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

    Gentle Bioanalysis of Proteins APPLICA 2017

    Patrick Endres, Judith Vajda, Egbert Mller Tosoh Bioscience GmbH September 7th, 2017

  • Tosoh Bioscience

    Demand on chromatographic analysis Fast

    Inexpensive (Column and buffer)

    Robust

    (Easy method development)

    (Non-denaturing)

    Is RPC always the best option? I think not!

    2

  • Tosoh Bioscience

    Small Molecules large Molecules

    Convential APIs Biologics

    *EvaluatePharma World Preview 2014, Outlook to 2020

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

    Sizing factor 100-1000

    3

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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

    4

    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

    5

    Fc

    Fab Antigen binding

    Biological activity

    Glycosylation site

    or light chain

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    Chromatography Toolbox

    6

    Characteristic LC method Size SEC (gel filtration)

    Charge IEX Hydrophobicity RPC/HIC

    Titer Analytical Protein A

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    MAb Heterogeneity

    7

    Characterization by Size Exclusion Chromatography

    Aggregation

    Fragmentation

    Incorrect disulfide bonds

    Met oxidation

    C-terminal Lys truncation

    N-terminal cyclization (Glu/Gln pGlu)

    Deamidation (AsnAsp)

    Isomerization (AspisoAsp)

    Glyco pattern

    Artificial modifications (PEG, isotopes)

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    Size Exclusion Chromatography

    8

    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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    Speedor 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

    there is always a trade-off between performance and time

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    y = 0,0565x + 0,0154 R = 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 fast analysis and high throughput. 4.6 mm ID x 30 cm 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 pGlu)

    Deamidation (AsnAsp)

    Isomerization (AspisoAsp)

    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 m) are alkaline resistant Non-porous particles have fast mass transfer properties Innovative surface design 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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    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 L Conc. : 0.5 g/L Samples: Therapeutic antibody, treated and

    untreated with carboxypeptidase B Procedure: To a 35 L of therapeutic antibody (10 g/L), 1 L 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 L of 20 mmol/L MES (pH 6.0) to dilute the antibody concentration of 0.5 g/L, 20 L 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

    13

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    UHPLC system HPLC system

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

    Comparison of Resolution and plate numbers

    - - - - - -

    - - - - - -

    - - - - - - - - -

    - - -

    7 m

    Why are UHPLC systems beneficial for IEX?

    14

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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 L Injection volume.

    15

    Peaks are sharper: higher resolution and plate numbers

    Due to smaller dead volume: Elution occurs earlier Increase Gradient delay volume to keep the same integration method

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    Artificial Modifications

    16

    Aggregation

    Fragmentation

    Incorrect disulfide bonds

    Met oxidation

    C-terminal Lys truncation

    N-terminal cyclization (Glu/Gln pGlu)

    Deamidation (AsnAsp)

    Isomerization (AspisoAsp)

    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

    17

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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

    18

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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 m, 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 L Sample: Herceptin; 0.24 g/L,

    ADC 2.2 g/L

    19

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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

  • Tosoh Bioscience

    Temperature

    21

    50C

    30C T

    y

    40C

    40C

    40C T

    y

    40C

  • Tosoh Bioscience

    Temperature

    22

    40C

    40C 40C

    50C

    30C 40C

    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.

    23

  • Tosoh Bioscience 24

    High Flow Rates for High Throughput Analysis

    20 L 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.

  • Tosoh Bioscience 25

    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 g) 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

    26

    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

  • Tosoh Bioscience

    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

  • Tosoh Bioscience

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

    Keegan Gyke (Protein A Data)

    PD Dr. Egbert Mller

    28

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    Questions?

    http://www.separations.eu.tosohbioscience.com/ E-mail: [email protected] Phone: +49 6155 7043700 Mail: Im Leuschnerpark 4

    64347 Griesheim Germany