dedicated uplc chemistries for biotherapeutics · 2016-05-18 · p.c. iraneta, k.d. wyndham, d.r....

©2016 Waters Corporation 1

Dedicated UPLC Chemistries for BioTherapeutics

Joe Walsh WITS BioPharm Session

2016


Market Drivers

In 2014, 5/10 of the top selling drugs globally were monoclonal antibodies (mAbs) (totaling $44B)1

In 2014, 4/5 of the top selling drugs globally were proteins - 3 mAbs 1 Over 50 mAb therapeutics have been approved by the FDA/EMA2

300+ mAbs are currently under development3

8 mAbs go off patent between 2013 and 20184

At least 4 mAbs/year-regulatory approval expected over the next several years5

1GEN Report 2014 2 https://www.antibodysociety.org/news /approved_mabs.php 3http://www.bptc.com/sites/default/files/articles/ ecker-2015-the_therapeutic_antibody_market-rprnt.pdf 4http://www.marketsandmarkets.com/ResearchInsight/biosimilars-market.asp 5 http://yalebooksblog.co.uk/2015/08/18/why-monoclonal-antibodies-mabs-are-the-future-of-medicine/

5

https://www.antibodysociety.org/news�


Biopharmaceuticals vs. Small Molecules Therapeutics

Herceptin mAb

Ibuprofen


Agenda

Size Exclusion Chromatography (SEC)

Ion Exchange Chromatography (IEX)

Novel Column Technologies for Peptide Mapping

Glycoprotien Analysis – Released N Glycan Analysis – Intact Analysis – Glycopeptide Mapping – Glycan Occupancy


Principles of Size Exclusion Chromatography Separates proteins by their size in solution (Stokes radius) NOT

Absolute Molecular Weight Separations are Isocratic Ideally no adsorption to surface of particles Analytes elute in CV ≤ 1 resulting in low peak capacities as compared

to other methods such as RP methods where analyte elutes in CV ≥ 1

Flow


Chromatographic Materials for Peptide and Proteins SEC Cross -Linked agarose (e.g.,GE Healthsciences Sephadex®)

– Poor mechanical strength – Low adsorption of proteins

Diol-Bonded, 100% silica (e.g., Tosoh’s TSKgel® SuperSW mAb)

– Good mechanical properties (strength and pore volume) – Residual silanol activity

Diol-Bonded, silica-hybrid (Waters BEH SEC HPLC and UPLC)

– Good mechanical properties (strength and pore volume) – Reduced silanol activity – Universally applicable


Comparative SEC Column Life (pH 6.8, 150 mM NaCl)

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Minutes 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

ACQUITY UPLC Protein BEH SEC 200Å, 1.7 µm, 4.6 x 300 mm Injection 19 Injection 618

Lysozyme, pKi = 10.7

Suggestive of DIOL Bleed AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Minutes 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection 618

Suggestive of DIOL Bleed

Lysozyme, pI = 10.7

BEH200 SEC shows minimal secondary interactions even after 600 injections

Buffer Peak

Buffer Peak


Narrow Peak Increased Sensitivity Increased Resolving Power

Requires Columns and Instrumentation to Minimize Band Spreading

Waters UPLC®

Technology Broad Band

Broad Peak Less Sensitivity Less Resolving Power

HPLC

Advantages of UPLC Technology for SEC Separations


UPLC-SEC vs HPLC-SEC of mAb monomer and aggregates

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes2.00 4.00 6.00 8.00 10.00

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes5.00 10.00 15.00 20.00 25.00 30.00

2.26 % Aggregate 2.24 %

Aggregate

HPLC 100% Silica-Diol

SEC 250Å 5µm 7.8 x 300 mm

ACQUITY BEH200 SEC, 1.7 µm 4.6 x 300mm

8.00 30.00 8.00 30.00


0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

UV

Abso

rban

ce (2

80 n

m)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

1b 2

3

4

5

250Å, 5 µm, Silica-Based SEC

200Å, 3.5 µm, XBridge SEC

200/250 Å Comparison

1a

Effect of Particle Size

UV

Abso

rban

ce (2

80 n

m)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Minutes 6.00 8.00 10.00 12.00 14.00 16.00

1b 2 3

4

5

1a

450Å, 8 µm, Silica-Based SEC

450Å, 3.5 µm, BEH SEC

450 Å Comparison

1a & 1b) thyroglobulin dimer & monomer (1.34 MDa & 669 KDa), 2) IgG (150 KDa), 3) BSA (67 KDa), 4) myoglobin (14 KDa), and uracil (112 Da).


AU

0.00

0.05

0.10

AU

0.00

0.05

0.10

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

2 1

3 4 5

6

ACQUITY UPLC BEH SEC 200Å, 1.7µm (300mm)

ACQUITY UPLC BEH SEC 200Å, 1.7µm + 450Å, 2.5µm (150mm + 150mm)

ACQUITY UPLC BEH SEC 450Å, 2.5 µm (300mm)

2 1

3 4 5

6

2

1

3 4 5

6

Selecting the Optimal Pore Size and Combining Pore Sizes

BEH200 SEC Standard: 1. Thyroglobulin Dimer (1,340 KDa), 2. Thyroglobulin (667 KDa), 3. IgG (150 KDa), 4. BSA (66 KDa), 5. Myoglobin (17 KDa), 6. Uracil (112 Da)


Agenda






Ion-Exchange Chromatography

Binds at Low Ionic Strength Elute with Step or Continuous

Gradients of Increasing Ionic Strength Separations are based on net surface charge on protein with oppositely

charged groups on ion-exchanger

Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing pH (less common)

Anion-Exchange Particle

Na +

Na +

Cl - Na +

Cl -

Cl -

Na +

Na +

Cl -

Na +

Cl - Na +

Na + Cl -

Cl -

Cl -

Na +

Cl -

Cl -

Cl -

Anion-Exchange Particle


Ion-Exchange

Advantages Moderate resolution

Concentrating technique

– Can load large volumes of a dilute sample

Non-denaturing protein elution techniques

Preferred first step when a cost-effective affinity purification is not available

Limitations Separation by charge and

charge distribution; insensitive to other properties

For salt gradients, time-consuming pH optimization required for best resolution.

Fractions may need to be

desalted prior to the next purification or characterization step (e.g., Mass Spectrometry)


AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

Minutes 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

Strong and Weak Cation Exchange Column Comparison

Strong cation exchanger

Weak cation exchanger

Ribonuclease A 1

cytochrome c 2

Lysozyme 3 1

1

2

2

3

3

Strong and weak cation exchange columns demonstrate different retention


Protein-Pak Hi Res IEX


Attributes of Protein-Pak™ Hi Res IEX Columns

Multi-layered network of ion-exchange groups (SP, CM or Q) o Effective diffusion and binding o High sample loading and resolution o Minimal non-desired interactions

No MW limitations: non-porous material

QC tested with protein samples for batch-to-batch reproducibility

High chemical stability: hydrophilic, polymer-based IEX particles

– Wide pH range (3- 10) – high salt concentrations (1M) – Standard pressures (up to 1450 psi for CEX and 2175 psi for AEX) – Can be cleaned with aggressive washing

eCord enabled for data tracking


12.5% 100mM NaH 2 PO 4

15% 1000 mM NaCl

60% H 2 0

12.5% 100mM Na 2 HPO 4

25mM Sodium Phosphate, 150mM NaCl pH 6.8

Waters Auto•Blend Plus™ Technology


Agenda






Reversed Phase Peptide Separations

Peptide separations – critically important – peptide mapping, bottom-up proteomics … – reversed phase – challenges remain

Peptide

TFA

TFA

LC performance MS performance

Still needed: – high resolution, high sensitivity peptide separations regardless of eluent additive – i.e. formic acid (FA) separations for LC-MS

Ionic analytes – peptides – Secondary interactions - Poor peak shape – Overloading at very low loads (<< neutrals) – MS signal suppressing ion pairing agents needed


0.0

1.0

2.0

3.0

0 10 20 30 40 50 60

A21

4

Time (min)

Competitor’s “Industry Standard” 5 µm Porous Silica C18

1 2

3

4

5

6

7 8

9

1 2

3

4

5

6

7

8

0.1% TFA

0.02% TFA 0.08% FA

Competitor’s “Industry Standard” C18 2.1 x 250 mm, Porous 5 µm, 300Å

ACQUITY UPLC® H-Class Bio

2% ACN for 1 min, then to 50% ACN over 60 min

0.3 mL/min

40°C

UV @ 214 nm / Xevo® G2 QTOF

5.6 µg MassPREP Peptide Mixture

Peptide Sequence

1 RASG-1 RGDSPASSKP

2 Angiotensin 1-7 DRVYIHP

3 Bradykinin RPPGFSPFR

4 Angiotensin II DRVYIHPF

5 Angiotensin I DRVYIHPFHL

6 Renin Substrate DRVYIHPFHLLVYS

7 Enolase T35 WLTGPQLADLYHSLMK

8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK

9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ

0.1% FA

9

1 2

3

4

5

6

7

8 9 – not detected

Ion Pairing

Strong

Weak

MassPREP™ Peptide Mixture


Charged Surface Hybrid (CSH) Technology

Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography.

P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter

Waters White Paper 720003929EN 2011

Peptide

Expands upon the robust BEH particle technology

CSH130 C18 = BEH130 base particle + low level of basic moieties + trifunctional C18/end cap Acidic pH Positive Surface Charge

patent pending


20

70

120

170

220

270

320

370

0.00 0.05 0.10

P c,4

σ

Percent TFA

MS Signal - FA vs TFA

CSH130 C18 1.7 µm

Competitor’s SPP “Peptide” C18 1.7µm

BEH130 C18 1.7 µm

0.05 0.05

0.10 0.00

0.00 0.10

% TFA % FA

Competitor’s “Industry Standard” C18 5 µm

12

10

8

6

4

2

0.00 0.05 0.10

Fold

Dec

reas

e in

MS P

eak

Are

a

Percent TFA 0.05 0.05

0.10 0.00

0.00 0.10

% TFA % FA

Peak Capacity MS Signal

90%

20%


-0.1

0.2

0.5

0.8

1.1

0 10 20 30 40 50 60 70 80 90 100 110

A21

0

Time (min)

-0.1

0.2

0.5

0.8

1.1

0 10 20 30 40 50 60 70 80 90 100 110

A21

0

Non-Reduced Lys-C Peptide Maps Trastuzumab

0.02% TFA 0.08% FA

BEH130 C18 Peak Capacity = 207

CSH130 C18 Peak Capacity = 394

97.1% Coverage

96.7% Coverage

1 2 3

4

5

5

4

3 2

1

Peak BEH130 C18 CSH130 C18

1 33.7 15.4

2 16.1 9.7

3 9.5 5.7

4 18.9 9.9

5 23.9 13.2

wh (sec)


Agenda






Glycosylation of Biotherapeutics

N-glycolylneuraminic acid

Fucose

GlcNAc

Mannose

Galactose

N-acetylneuraminic acid

Immunogenic (αGal / N-glycolylneuraminic acid)

Low Half Life (high mannose)

Anti-Inflammatory (sialylation)

Effector Functions (ADCC/CDC) (fucosylation/galactosylation)

Overall profile sensitive to manufacturing conditions

N-glycosylation is a quality attribute of biotherapeutics N-glycan profiles are frequently characterized in detail and routinely monitored

Anal Chem 2013, 85 (2), 715-36.


Intact Protein

Middle Up/Down

Released Glycan

Glycopeptide

Monosaccharide/Sialic Acid

Multiple Strategies– Complementary Information

HILIC Hydrophilic Interaction

Chromatography


10 min 5 min 10 min

30 min

Patent Pending

Simplified Workflow

Total Sample Prep Time

Conventional 5 Hours

to 2 Days

GlycoWorks RapiFluor-MS N-Glycan Kit

Direct Analysis (Organic Solvent Dilution)


0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Fluorescence MS (BPI)

Instant AB Labeled

RapiFluor-MS Labeled

2-AB Labeled

Rel

ativ

e Pe

rfor

man

ce (

%)

52.5

7.0 0.1 0.6

Procainamide Labeled

30.0*

7.0*

(*) Comparative result extrapolated from a published comparison of N-glycans, wherein it was found that procainamide provided comparable fluorescence and up to 50 fold greater ESI-MS sensitivity when compared to 2-AB(Klapoetke et al. 2010).

Revolutionizing N-Glycan Sample Prep Sensitivity Comparison


Intact Protein

Middle Up/Down

Released Glycan

Glycopeptide

Monosaccharide/Sialic Acid

Multiple Strategies– Complementary Information

HILIC Hydrophilic Interaction

Chromatography


0

0.1

0.2

0.3

0.4

0.5

0.6

5 6 7 8 9 10 11 12 13 14 15

A 214

Time (min)

Unbonded BEH 130Å

Amide Bonded BEH 130Å

Amide Bonded BEH 300Å

Improved Resolution for Large Analytes

Highly Branched Glycans

Intact mAbs

Patent pending

Separation of RNase B Glycoforms On Different HILIC Stationary Phases


Orthogonality and LC-MS (Glycoforms)

0

0.09

0.18

0.27

A 214

RNase Baglycosylated

+Man5+Man6+Man7+Man8+Man9

Reversed PhaseBEH C4300Å

0

0.02

0.04

0.06

6 8 10 12 14 16 18 20

A 214

Time (min)

+Man5

+Man6

+Man7

+Man8

+Man9


HILICBEH Amide300Å


0

0.02

0.04

0.06

6 8 10 12 14 16 18 20

A 214

Time (min)

+Man5

+Man6

+Man7

+Man8

+Man9


HILICBEH Amide300Å

Orthogonality and LC-MS

0

0.09

0.18

0.27

A 214


+Man5+Man6+Man7+Man8+Man9

Reversed PhaseBEH C4300Å

Patent pending

13500 14000 14500 15000 15500 16000

m/z

Man9 Glycoform


Trastuzumab Native

5.0 10.0 min

2 N-Glycans

1 N-Glycan 1.0%

2 N-Glycans

1 N-Glycan 21.5%

0 N-Glycans 3.1%

Trastuzumab Partially Deglycosylated

Assaying Deglycosylation and Glycan Occupancy on Intact mAbs

Intact

Intr

insi

c Fl

uore

scen

ce D

etec

tion

Patent pending

5.0 10.0 min

2 N-Glycans

1 N-Glycans

0 N-Glycans


Orthogonal Peptide Mapping Techniques Trastuzumab Lys-C Digest

0.0

0.5

1.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

A 214

0.0

0.5

1.0

0 5 10 15 20 25 30 35 40 45

A 214

Time (min)

Peptide BEH C18 300Å 1.7 µm

Glycoprotein BEH Amide 300Å 1.7 µm

Non-glycosylated Peptides

Glycopeptides

Non-glycosylated Peptides

Peptide Reversed

Phase

HILIC


Middle-Up Analysis Orthogonality

0.00

0.04

0.08

0.12

0.16

6 7 8 9 10 11 12 13 14 15 16

A 214

Time (min)

0.00

0.06

0.12

0.18

0.24

6 7 8 9 10 11 12 13 14 15 16

A 214

Time (min)

Fc/2 Glycoforms

Fd’

LC

Protein BEH C4 300Å 1.7μm

LC Fd’

Glycoprotein BEH Amide 300Å 1.7μm

Fc/2 Glycoforms

Patent pending

‘Middle-Up’


Summary

Advanced chromatographic technologies are required for the analysis of complex analytes such as Biopharmaceuticals

Size Exclusion – Smaller particle size columns provide improved resolution and

throughput – Physical and chemical column lifetime is improved with hybrid particles

(BEH)

Ion Exchange – Auto•Blend Plus™ Technology allows for change of pH and ionic

strengths without preparation of different buffers to simplify methods development

– Combination of the Protein-Pak Hi Res IEX column benefits and H-Class Bio ACQUITY UPLC o Consistent batch-to-batch performance (tested with protein standards) o Minimal column related carryover o Stable over a wide pH range


Summary cont.

Peptide Analysis – Novel Charged Surface Hybrid (CSH) Columns provide improved

chromatographic performance under MS friendly conditions – Balanced peak capacity and MS sensitivity for peptide mapping

Glycoprotein Characterization

– RapiFluorMS can simplify sample preparation and improve MS sensitivity for released N glycan analysis

– Novel Widepore BEH Amide HILIC column provides high resolution and unique selectivity for analysis of intact glycoproteins, glycoprotein subunits and glycosylated peptides

dedicated uplc chemistries for biotherapeutics · 2016-05-18 · p.c. iraneta, k.d. wyndham, d.r....

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