Breakthrough applications to expand and speed ... · Small ubiquitin-related modifier 1 [SUMO-1]...

Breakthrough applications to expand and speed biopharmaceutical characterization

Bryan Fonslow, PhD – Applications Scientist, SCIEX Separations

Eric Johansen, PhD – Global Scientific Manager,

BioPharmaceutical Development, SCIEX June 3rd, 2015

2 © 2015 AB Sciex

Presentation Outline

• Capillary electrophoresis (CE) technology in BioPharma

• CESI technology and application in BioPharma

‒ New applications at ASMS

‒ Intact biopharmaceutical analysis

‒ Peptide mapping of biopharmaceuticals

• Host cell protein detection technology

• SelexIONTM technology for reduced and intact mAb detection selectivity

• Summary

Sensitive and comprehensive characterization of biopharmaceuticals

3 © 2015 AB Sciex

mAbs, biosimilars, biobetters, and ADCs

Beck et al., Nature Reviews Immunology, 2010, 10, 345-352.

4 © 2015 AB Sciex

Capillary Electrophoresis – an analytical separation science

• Basis of separation is the differential migration of molecules in an applied electric field

• Electrophoresis, not chromatography! Orthongonal to LC

• Exceptional Resolving Power Peak efficiencies > 1,000,000 theoretical plates

• Small injection volumes

• Minimal run buffer requirements

• Separation flexibility capillary environment

buffer selection and compatibility

• Automated, quantitative technique

5 © 2015 AB Sciex

Established Assessment of IgG Purity and Heterogeneity

• In-process monitoring ‒ clone selection / cell line development

‒ product / manufacturing consistency

‒ process / product related impurities

• Characterization ‒ molecular size

‒ heterogeneity

‒ charge & size

‒ identity

• Molecular stability program ‒ effect of environment over time

‒ oxidation

• Final lot release ‒ purity

‒ quality / heterogeneity

‒ identity

Capillary Electrophoresis

Focused on Development & Production

SDS-Gel - CGE

Glycan Analysis Isoelectric Focusing

6 © 2015 AB Sciex

“Capillary electrophoresis uses one or

more capillaries as migration channels for

electrophoresis and increasingly has

become the procedure of choice when an

electrophoretic separation method is

needed. This is because CE is easier to

perform, requires less time, and allows

better precision and robustness than

PAGE.”

USP Guideline for Submitting Requests for Revision

to USP-NF, v3.1

2. CIEF & CZE = charge

heterogeneity analysis

3. Glycan analysis =

microheterogeneity determination

1. CE-SDS = Purity analysis

Established Assessment of IgG Purity and Heterogeneity

7 © 2015 AB Sciex

Combining CE & ESI-Mass Spectrometry

Ultra-low flow rates of

< 30nL/min High resolution,

ultra-low flow rate

CE separations

coupled with

high resolution,

high sensitivity MS

CESI 8000 High Performance Separation-ESI Module

TripleTOF® 5600+

and 6600 Systems

CESI - “The Integration of Capillary Electrophoresis (CE) with Electrospray

Ionization (ESI) Into a Single Dynamic Process Within the Same Device”

8 © 2015 AB Sciex

Implementation of CESI-MS Through a Commercial Interface

A single capillary with no fluidic connections simplifies nanoflow

separations with mass spectrometry.

Intact biopharmaceutical analysis using CESI-MS

10 © 2015 AB Sciex

CESI-MS for automated mAb infusion experiments

50% acetic acid dissolution facilitates ESI-MS and capillary rinsing

20 psi infusion

5 min rise time

100 psi rinse

2 min fall time

20 psi infusion

8 min stable spray

500 mg/mL

Non-desalted

mAb

11 © 2015 AB Sciex

Upcoming PEI capillary coating Tech Note

12 © 2015 AB Sciex

Beck et al., Anal. Chem. 2012, 84, 4637-4646.

Average mass: 148,057 Da (1,328 a.a.)

LC : -N30T – (D/isoD, +1 Da)

HC : -N55T – (D/isoD, +1 Da)

HC : -N387T – (D/isoD, +1 Da)

Intact Trastuzumab characterization

13 © 2015 AB Sciex

CESI-TripleTOF® analysis with BioPharmaViewTM software

14 © 2015 AB Sciex

CESI-TripleTOF® intact analysis with BioPharmaViewTM

1 mg/mL Trastuzumab

3.5 ng injection

19 © 2015 AB Sciex


Approx. 0.5%

relative abundance

22 © 2015 AB Sciex

IgG1, 2, & 4 Peptide mapping

Reduce, alkylate, & digest

Alkylate & digest Disulfide mapping

Intact IgG

Charge Heterogeneity

& Reduced Analysis

Oral presentation: Wednesday 2:30-2:50 PM, Ballroom 220/221

Comprehensive characterization of three IgG forms using CESI-MS Bryan Fonslow*1; Olga V. Friese2; K. Steven Cook2; 1SCIEX; 2Pfizer

SAME CZE METHOD &

REAGENTS USED FOR

ALL ANALYSES

Complete Characterization of Intact mAbs

CESI 8000 – TripleTOF® 6600 System Applications

23 © 2015 AB Sciex

Rapigest denaturation,

DTT reduction,

iodoacetamide alkylation,

and trypsin digestion

Tryptic peptides

(~50 ng analyzed)

Fab-linker-drug conjugates

(~10 ng analyzed)

Fragment antigen

binding (Fab)

domain

(~10 ng analyzed)

Drug-linker-

NHS

(~0.2 pmol

used)

1. Infusion of the

drug-linker molecule

with an uncoated

capillary

2. Separation and MS

analysis of the intact ADC

with a PEI coated capillary

3. Peptide mapping the

digested ADC with an

uncoated capillary

Conjugation

reaction

Trifunctional analysis of ADC-like molecule


Poster - Wednesday # 663

Comprehensive characterization a representative antibody-drug conjugate-like molecule Hans Dewald, Bryan Fonslow, Eric Johansen; SCIEX

24 © 2015 AB Sciex

Drug-linker-modified

~50% relative abundance

Unmodified

Linker-modified

Drug-linker-peptide

Trifunctional analysis of ADC-like molecule


Automatic DAR calculation

Drug Drug-linker

Drug-linker-NHS

Small molecule infusion characterization Intact ADC characterization

ADC peptide relative quantification

Peptide mapping biopharmaceutical analysis using CESI-MS

26 © 2015 AB Sciex

Trastuzumab – A Well Characterized mAb

• Approved by FDA 1998

• Humanized IgG1 to HER2/neu receptor

• HER2/neu overexpressed in 20-30% invasive breast

cancer patients

• 2 Heavy Chains (HC) – 449 amino acids

• 2 Light Chains (LC) – 214 amino acids

• HC N-glycosylation consensus site Asn 300

• Well characterized PTMs

‒ Degradation Hot Spots – Stability Indicating

‒ Glycovariants

Beck et al., Anal. Chem. 2012, 84, 4637-4646.

27 © 2015 AB Sciex

Current CESI-MS Tech Notes for biologic characterization

28 © 2015 AB Sciex

mAb peptide mapping sample preparation and analysis

Denaturation,

Reduction,

& Alkylation

Trypsin

digestion

29 © 2015 AB Sciex

Complete sequence with 1 digest, 1 injection

Amino acid sequence characterization

mAb Sequence Coverage

100 % 100 %

Heavy Chain Light Chain

Gahoual et. al., mAbs 5:3, p. 479-490; May/June 2013.

30 © 2015 AB Sciex

Variable domain retraced on

120/120 AAs for the HC

Variable domain retraced on

105/107 AAs for the LC

MS/MS spectra obtained through the CESI interface allowed

characterization of almost the entire variable domain

Trastuzumab MS/MS peptide mapping variable domains

Gahoual et. al., mAbs 5:3, p. 479-490; May/June 2013.

31 © 2015 AB Sciex

Trastuzumab and Biosimilar MS/MS peptide mapping

Biosimilar amino acid substitution characterization

MS/MS spectrum of ion 517.3095 (1+) MS/MS spectrum of ion 314.6937 (2+)

MS/MS spectra allowed to determine unambiguously biosim.

amino acid substitution compared to trastuzumab V D K R217 V E P K

Courtesy Yannis-Nicolas François, University of Strasbourg, France

32 © 2015 AB Sciex

Trastuzumab degradative PTM hot spot characterization

Differentiating Asp and IsoAsp from deamidation

Gahoual et al, Anal Chem. 86(18):9074-81, 2014.

33 © 2015 AB Sciex

A. Beck et al., Anal. Chem. 2012, 84, 4637-4646

Average mass: 148,057 Da (1,328 a.a.)

LC : -N30T – (D/isoD, +1 Da)

HC : -N55T – (D/isoD, +1 Da)

HC : -N387T – (D/isoD, +1 Da)

Trastuzumab N300 glycosylation hot spot characterization

34 © 2015 AB Sciex

Analyte suppression decreases logarithmically

below 50 nL/min

Reducing Ion Suppression Bias at Low Flow Rates

Monitoring maltotetraose suppression in the presence of neutrotensin

Maltotetroase Intensity

Neurotensin Intensity

Poster - Wednesday # 420

Comprehensive characterization a representative antibody-drug conjugate-like molecule Hans Dewald, Bryan Fonslow, Eric Johansen; SCIEX

35 © 2015 AB Sciex

Separation and relative quantification of glycopeptides

G0F

G1F

G2F

G0

A2BG2

G1B

G1FB

Man8

36 © 2015 AB Sciex

Biosimilar glycosylation characterization

Courtesy Yannis-Nicolas François, University of Strasbourg, France

Tools to Detect sub-1ppm impurities in Biological Products at Every Stage of Their Development

38 © 2015 SCIEX

Which HCPs Can Humans Tolerate and Which Would Harm Human Life?

• HCP immunogenicity varies by species.

• Serum raised in rabbits/ goats may fail to detect an HCP that is highly immunogenic in humans.

• EMA Comment: “The predictive value of animal models for evaluation of immunogenicity is low due to inevitable immunogenicity of human proteins in animals”. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003947.pdf

39 © 2015 SCIEX

SCIEX has a Strong Track Record with HCP Detection

In a Single-One Hour Run, SCIEX can:

PROFILE the HCP complement up to 1000s of proteins to ppm level

IDENTIFY HCPs without bias [without inclusion/ exclusion lists]

Provide a CATALOG of HCPs for a process

Provide precursor and fragment information to allow easy MONITORING

(MRM) and ABSOLUTE QUANTITATION of HCPs

40 © 2015 SCIEX

HCP

SVA

Modified peptide

Nothing Hides

from SWATH®

Host Cell Protein Studies Using Unbiased Methodology

One data set

can be used for

multiple tasks

41 © 2015 SCIEX

Time ROI for Single-Digit PPM Detection

Post Purification

Fraction 01 – 2Hrs

Fraction 02– 2Hrs

Fraction 03– 2Hrs

Fraction 04– 2Hrs

Fraction 05– 2Hrs

Fraction 06– 2Hrs

Fraction 07– 2Hrs

Fraction 08– 2Hrs

Fraction 09– 2Hrs

Fraction 10– 2Hrs

Post Purification One Run – 60 Minutes

Total Time: ~ 1 Hour Total Time: ~ 20 Hours

2D Method

42 © 2015 SCIEX


Total Time: ~ 3 Hours Total Time: ~ 2.5 Days

2D Method

Pre Purification

Post Purification 1

Post Purification 2

43 © 2015 SCIEX


Total Time: ~ 5 Hours Total Time: ~ 4 Days

50/50 #1

50/50 #2

Pre Purification

Post Purification 1

Post Purification 2

2D Method

44 © 2015 SCIEX


Total Time: ~ 15 Hours Total Time: ~ 12.5 Days

50/50 #1 50/50 #1

50/50 #1

50/50 #2 50/50 #2

50/50 #2

Pre Purification Pre Purification

Pre Purification

Post Purification 1 Post Purification 1

Post Purification 1

Post Purification 2 Post Purification 2

Post Purification 2

2D Method

45 © 2015 SCIEX

Return on Investment Based on Representative Example

System Cost of MS per

Day (Instrument,

service, operator,

overhead)

# of cell

lines (Assume

~1000

Proteins)

# of Runs Time Taken Analysis cost

2D

Method

1000 USD 10 10 Cell lines

X 5 Samples

X 3 replicates

X 10 fractions

= 1500 Runs

1500 Runs

X 2Hr Ea

=3000 Hr

=125 Days

1000 USD/Day

X125 Days

= $125,000

SCIEX

1D

Method

1000 USD 10 10 Cell lines

X 5 Samples

X 3 replicates

= 150 Runs

150 Runs

X 1 Hr Ea

=150 Hr

=6.25 Days.

1000 USD/Day

X6.25 Days

=$6,250

46 © 2015 SCIEX

HCP analysis via SWATH® - Study Design

• 48 model HCPs digested and analyzed by IDA MS and MS/MS to

generate a peptide library via ProteinPilot™ software searches at

upper concentration.

• SWATH® acquisition used for all subsequent concentration levels

• A range of concentrations of model proteins were spiked into 10 ug of

IgG1 product digest.

Digest LC-MS/MS Run (45’)

Generate Library

Analyze Dilution Series

47 © 2015 SCIEX

UniProt Protein Name [Synonym] MW (Da) PPM At Lowest

Dilution UniProt Protein Name [Synonym] MW (Da)

PPM At Lowest Dilution

Gelsolin 82,954 7.56 Ubiquitin-conjugating enzyme E2 C [UbcH10] 20,473 1.87 Lactotransferrin 78,289 7.14 Peptidyl-prolyl cis-trans isomerase A [Cyclophilin A] 17,947 1.64 Serotransferrin [Apotransferrin] 75,143 6.85 Tumor necrosis factor [TNF-alpha] 17,350 1.58 Serum Albumin 66,393 6.05 Myoglobin C 17,051 1.55 Catalase 59,583 5.43 Interferon gamma (IFN-gamma) 16,879 1.54 Histidyl-tRNA synthetase [Jo-1] 58,223 5.31 Leptin 16,024 1.46 Antithrombin-III 49,033 4.47 Cytochrome b5 16,021 1.46 Microtubule-associated protein tau [Tau protein] 46,810 4.27 Hemoglobin beta chain 15,867 1.45 Creatine kinase M-type [CK-MM] 43,070 3.93 Superoxide dismutase [Cu-Zn] 15,800 1.44 Small ubiquitin-related modifier 1 [SUMO-1] 37,420 3.41 Gamma-Synuclein 15,363 1.40 Annexin A 5 35,782 3.26 Hemoglobin alpha chain 15,127 1.38 NAD(P)H dehydrogenase [quinone] 1 [DT Diaphorase] C 30,984 2.82 Fatty acid-binding protein 14,716 1.34 Carbonic anhydrase 2 29,095 2.65 Lysozyme C 14,692 1.34 Carbonic anhydrase 1 28,738 2.62 Alpha-lactalbumin 14,070 1.28 Ribosyldihydronicotinamide dehydrogenase [quinone] [Quinone oxidoreductase 2] [NQO2] 25,817 2.35 Thioredoxin 12,424 1.13 Glutathione S-transferase A1 [GST A1-1] 25,482 2.32 Platelet-derived growth factor B chain 12,286 1.12 Glutathione S-transferase P [GST] 23,220 2.12 Beta-2-microglobulin 11,729 1.07 C-reactive protein 23,030 2.10 Cytochrome c[Apocytochrome c] 11,608 1.06 Ubiquitin-conjugating enzyme E2 I [UbcH9] 22,907 2.09 Ubiquitin 9,387 0.86 Ubiquitin-conjugating enzyme E2 E1 [UbcH6] 22,222 2.03 Neddylin [Nedd8] 9,071 0.83 Peroxiredoxin 1 22,106 2.01 Interleukin-8 8,381 0.76 BH3 Interacting domain death agonist [BID] 21,978 2.00 Complement C5 [Complement C5a] 8,266 0.75 GTPase HRas [Ras protein] 21,292 1.94 Insulin-like growth factor IA 7,643 0.70 Retinol-binding protein 21,065 1.92 Insulin-like growth factor II 7,464 0.68 Ubiquitin-conjugating enzyme E2 C [UbcH10] 20,473 1.87 Epidermal Growth Factor 6,211 0.57

48 Proteins Ranging From

7.56 ppm Down to 0.57

PPM at the Lowest

Dilution Level

The Number of Proteins is Not Limited

48 © 2015 SCIEX

SWATH® HCP Analysis - Optimized LC Method

LCMSMS Run (45’)

Divert

Valve

50 Micron Peeksil

25 Micron Peeksil

To Waste

Pump

Column Oven@ 55℃

Autosampler

Time %B

0 5

4 5

49 35

50 90

55 90

56 5

60 5

Flow Rate 7 ul/min Easily Transferable Method!

0.3x 150mm ChromXP™ C18 Column

10 ug Product on Column

49 © 2015 SCIEX

SWATH® Acquisition: 45 Minute Run @ 7 uL/min on 0.3mm x 15cm column

visualized in PeakView® software

HCP Analysis via SWATH® - Visualisation Informatics

Ion Library

Listing - Proteins

Replicates of

Chromatographic

Runs

Overlaid Fragment

Ion Display Linked

to Peptide Display

Spectrum Display

Linked to Peptide

Display

Ion Library Listing

- Peptides

50 © 2015 SCIEX

Statistical Analysis for Tracking and Trending

MarkerView™ software: trending HCPs across different runs

51 © 2015 SCIEX

8.55 ppm 4.28 ppm 2.14 ppm 1.07 ppm

Beta-2 Microglobulin

PEPTIDE: VNHVTLSQPK

137 ppm 68.4 ppm 34.2 ppm 17.1 ppm

52 © 2015 SCIEX

Complement C5 [Complement C5a]

90 ppm 45 ppm 22.5 ppm 11.25 ppm

5.63 ppm 2.81 ppm 1.40 ppm 0.70 ppm

PEPTIDE: AFTEC[MSH]C[MSH]VVASQLR

55 © 2015 SCIEX


• Typically load 20-30 ug product per run

• 2mm x 250mm C18 Column or UPLC

column

• Gradients between 60 and 90 min long

56 © 2015 SCIEX


• Typically load 30-40 ug product per run

• 2mm x 250mm C18 Column or UPLC

column

• Gradients between 60 and 90 min long

Poster presented at WCBP 2015, Washington DC

57 © 2015 AB Sciex

CESI 8000 – TripleTOF® 5600+ System Application

Achieve same sensitivity using

far less sample (~10 ng vs. 10 mg)

Poster-Wednesday # 599

Ultra-sensitive Host Cell Protein Quantification Using CESI-SWATH® Acquisition MS Edna Betgovargez*; Bryan Fonslow; Eric Johansen SCIEX

HCP detection with SWATH® acquisition

58 © 2015 SCIEX

Conclusions

Unbiased and Comprehensive Analysis

Sub-Single Digit PPM HCP Detection

Simple, 1D Generic Methodology

Substantial Return on Investment

New Tools: SelexION™ Technology and BioPharmaView™ Software 1.5

60 © 2015 SCIEX

SelexION™ Technology and BioPharmaView™ Software

Challenge

New Feature

Feature Benefits

Development samples usually contaminated,

cleanup wastes time and $$$.

SelexION™ Technology

Compatibility.

SelexION compatibility accelerates method

development and reduces sample prep, saving time

and $$$.

Calculating ADC Drug-Antibody Ratio involves

many steps/opportunities for human error.

Automated drug load

assignment and DAR or

% Mod Calculator.

Software assignment and calculation reduces the

opportunities for human error and greatly speeds processing

saving time/$$$

61 © 2015 SCIEX

Large number of ‘interfering’

species over wide m/z range

Antibody-Drug Conjugate A

Add SelexION™ Technology

Contaminants Are Gone

62 © 2015 SCIEX

How does SelexION™ Technology separate Ions?

Separation waveform (SV):

Radially displaces ions towards

one or the other electrode,

depending upon high and low

field mobility characteristics

Compensation voltage (COV):

Restores the trajectory for a

given ion to allow them to

transmit through the DMS device

and enter the mass

spectrometer

SV COV

To

MS Gas

flow

Differential Mobility Spectrometry (DMS) is the term used for planar geometry

63 © 2015 SCIEX

Antibody-Drug Conjugate A – With DMS

3.) Automatically Process Data in BioPharmaView™ Software

2.) Select Experiment with best Data (-5 CoV in this case).

1.) Profile a sample using several MS experiments with different CoV to optimize separation.

Figure 6. Intact ADC-A Spectra at Four Separate DMS CoV Values. This Lysine-

linked ADC also selectively elutes from the DMS device at a CoV around -5. Smaller

contaminants and contaminating fragments elute from the DMS device approaching

positive CoV values. Retention time and all other instrument parameters were held

constant for each of the four spectra.

-10 COV

0 COV

-5 COV

+10 COV Ability to look at other ‘impurities’

Main peak of

Interest only

Ability to look at large ‘impurities’

64 © 2015 SCIEX

Mass-Reconstruction of ADC-A With SelexIon™in BioPharmaView™

Software

Metadata

Obscured

(Drug in

Development)

-5 CoV

More Species

Revealed

66 © 2015 SCIEX

Mass-Reconstruction of ADC-A With SelexIon™ in BioPharmaView™

Software

-5 CoV

Detailed DAR

Calculation and

Distribution Chart

67 © 2015 SCIEX

Baseline Resolution of Co-Eluting Heavy and Light Chain

+2 COV -13 COV

5 Minute Gradient, Light

and Heavy Co-Elute

Seperated by SelexION™

Technology!

68 © 2015 AB Sciex

Summary

• CESI – effectively integrates capillary electrophoresis with electrospray

ionization serving to increase ionization efficiency and sensitivity for

biopharmaceutical characterization

• CESI-MS trifunctional BioPharma workflows:

‒ Infusion - Small molecule drugs and intact mAbs

‒ Peptide mapping – Potential CQA’s, Deamidation (Asp/IsoAsp), Glycopeptides

‒ Protein characterization – Charge heterogeneity, reduced, and intact analysis

• SWATH™ acquisition enables sensitive HCP detection

• SelexION technology provides biopharmaceutical analysis selectivity

70 © 2015 SCIEX

Trademarks/Licensing

• For Research Use Only. Not for use in diagnostic procedures.

• © 2015 AB Sciex. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

• © 2015 SCIEX. All rights reserved. Information subject to change without notice.

Breakthrough applications to expand and speed ... · Small ubiquitin-related modifier 1 [SUMO-1]...

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Transcript of Breakthrough applications to expand and speed ... · Small ubiquitin-related modifier 1 [SUMO-1]...