Define a method with an internal gradient analytical gradient repeated, without any injection being made. o If peak appears in the second gradient at the same time after beginning of gradient, then carryover is memory effect, due to the column o If protein only appears when an injection is made, then carryover is due to adsorption onto a system component 2011 Waters Corporation 26

27 Effect of Temperature on Carryover ºC nd gradient start Protein Mix carryover ºC Waters Corporation 27

28 5µg injection of mabs Effect of Organic Solvent on Carryover 1.20 IPA, 0.1% TFA, 80ºC <0.1% carryover nd gradient start 1.20 MeOH, 0.1% TFA, 80ºC ~30% carryover Waters Corporation 28

29 Observed Carryover Protein Mix; 0.1% TFA Approximate Carryover Mobile Phase B1 100% ACN 7:3 IPA:ACN 100% IPA *100% MeOH Protein 40ºC 80ºC 40ºC 80ºC 40ºC 80ºC 40ºC 80ºC Ribonuclease ND ND ND ND ND ND 2 3 Cytochrome c ND ND ND ND ND ND 1 1 Bovine Serum Albumin 0.4 <0.1 ND <0.1 ND ND 5 18 Myoglobin 0.6 ND 0.6 ND 7 ND Enolase 0.1 ND 0.1 ND 15 ND ND ND Phosphorylase b ND ND <0.1 ND 0.2 ND ND 6 ND Not detected * Further testing required to confirm accuracy 2011 Waters Corporation 29

30 Effect of Gradient Slope on Carryover 0.1% TFA, IPA, 20-37% B, 0.2 ml/min, 3.3 µl injection Gradient Slope 1st Gradient Area Relative Area Column temperature 80 C TUV at 220 nm 2nd 3rd Total Gradient Gradient Total Area Relative Area Area Area 3%/CV % Carryover %/CV % Carryover %/CV % Carryover Waters Corporation 30

31 Effect of Mass Load on Carryover µg Humanized IgG4 Mass loads (µg) % Carryover 0.5 N/A 1 N/A A: 0.1% TFA in water; B: 0.1% TFA in IPA; 0.2 ml/min 20-37%B in 14.7min (1% per CV), with 2 nd internal gradient; 3.3 µl injection; TUV at 280 nm; 80 C column tem BEH300 C4 1.7 µm 2.1x50 mm 35 2 nd gradient carryover < 20% of LLOQ 00 2 nd gradient start Waters Corporation 31

32 Minimizing Column-related Carryover Minimizing memory effect Raising temperature can minimize incomplete elution of protein Replace part or all of organic solvent Decrease gradient slope Decrease flow rate (increase gradient time accordingly) Decrease mass load of protein on column, if possible Include a series of fast regeneration gradients (sawtooth gradients) 2011 Waters Corporation 32

33 Method Development and Quantitation Summary ACQUITY UPLC H-Class Bio and BEH300 C4 columns Exploring % acid modifier, temperature, organic solvent mixtures, gradient slope and flow rate Develop protein separations with good resolution, good peak shape, wide dynamic range, good sensitivity & low carry-over Column and system design optimised for proteins Reproducible quantitation across three orders of magnitude can be obtained for API s and low-level l l impuritiesiti 2011 Waters Corporation 33

34 Relevant literature downloads Search waters.com for these literature titles: Reversed-Phase Analysis of Proteins Using ACQUITY UPLC H- Class Bio and AutoBlend Plus Quantitation of Monoclonal Antibodies Using Reversed-Phase Liquid Chromatography Using the BEH300 C4 Column Chemistry Developing Separations of Monoclonal Antibodies And Other Proteins Using Reversed-Phase UPLC Protein Separation Technology Columns Brochure ACQUITY UPLC H-Class Bio System 2011 Waters Corporation 34