Ion Exchange Chromatography for Protein Characterization Exchange Chromatography for...Principles and Practice of Ion Exchange Chromatography for Proteins – Anion vs. Cation Exchangers

©2019 Waters Corporation 1COMPANY CONFIDENTIAL

Ion Exchange Chromatography

for Protein Characterization


10 of Top 15 are Biopharmaceuticals

(2016)

50 of Top 100 are Biopharmaceuticals

(2014)

Lots of Growth in Biotherapeutics

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj99JbPmfzTAhXGHxoKHfYQCxMQjRwIBw&url=https://www.pharmacompass.com/radio-compass-blog/top-drugs-by-sales-in-2016-who-sold-the-blockbuster-drugs&psig=AFQjCNEBEJPrurRpPJxdit-VjzYXRLYB1A&ust=1495291645512395


Monoclonal Antibody Based Protein Therapeutics

Aspirin

50 Approved mAbs500 Candidates

3 Approved ADCs80 Candidates

10 Approved Fc-Fusion Proteins40 Candidates

2 Approved Bispecific Abs60 Candidates

150 mAb Biosimilars in Development Market to be$23B by 2020

+cytotoxin

Understanding Complexity = Analytically Challenging

Protein Cell. 2018 Jan; 9(1): 86–120


Quality Attributes to Consider - Biotherapeutic

Monoclonal Antibodies (mAb)

Protein Cell. 2018 Jan; 9(1): 86–120


Waters Offerings to Address mAb Characterization

and Compound Heterogeneity

Reversed Phase

• Separate oxidized, fragmented species

• Intact Mass

• Reduced mAb characterization

Peptide Mapping

• mAb Identity

• PTM characterization

• Disulphides

• MAM

Amino Acid Analysis

• Understanding molar absorptivity

• Protein Concentration

• (Quantitative AAA)

Hydrophobic Interaction

• Separate based on hydrophobicity under non denaturing conditions

Size Exclusion

• Separation Based on size of molecule

• Dimers/Aggregation

• High Molecular Weight

• Low Molecular Weight

• Clips

Glycan HILIC

• Glycosylation Pattern

• Released N-Glycan

• Orthogonal methods for Intact, Subunit, Glycopeptide Mapping

Ion Exchange

• Separation Based on charge

• Monitor deamidation, sialyaltion, pyroglutamate

Higher Order

Structure

HDX-MS

SEC-MALS

Impurities

Host Cell Protein

Excipients Analysis (e.g.,

Polysorbates

Understanding

Primary Structure

Purity,

Content,

Glycosylation


Importance of Understanding Charge Variants in Biotherapeutic Monoclonal Antibodies (mAb)

Principles and Practice of Ion Exchange Chromatography for Proteins– Anion vs. Cation Exchangers

– Isocratic vs. Gradient Separations

– Protein Isoelectric Points

– Optimizing IEX Protein Separations

– Auto•Blend Plus™ to Assist in IEX Method Development and Optimization

Designing a Solution to Meet Today’s Challenges– New Purposefully Designed Stationary Phase

– New Guard Equipped Column Design

– New pH Gradient Separation Platform

– IEX-MS Capabilities

Agenda


IEX can determine and monitor protein charge heterogeneity

– Oxidation

– Asparagine deamidation

– Aspartic acid isomerization

– C-terminal Lysine truncation

– Glycan modifications (sialylation)

– Tyrosine sulfation/phosphorylation

– Tertiary Structure , etc.

Many of these Critical Quality Attributes (CQA’s) monitored from DISCOVERY, DEVELOPMENT,

and Manufacturing of mAb drugs.

Each attribute can effect efficacy and immunogenicity

Why is Ion Exchange Chromatography (IEX) used for

biomolecule characterization?


Protein Isoelectric Points and IEX

pH is ≤1 unit lower than protein pI

+

+

+

+

+

++

++

+

++

+

++

+

++

+--- --

--- --

-

----

-

-

--

-

--

--

++

+

++

++

++++

pH is ≥ 1 unit higher than protein pI

Anion

Exchanger

Cation

Exchanger

--Stationary Phase

Stationary Phase

+

+

+

+

--

--

+-+-- +Zero Net

Charge


Typical Gradient Profile

5-10 cv

~ 5 cv

5-10 cv

10-20 cv

unbound proteins

EquilibrationSample Injection

GradientHigh Salt

WashRe-

Equilibration

Sample Injection

CV = Column Volume


Salt Gradient: increasing salt concentration with time

– Increasing ionic strength of the mobile phase affects the charge interaction of the protein

and the stationary phase.

pH Gradient: varying mobile phase pH as a function of time

– The charge on the molecule is changed by pH, thus affecting the binding of the molecule

to the stationary phase.

Simultaneous Salt / pH Gradient:

IEX Separation Methods


Optimizing IEX Protein Separations

Ionic Strength

pH

Buffer

& Counter ion

Temperature

https://tinyurl.com/ycolakoz

Ion-Exchange Chromatography for Protein CharacterizationFREE, ON-DEMAND, Webinar




Selectivity altered with pH

pH 6.2

pH 6.0

pH 5.8

pH 5.6

pH 5.4

pH 5.2

Lysozyme, pI = 11.4

Acidic variant

A

B

C

MES Buffer SystemA: 100 mM MESB: 100 mM MES/NaC: 1000 mM NaClD: H2O

Cytochrome C 1

Ribonuclease A 2

Lysozyme 3

Waters IEX Cation Standard(P/N 186006870)

Column: Protein-Pak Hi Res CM 4.6 mm x 100mm (P/N 186004930)


TO

COLUMN

Solvent A:

Low pH Buffer

Solvent C:

Salt Solution

Solvent B:

High pH Buffer

Solvent D:

Water

IEX AutoBlendTM Methodology

Precise Eluent

Proportioning Valve


Enter Buffer Concentration Enter Buffer System

Auto•Blend Plus™ Technology

Enter pH andSalt Gradient


IEX Gradient Separation Types


IEX Separation Gradient for mAb Charge Variants

25 therapeutic

mAbs

Source:

Goyon, A. et al. Journal of chromatography. B,

Analytical technologies in the biomedical and life

sciences 2017, 1065-1066, 119-128.


IEX Separation Gradient for mAb Charge Variants

Source:

Goyon, A. et al. Journal of chromatography. B,

Analytical technologies in the biomedical and life

sciences 2017, 1065-1066, 119-128.

25 therapeutic mAbs and their isoelectric points

25 therapeutic

mAbs and their

isoelectric points


Confirmation of c-terminal Lysine Variants of Infliximab

Using Auto•Blend Plus™ Technology

Protein Pak Hi Res SP 7µm, 4.6 x 100 mm

Solvents: A: 100 mM Sodium Phosphate monobasic, B: 100 mM Sodium phosphate dibasic, C: 500 mM Sodium chloride, D: WaterMobile phase pH: 6.4


AU

0.00

0.02

0.04

0.0 10.0 20.0 30.0 40.0 50.0

AU

0.00

0.02

0.04

0.0 10.0 20.0 30.0 40.0 50.0

AU

0.00

0.02

0.04

0.0 10.0 20.0 30.0 40.0 50.0

AU

0.00

0.04

0.08

0.0 10.0 20.0 30.0 40.0 50.0

AU

0.00

0.03

0.06

0.0 10.0 20.0 30.0 40.0 50.0

AU

0.00

0.03

0.06

0.0 10.0 20.0 30.0 40.0 50.0

panitumumab

pI 6.8

IgG2

infliximab

pI 7.6

IgG1

trastuzumab

pI 9.1

IgG1

adalimumab

pI 8.9

IgG1

NIST mAb

pI 9.2

IgG1

rituximab

pI 9.4

IgG1

Pc* 11.7 Rs 8.1p/v 7.1 p/v 7.0

All the chromatograms were acquired with a generic

gradient of increasing pH on a BioResolve SCX mAb

column (4.6×50mm) and BioResolve CX pH Buffer

Concentrates.

Cation Exchange Gradient Separation of Three

Commercially Available mAb Biotherapeutics

Gradient of Increasing pH


Cation-Exchange Gradient Separation of Three

Commercially Available mAb Biotherapeutics

Gradient of Increasing Ionic Strength and Constant pH


Cation-Exchange Gradient Separation of Trastuzumab (pI 9.1)

pH gradient vs. salt gradient

Gradient of Increasing Ionic Strengthand Constant pH

Gradient of Increasing Ionic Strengthand Constant pH

Gradient of Increasing pH and Constant Ionic Strength

60 - 110 mM NaCl in 15 min,

20mM MES, pH 6.2

Flow rate: 1.2 mL/min

pH 6.0 – 7.1 in 15 min,

70 mM NaCl

Flow rate: 1.2 mL/mi


Importance of Understanding Charge Variants in Biotherapeutic Monoclonal Antibodies (mAb)

Principles and Practice of Ion Exchange Chromatography for Proteins– Anion vs. Cation Exchangers

– Isocratic vs. Gradient Separations

– Protein Isoelectric Points

– Optimizing IEX Protein Separations

– Auto•Blend Plus™ to Assist in IEX Method Development and Optimization

Designing a Solution to Meet Today’s Challenges– New Purposefully Designed Stationary Phase

– New Guard Equipped Column Design

– New pH Gradient Separation Platform

– IEX-MS Capabilities

Agenda


New BioResolve SCX mAb Columns and Consumables Simplify and Improve mAb Characterization and Monitoring through Holistic Approach

MILFORD, Mass. – January 29, 2019 – Waters Corporation (NYSE:WAT) today introduced a new cation exchange column line with specialized consumables to simplify and improve the characterization and monitoring of monoclonal antibody (mAb) therapeutics. The new BioResolve™ SCX mAb Columns and Vanguard™ FIT Cartridge technologies, together with a suite of complementary consumables, enable mAb charge-variant analyses as required by the World Health Organization, the U.S. Food and Drug Administration, and the International Conference on Harmonization for confirming the efficacy and safety of biologics and biosimilars within discovery, development, and manufacturing applications.

http://wvmc.waters.com/bioresolve-2018/?alias=bioresolve


Challenges in LC IEX for mAb Charge Variant Analysis

Resolution of Variants Reproducibility of MethodComplexity of Method Dev.

Minimal CarryoverLoadability Lifetime MS Compatibility


BioResolve SCX (Strong Cation Exchange) mAb

Columns:2.1 and 4.6 mm

by50 or 100mm

BioResolve SCX mAb:2.1 and 4.6 mm

by50 or 100mm

with Vanguard FIT (Fully

Integrated Technology)

Novel, Patent Pending, Integrated Guard Design

mAb Charge Variant Standard

andBioResolve CX pH Buffer

Concentrates

Setting a New Standard for LC IEXBioResolve SCX mAb Consumables


Novel Stationary Phase

TrastuzumabIntermediate pI

AdalimumabBasic pI

NIST mAbBasic pI

p/v

p/v

InfliximabIntermediate pI

Effective

Peak Capacity

(Pc*)

“Rs”

• Novel, specialized polymerization reactions for an optimized

hydrophilic surface and sulfonic acid ligand

• 3 µm non-porous particle for optimal diffusion kinetics, high

pressure capability and amenability to HPLC, UHPLC and

UPLC

• Developed through purposeful prototyping and comprehensive

testing with a wide range of mAbs and separations and both salt

and pH gradient chromatography

Patent Pending

Non-porous

polymeric

particle

Multi-component

surface chemistry

Hydrophilic coating

See: “Designing a New Particle Technology for Robust Charge Variant

Analysis of mAbs” Waters Application Note: 720006475EN (January 2019)

http://www.waters.com/webassets/cms/library/docs/720006475en.pdf



Salt Gradient

Ad

alim

um

ab

NIS

T m

Ab

Tra

stu

zu

ma

bIn

flix

ima

b

Representative Chromatograms

Salt GradientsA

U

0.00

0.12

0.24

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.10

0.20

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.15

0.30

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.06

0.12

0.0 2.0 4.0 6.0 8.0 10.0

Thermo MAbPac SCX

3 µm

AU

0.00

0.20

0.40

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.15

0.30

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.07

0.14

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.15

0.30

0.0 2.0 4.0 6.0 8.0 10.0

YMC BioPro SP-F

3 µm

Sepax Proteomix SCX

3 µm

AU

0.00

0.07

0.14

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.15

0.30

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.10

0.20

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.05

0.10

0.0 2.0 4.0 6.0 8.0 10.0

Protein-Pak Hi-Res

7 µm

AU

0.00

0.07

0.14

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.15

0.30

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.10

0.20

0.0 2.0 4.0 6.0 8.0 10.0

AU

0.00

0.05

0.10

0.0 2.0 4.0 6.0 8.0 10.0

BioResolve SCX mAb

3 µm

4.0 5.0

4.0 6.0

3.0 4.0 5.0

2.0 4.0

4.6 or 4 x 50 mm columns

20mM MES pH 7.0

0.72 mL/min (for 4.6 mm) / 0.54 mL/min (for 4.0 mm)

10 min gradient; ~10 to 200 mM NaCl in 10 min

Note: Data Generated by Waters. Results could vary with different samples and separation conditions


A New, Purposefully Designed

Stationary Phase Salt Gradient

4.6 or 4 x 50 mm columns

20mM MES pH 7.0

0.72 mL/min (for 4.6 mm) / 0.54 mL/min (for 4.0 mm)

10 min gradient; ~10 to 200 mM NaCl in 10 min

10.07

6.45

1.31

2.94

8.32

5.80

2.74

4.62

10.35

5.36

1.93

2.66

7.73

4.48

2.79

2.32

0

2

4

6

8

10

12

Infliximab(Pc*)

Trastuzumab(Rs)

NIST mAb(p/v)

Adalimumab(p/v)

系列1

系列2

系列3

系列4

3801

2355

852

673

0

1000

2000

3000

4000

5000

6000

Pressure(psi)

Thermo MAbPac SCX-10, 3 µm

YMC BioPro SP-F, 3 µm

Sepax Proteomix, 3 µm

Protein-Pak Hi-Res SP, 7 µm

Reso

lution (

Pc, p

/v,

Rs)

Ma

x C

olu

mn P

ressu

re (

psi)

7 Verification Batches of

BioResolve SCX mAb

columns ± 1 Std Dev

Note: Data Generated by Waters. Results could vary with different samples and separation conditions


Batch DBatch CBatch A Batch E Batch FBatch B

NIS

T m

Ab

Ad

alim

um

ab

Tra

stu

zu

ma

bIn

flix

ima

b

4.0 5.0

4.0 6.0

3.0 4.0

1.5 3.0

2.3

2.0

8.0

10.4

2.1 2.22.3 2.0 2.2

1.92.0 1.9 1.81.9

7.67.7 7.0 7.17.6

10.610.3 10.3 10.29.9

4.0 5.0 4.0 5.04.0 5.0 4.0 5.0 4.0 5.0

4.0 6.0 4.0 6.04.0 6.0 4.0 6.0 4.0 6.0

3.0 4.0 3.0 4.03.0 4.0 3.0 4.0 3.0 4.0

1.5 3.0 1.5 3.01.5 3.0 1.5 3.0 1.5 3.0

Batch G

4.0 5.0

4.0 6.0

3.0 4.0

1.5 3.0

2.2

2.0

7.9

10.6

Rs*

Rs

Rs

Pc*

1

2

NIST mAb, Rs 3

4

5

NIST mAb, Main Peak, RT

1.9%C

4.3%B

0.2%C

1.1%B

1

2

Adalimumab, Rs

RSD

1.2%C

3.5%B3

4

5

Adalimumab Main Peak

RSD

0.4%C

2.1%B

4

6

8

Trastuzumab, Rs

2.4%C

4.9%B

3

4

5

Trastuzumab Main Peak

0.2%C

1.1%B

1

2

3

Infliximab Main Peak

0.9%C

3.0%B

7

9

11

Infliximab, Pc*

0.7%C

2.3%B

BioResolve SCX mAb Columns:Improved Resolution with Outstanding Reproducibility

Resolution RetentionTime

Minimal batch-to-batch and column-to-column variation


Peak 1

Peak 2

Peak 3

Peak 4

Peak 5

Peak 6

Peak 7

0.000

0.025

0.050

0.0 5.0 10.0 15.0 20.0 25.0 30.0

mAb Charge Variant Standard A Mixture of Tryptophan, Conalbumin, and NIST mAb

Conalbumin NIST RM 8671

(NIST mAb)

Tryptophan

QC batch testing for a rigorous check on

suitability for biopharm applications

Available for proficiency checks and system

suitability

Peak 3

Peak 4

Peak 5

Peak 6

Peak 7

0.000

0.007

0.014

11.0 12.0 13.0 14.0 15.0 16.0 17.0

• Retention

• Selectivity

• Resolution

• Recovery

Column dimension 4.6 × 50 mm

TimeFlow

(mL/min)%A %B Curve

Initial 1.44 100 0 Initial

1.0 1.44 100 0 6

23.6 1.44 0 100 6

24.6 1.44 0 100 6

25.6 1.44 100 0 6

30.0 1.44 100 0 6

pH Gradient


Low Injection to Injection Carryover

Low injection-to-injection carryover

BioResolve SCX mAb., 3 µm

4.6 x 50 mm

20mM MES pH 7.0

0.72 mL/min

10 to 200 mM NaCl in 10 min

As Observed by Simple Sample Injection Followed by

Two, Gradients of Increasing Ionic Strength


“Biocompatible” Hardware

Corrosion-Resistant Hardware and Frits Titanium has exceptional corrosion resistance

(Example: 1 week 1M NaCl pH 2)

Shown here are images of solvent line sinkers (Ti and SS Versions) exposed to high salt under acidic

conditions. Even at 1 week, the stainless components are seen to tarnish under these conditions.


BioResolve SCX mAb Column Hardware DesignMitigate corrosion risk, while maintaining UPLC pressure compatibility,

column packing performance, and repeatability

Shelf life testing of the improved design has also shown no

degradation in separation performance. Columns were

evaluated with salt gradients, flushed, and stored for 1 month.

Accelerated corrosion testing of 2.1mm column frits. The stainless steel hardware shows visual rusting in as little as one week, where the BioResolve design has

no noticeable corrosion.

BioResolve SCX mAb

Titanium Hardware

Stainless Steel

Hardware


pH Gradient Separation PlatformBuffer Concentrates for A Novel pH Gradient Method

BioResolve CX pH Concentrates Patent pending pH gradient mobile phase

system for simple-to-implement charge

variant profiling

Carefully optimized pH 5.0 to 10.2 gradients

for use with BioResolve SCX mAb columns

to achieve high resolution, robust

separations

Easy to use: 100 mL 10x concentrates for

the preparation of mobile phase using a

simple 10-fold water dilution

Patent PendingN

IST

mA

badalim

um

ab

trastu

zum

ab

inflix

imab

20.0

15.0

10.0 15.0

10.0

7.0

10.6

7.9

10.6

Rs*

p/v

p/v

Pc*

See: “Development of Monoclonal Antibody Charge Variant Analysis Methods Using a

BioResolve SCX mAb Column” Waters Apps Note: 720006477EN (January 2019)

and

“Development of pH Gradient Mobile Phase Concentrates for Robust, High Resolution mAb Charge Variant Analysis” Waters Apps Note: 720006491EN (January 2019)

Note: These Buffers

are NOT Volatile so

cannot be used for

LC/MS




Traditional ion exchange chromatography is incompatible with

mass spectrometry, due to high concentrations of non-volatile

salts needed to achieve separation.

Previous solutions include fractionation with subsequent RP-

LCMS analysis or a 2D-LCMS approach.

Ion Exchange Chromatography Coupled With

Mass Spectrometry

Using the new BioResolve SCX mAb column, along with a dual pH/salt gradient

which employs volatile salts, we can now directly couple an ion exchange

separation with ESI-MS detection.UV @ 280nm

Trastuzumab

IdeS Digest


Infliximab C-terminal mAb Lysine Variants Separated and Identified Without Fractionation or 2D-LC

UV@280nm AB

C

C

B

A

Infliximab Expected Mass: 148,511.9 Da (G0F/G0F) (avg)

Infliximab, 0K

Infliximab, 1K

Infliximab, 2KC

B

A

*5mg intact mAb on column

~27-32ppm accuracy in MaxEnt 1 deconvolutions

G0F/G0F

G0F/G1F

G1F/G1F

G1F/G2F

BioResolve SCX mAb column


Reproducibility of BioResolve SCX mAb Separations in LC/MS Injection to injection

Gradient: IdeS Digests

Time

(min)

Flow rate

(ml/min)

A1 % B1 %

0.00 0.10 100 0

1.00 0.10 100 0

21.00 0.10 0 100

22.00 0.10 0 100

23.00 0.10 100 0

30.00 0.10 100 0

Gradient : NR mAbs

Time

(min)

Flow rate

(ml/min)

A1 % B1 %

0.00 0.10 60 40

1.00 0.10 60 40

21.00 0.10 0 100

22.00 0.10 0 100

23.00 0.10 60 40

30.00 0.10 60 40

Infliximab Nonreduced

Triplicate Injection

NIST mAb

IdeS Digestion

Triplicate Injection

0K

1K

2K


100% Aqueous Mobile Phases at physiological pH

and ionic strength can support microbial growth.

Microbes can attach to surfaces such as LC tubing,

solvent sinkers, frits, or top of packed column.

Top of SEC or IEX Column Bed will capture

microbes

and other particulates.

Result: Short column lifetime

– Poor flow distribution –

o split or tailing peaks

– Increasing system pressure sometimes is

observed

The Number One Cause for Short SEC or IEX Column LifeMicrobial Growth


Bacterial Contamination in

a “Poor Performing” SEC Column

Scanning Electron Micrograph of the Inlet Frit Removed from an ACQUITY UPLC Protein BEH SEC 200Å, 1.7 µm

Column Contaminated with Bacteria


Finding a SolutionBioResolve SCX mAb VanGuard Fit (Fully Integrated Technology)

No Scattered

Light Seen

Significant

Light

Scattering

Detected

Inoculation onto Microbe Supporting Blood Agar

Plates confirms presence of lactobacillus in Flask B

No growth

seen

- 100 ul from

bottle applied to

plate.

- Bacterial

colonies seen

after overnight

incubation

562nm

Green

Laser


Question: Could Waters VanGuard Cartridges Trap Microbes ?

Far Greater

than 10^6

lactobacillus

CFUs / 10mL

since

microbes

continued to

grow in flask

Pressure traces from six, 20min runs at 0.5mL/min flow

Day 2

Injection 6

Injection 4Injection 5

Injection 2

Injection 3

Injection 1

No Growth Seen


No Compromise Column Protection and Extended

Lifetimes with the Use of VanGuard FIT Cartridges

BioResolve SCX mAb Column, 4.6 x 50 mm column

with integrated VanGuard FIT. Replaceable cartridge

screws directly into the column inlet end nut. Optimal

installation has never been easier nor more efficient in

terms of maintaining analytical columns resolving power.

Charge variant profiles1 for NISTmAb. No significant difference in chromatographic

performance on BioResolve SCX mAb columns without (top) or with (bottom) VanGuard FIT.

AU

0.00

0.05

0.10

0.15

AU

0.00

0.05

0.10

0.15

Minutes

4.00 4.20 4.40 4.60 4.80 5.00 5.20

BioResolve SCX mAb, 3µm

with VanGuard FIT

BioResolve SCX mAb, 3 µm

without VanGuard FIT

Peak 1

Peak 2

Rs = 2.49

Rs = 2.41

mAb in 100%

Aqueous IEX Brf


0.00

0.15

0.30

4.0 5.0

0.00

0.15

0.30

4.0 5.0

0.00

0.15

0.30

4.0 5.0

0.00

0.15

0.30

4.0 5.0

Initial

USP Half Height (HH) =

Rs 2.03

Moderate Chemical Fouling

USP HH Rs 1.70

Replaced VanGuard FIT

USP HH Rs 2.07

Severe Chemical Fouling

USP HH Rs n/a

(A) A suspension of 0.1 µm polystyrene beads was repeatedly injected on a 4.6 x 50 mm BioResolve SCX mAb

3 µm column with VanGuard FIT until ~30 % backpressure increase was realized. Backpressure returned to normal upon replacement of the VanGuard FIT.

(B) Rat plasma was injected into salt gradients (H-Class Bio, 20 mM MES pH 7.0, 10 min gradient, 10 to 200 mM NaCl, 30 °until loss of resolution was

observed. Resolution was recovered upon VanGuard FIT replacement.

A B

1700

1900

2100

2300

2500

2700

0 10 20 30 40 50 60

Co

lum

n p

si

Injection

VanGuard

Cartridge

replaced

ACQUITY H-Class Bio

20 mM Phosphate pH 6.8

0.50 mL/min, isocratic

Column Temp. = 30 °C

Extension of BioResolve SCX mAb Column (4.6 x 50mm) by Replacement

of VanGuard Fit Cartridge on Particulate and Chemically Fouled* Column

mAb in plasma


Novel InnovationsImproved Performance

Increased resolution between major mAb

species and charge variants

– Longer column does not always lead to more resolution

Batch to batch and column to column

reproducibility

Better sample recovery and less injection to

injection sample carryover

Flexibility to obtain “higher” throughput (4.6 x

50mm) vs. “higher” resolution (4.6 x 100mm)

Ability to collect fractions and “reasonable

amounts” of sample for additional testing (e.g.,

ligand binding)

Summary of New BioResolve SCX mAb

Charge Variant Solution Offerings

A “Platform IEX Method” that could be used to

adequately separate a diversity of mAbs and

ADC’s (with CX pH buffers)

Transferability of methods from development to

QC using same column / particle (various LC

platforms)

Corrosion Resistance: reassurance that the

separation won’t be fouled by corrosion/metal

ions

Extended column life via use of a Guard

Equipped Column Design that does NOT

degrade separation

Obtaining MS data with on-line IEX-MS


www.waters.com/BioResolve

For more information



CE / cIEF techniques also used for mAb charge variant

analysis since a COMPLEMENTARY Technique

ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: https://www.tandfonline.com/loi/kmab20

Figure 1. cIEF electropherograms of COMBO with various mAb-A: mAb-B ratios.

Figure 2. IEC charge variant profiles of non-stressed and stressed COMBO with various mAb-A: mAb-B ratios. The main peak split in mAb-B is caused by IgG2 disulfide bond isoforms (mainly B and A/B forms).


CE / cIEF techniques also used for mAb charge variant

analysis since a COMPLEMENTARY Technique

Regulatory agencies want structure / function information from isolated peaks.


AU

0.00

0.08

0.16

0.0 10.0 20.0

AU

0.00

0.07

0.14

0.0 10.0 20.0 30.0

Competitor A

3 µm Column

and Buffer System

BioResolve SCX mAb Column

and

BioResolve CX pH Brf

Concentrates

Adalimumab

p/v

1.4

p/v

5.8

Bioresolve SCX mAb Challenges

the Resolving Power of CE pH Gradient / IEX Platform Method

BioDrugs 2016. 30, 321–338.

Beckman PA800 CE

30 minute run

AcidicBasic

Comparable Resolution


Moderate to high resolution

Fast, 15 minute “turn-key” separations

from CE companies such as Beckman

and ProteinSimple

Frequently used in Drug Discovery for

candidate screening since complete

characterization not needed.

Capillary Electrophoresis (e.g., cIEF)

▪ Regulatory agencies want structure / function

information from isolated peaks

- Matrix used in cIEX NOT volatile so no MS

- Matrix used with CE frequently contains urea

to avoid mAb precipitation. Again, no MS

- CE can be performed without urea in matrix

using acetic acid electrolytes. While this can be

coupled to a MS, component resolution NOT as

good as cIEX or IEX.

- Too little mAb material injected with CE / cIEF

so impossible to collect fractions for subsequent

ligand binding or biological studies looking at

activity.

Advantages Limitations


Acknowledgements

Matthew Lauber

Susan Rzewuski

Mike F. Morris

Jessica A. Field

Mingcheng Xu

Darryl Brousmiche

Justin McLaughlin

Steven Byrd

Stephen Shiner

Mike Savaria

Mathew DeLano

Jonathan Belanger

Bei Niu

Yuehong Xu

Tom Walter

Kevin Wyndham

Pam Iraneta

Bill Warren

Jen Fournier

Wray Johnson

Chemistry

Yiting Zhang

Ed Grover

Weiqiang Gu

Koley Hall

Kevin Thompson

Pat Curtis

Alan Carley

Yiting Zhang

Manufacturing

Steve Koza

Samantha Ippoliti

Ying Qing Yu

Henry Shion

Weibin Chen

Scientific Operations

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