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Gentle Bioanalysis of Proteins APPLICA 2017

Patrick Endres, Judith Vajda, Egbert Müller

Tosoh Bioscience GmbH September 7th, 2017

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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!

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Small Molecules – large Molecules

•  Convential API‘s Biologics

*EvaluatePharma World Preview 2014, Outlook to 2020

aspirin 180,16 g/mol IgG > 150.000 g/mol

Sizing factor 100-1000

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Biological Products are complex!

Quaternary structure Spherical arrangement of different domains

Amino acid sequence Secondary structure Alpha, beta

Tertiary structure Protein folding

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Plus: glycosylation, deamidation, hydroxylation… etc.

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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

Characterization: •  Size •  Charge variants •  Glycosylation •  Peptides after enzymatic digest •  Synthetic modifications

*Zahlen 2017, FDA & Wikipedia

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Fc

Fab Antigen binding

Biological activity

Glycosylation site

λ or κ light chain

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Chromatography Toolbox

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Characteristic LC method Size SEC (gel filtration)

Charge IEX Hydrophobicity RPC/HIC

Titer Analytical Protein A

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MAb Heterogeneity

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Characterization by Size Exclusion Chromatography

Aggregation

Fragmentation

Incorrect disulfide bonds

Met oxidation

C-terminal Lys truncation

N-terminal cyclization (Glu/Gln àpGlu)

Deamidation (AsnàAsp)

Isomerization (AspàisoAsp)

Glyco pattern

Artificial modifications (PEG, isotopes)

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Size Exclusion Chromatography

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•  Separation of molecules according to the hydrodynamic radius •  Larger molecules have no or limited pore access •  Non-adsorptive

0,1

1

10

100

1000

6 8 10 12 14 16

MW

[kD

a]

retention time [min]

Column Calibration TSKgel SuperSW mAb HR

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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-SW3000 30 cm L

àthere is always a trade-off between performance and time

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y = 0,0565x + 0,0154 R² = 0,98805

0

0,01

0,02

0,03

0,04

0,05

0,06

0 0,2 0,4 0,6 0,8 H

[mm

] superficial velocity [mm/s]

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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

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Increase of efficiency: time-wise or performance-wise

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Charge Variants

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Aggregation

Fragmentation

Incorrect disulfide bonds

Met oxidation

C-terminal Lys truncation

N-terminal cyclization (Glu/Gln àpGlu)

Deamidation (AsnàAsp)

Isomerization (AspàisoAsp)

Glyco pattern

Artificial modifications (PEG, isotopes)

Various changes at amino acids and a varying sialinic acid content can lead to charge variants of a mAb.

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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 non-

porous resin •  Fast, high-resolution power for the analysis of proteins and peptides •  TSKgel CM-STAT – Carboxymethyl ligand (WCX) •  TSKgel SP-STAT - Sulfopropyl ligand (SCX)

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- - - - - -

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- - - - - - - - -

- - -

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Example: C-terminal Lysine

Column: TSKgel CM-STAT (4.6 x 100 mm) Eluent A: 20 mmol/L MES (pH 6.0) Eluent B: 20 mmol/L MES + 0.5 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 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.

min0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

mAU

-5

0

5

10

15

20

25

30

35

40

45

50

55

before digestion

digested with carboxypeptidase B

K KK

Varying number of positive charges at a mAb (lysine) can be resolved by CEX

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UHPLC system HPLC system

mAb separation (TSKgel SP-STAT 4.6 x 100 mm)

Comparison of Resolution and plate numbers

- - - - - -

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- - - - - - - - -

- - -

7 µm

Why are UHPLC systems beneficial for IEX?

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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.

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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

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Artificial Modifications

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Aggregation

Fragmentation

Incorrect disulfide bonds

Met oxidation

C-terminal Lys truncation

N-terminal cyclization (Glu/Gln àpGlu)

Deamidation (AsnàAsp)

Isomerization (AspàisoAsp)

Glyco pattern

Artificial modifications (PEG, ADCs)

Artifical modifications can be introduced for diagnostic use, to prolong half-life or to increase potency of a therapy

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Lysine Conjugation Cysteine Conjugation Site directed conjugation

*Genentech, World ADC Summit US 2011

Modern MAb formats

Antibody - Drug - Conjugates

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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

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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

AD CDAR=0

DAR=2 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: 0 - 100 % B (20 minutes) Flow rate: 0.5 mL/min Detection:: UV @ 280 nm Injection: 10 µL Sample: Herceptin; 0.24 g/L, 　

ADC 2.2 g/L

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Things to consider •  Low pressure or high pressure gradient?

20 http://www.ddbst.com/en/EED/VE/Images/VE0%202-Propanol;Water_001.png

Flowrate is not constant with high pressure gradient due to excess volume

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Temperature

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50°C

30°C T

y

40°C

40°C

40°C T

y

40°C

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Temperature

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40°C

40°C 40°C

50°C

30°C 40°C

Band broadening due to laminar flow profile

Sharper peak due to combined effect of laminar flow profile and viscosity

Temperature Laminar flow profile Combined effect

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Analytical Protein A Chromatography •  Most of the monoclonal antibody biotherapeutics on the market today

are based on IgG1. Interest in IgG2 and IgG4 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.

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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.

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Dynamic Range and Linearity

Sample: Purified polyclonal IgG

TSKgel Protein A-5PW column shows a wide dynamic range from 0.1 - 10 g/L (2 - 200 µg) with good linearity (R2 > 0.999) for IgG.

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• 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

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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 6.5

50 CV: 20mm sodium phosphate pH 7.4

Durability study using CHO crude Feedstock containing IgG1

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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

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Acknowledgements •  Dr. Judith Vajda (All Data besides Protein A)

•  Keegan Gyke (Protein A Data)

•  PD Dr. Egbert Müller

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Questions?

http://www.separations.eu.tosohbioscience.com/ E-mail: info.tbg@tosoh.com Phone: +49 6155 7043700 Mail: Im Leuschnerpark 4

64347 Griesheim Germany