development of analysis methods for therapeutic … 16, 2016 1 development of analysis methods for...

November 16, 2016

1

Development of Analysis Methods for Therapeutic Monoclonal Antibodies Using Innovative Superficially Porous Particle Biocolumns

Anne Blackwell Bio Columns Product Support Scientist Suresh Babu Senior Application Scientist

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

Outline of Talk

November 16, 2016

2

• Introduction – mAbs, Characterization

• Workflow Solutions

• Reversed-Phase Challenges

• RP Method Development/Optimization

• mAb Intact and Fragment Solutions

• Summary


Critical Quality Attributes and Analytical Methods

Pyro- Glutamate

Deamidation /Oxidation

Fragmentation (Hinge)

Glycosylation (G0, G1, G2)

Truncation (Lys 0, 1, 2)

Disulfide Shuffling

Aggregation

HILIC

IEX

IEX

IEX

RP

SEC

RP

November 16, 2016

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

November 16, 2016

4

Glycans Aggregation Molecular Weight Determination

Charge Variants

Peptide Mapping Oxidation Amino Acids Protein PEGylation

Comprehensive Portfolio of Analytical Instrumentation & Solutions for Biopharma & Biosimilars Discovery > Development > QA/QC

CE/LIF/MS, CE/MS LC/FLD, LC/MS

LC/UV Size Exclusion CE/UV Field-flow fractionation

CE/SDS-PAGE, CE/MS Microfluidic SDS-PAGE LC/UV or LC/MS

IEF analyzer (iCE 280) CE (cIEF), CE/MS Bio-LC/UV Ion Exchange

LC/UV LC/MS CE/UV, CE/MS

CE/MS LC/UV LC/MS HIC and RP

LC/UV LC/MS CE, CE/MS

LC/UV SEC / Cation Exchange Microfluidic SDS-PAGE


Characterization of Monoclonal Antibodies

November 16, 2016

5

Titer determination and purification Affinity Chromatography

Protein identification and impurity profiling Reversed-phase chromatography (RP)

Glycan analysis Hydrophilic interaction chromatography (HILIC)

Charge variant analysis Ion exchange chromatography (IEX)

Aggregation analysis Size exclusion chromatography (SEC)


November 16, 2016

6

Agilent Bio-LC Column Portfolio

Agilent Bio-LC Columns

Affinity

Bio-Monolith Protein A Protein G

Multiple Affinity Removal System

Reversed Phase

AdvanceBio Peptide Mapping ZORBAX RRHD

300A 1.8um

Poroshell 300

AdvanceBio RP mAb

ZORBAX 300SB

ZORBAX Amino Acid Analysis

PLRP-S polymeric reversed phase

AdvanceBio Desalting-RP

Cartridges

HILIC

AdvanceBio Glycan Mapping ZORBAX RRHD

300-HILIC

Size Exclusion

AdvanceBio SEC

Bio SEC-3

Bio SEC-5

ProSEC 300S

ZORBAX GF-250

ZORBAX GF-450

Ion Exchange

Bio-Monolith (QA, DEAE, SO3)

Bio mAb

Bio IEX (SAX, SCX, WAX, WCX)

PL SAX

PL SCX


AdvanceBio Oligonucleotide

AdvanceBio RP mAb

Reversed-Phase Columns: mAb Characterization

Primary Structure Characterization Workflows

7

November 16, 2016

Intact mAb LC/MS

Heavy / Light Chains

LC/MS, LC/UV

Reduction / alkylation

Peptides LC/UV

Enzymatic digestion

Fab / Fc Regions LC/MS, LC/UV

Enzymatic digestion


Challenge Disadvantage Consequence

Long analysis times

Low number of samples run per day

Low throughput

Insufficient Resolution Poor accuracy and precision

for results

Lack of confidence in

analysis results

Short column lifetimes

Must purchase new columns often

Poor use of resources

Method transfer to multiple columns

Different columns for UV and MS

Time consuming

The Challenges

November 16, 2016

8

I M P A C T

C O S T !


AdvanceBio RP-mAb Column with Poroshell Technology

November 16, 2016


Particle

• 3.5 μm SP particle

• 0.25 μm porous layer depth

• 450Å pore diameter

The optimum large molecule resolution for use with both HPLC and UHPLC systems

3.5 um 3.0 um

0.25 um

The most popular phases for proteins, plus a unique selectivity

Bonded Phases

• C4

• SB-C8

• Diphenyl

November 16, 2016

10

Method Development: Factors Affecting the Protein Separation AdvanceBio RP-mAb

• Column packing (silica type and bonded phase)

• Column dimension

• Mobile phase

• Column temperature

• Linear velocity

• Gradient profile

• Sample


November 16, 2016

11

Choose The Initial Bonded Phase: C18, C8, C4

Buy Them All and Try Them All…..

Not really feasible, right? How do you narrow it down?


November 16, 2016

12

Selection of Bonded Phase:

Application RP column Peptides and proteins, < 5000 MW C18 Hydrophobic polypeptides and proteins, Antibodies, > 5000 MW C8

Larger, hydrophobic peptides and proteins, Antibodies, > 5000 MW C4

Proteins with aromatic sidechains, Antibodies, Fusion proteins, > 5000 MW Diphenyl


C4 C8 C18 Diphenyl

Increasing protein size and hydrophobicity

November 16, 2016

13

Pore size

• Generally speaking, the “go to” pore size for most protein applications using RP is > 300Å.

≤ 4000 MW 4000 – 500,000 MW

Use smaller pore size columns to maximize loading capacity and retention Use > 300Å pore columns to maintain high efficiency. Increase column diameter to increase loading capacity


November 16, 2016

14

Mobile phase

• In order of increasing eluotropic force and decreasing polarity: Water, Methanol, Acetonitrile, isopropanol, THF

Acetonitrile: • Low UV Cutoff, 190 nm • Volatile • Good solubilization, denaturant • Selectivity differences and sample retention will vary significantly between mobile

phases

• pH and ionic strength of the aqueous portion of mobile phases are important in developing rugged methods

• It is very important to control pH to stabilize retention and selectivity

• % of organic solvent –there is a pressure maximum and minimum for organic:aqueous mobile phases and it differs depending on the organic

• Use solvents that are compatible with the shipping solvent For Research Use Only. Not for use in diagnostic procedures.

November 16, 2016

15

Ion-pairing agent

TFA

• Most common ion pairing agent for reversed-phase analysis

• Low UV absorbance

• Volatile

• Good solubilization, denaturant

• Acidic - improves peak shape

• Mild anionic ion pair - more retention of Lys, other free amines

TFA / Water : TFA / ACN mobile phase

• Low pH (0.1% TFA, pH 1.9) suppresses silanol interactions

• Relatively low viscosity - high efficiency, low pressure

If LC/MS is used, can substitute Formic or Acetic acid


November 16, 2016

16

Column Temperature • Higher column temperature can dramatically improve resolution and recovery • Column temperature can be used to fine tune a separation by affecting both the

retention k and selectivity • Check manufacturer specs for compatibility • Agilent AdvanceBio RP-mAb columns are rated to 90 oC

40º C

65º C

10 0 5

Time in Minutes

A B

A B

• About a 1% increase in T leads to a 1 to 2% decrease in k.

• Increase in T leads to decrease in pressure due to decrease in mobile phase viscosity.

• Increase in T also leads to decrease in peak widths.

• Also, use of a thermostat column compartment improves retention time precision.


November 16, 2016

17

min1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7

mAU

0

200

400

600

800

1000

Impact of Column Temperature on Intact mAb Analysis AdvanceBio RP-mAb

80 °C 70 °C 60 °C 50 °C 40 °C

• Temperature has a significant impact on run time, peak shape and recovery.

• Higher temperatures better with optimum at ~ 80 °C for this example

AdvanceBio RP-mAb SB-C4, 2.1 x 100 mm


18

70°C

80°C

90°C

Agilent-RIC collaboration biopharma - Nov 2014

Light chain Heavy chain

• High resolution separation of different variant peaks at higher temperature

AdvanceBio RP-mAb C4, 4.6 x 50 mm


Impact of Temperature on mAb Fragment Analysis AdvanceBio RP-mAb

Reduction

LC HCIntact

November 16, 2016

19

Linear Velocity/Flow Rate

Van Deemter Curve

The smaller the plate height, the higher the plate number and the greater the chromatographic resolution


November 16, 2016

20

Linear Velocity/Flow Rate

• Shorten analysis time by:

- Reduced column length

- Increase flow rate

• Key Chromatographic Parameters Affected by Flow Rate

- Resolution - Efficiency - Peak shape - Pressure


November 16, 2016

21

min0 0.5 1 1.5 2 2.5

mAU

0

200

400

600

800

1000

1200

1400

1600 • Linear velocity has an impact on peak shape and resolution

• Higher velocities better with optimum at ~ 1mL/min for 2.1 mm id for this example

min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

-100

-75

-50

-25

0

25

50

75

100

125

Impact of Linear Velocity on Intact mAb Analysis AdvanceBio RP-mAb


0.5 mL/min 0.8 mL/min 1.0 mL/min 1.5 mL/min


November 16, 2016

22

mAU

0

200

400

600

mAU

0

200

400

600

min1.8 2 2.2 2.4 2.6 2.8 3 3.2

mAU

0

200

400

600

0.5 mL/min

0.8 mL/min

1.0 mL/min

Impact of Linear Velocity on mAb Fragment Analysis AdvanceBio RP-mAb

• Linear velocity has strong impact on resolution.

• Higher velocities better with optimum at ~ 1 mL/min for 2.1 mm id for this example



Papain digestion

IntactFc

2 * Fab

Fab Fab/2

Fc Fab/2

Fc/2

November 16, 2016

23

Gradient Slope

• Gradient steepness effects retention (k*)

and resolution

• Adjust gradient slope to optimize

resolution - accomplished by changing:

- gradient time tG (most common way to

change gradient steepness)

- % change in organic modifier over time


November 16, 2016

24

min1.5 2 2.5 3 3.5 4

mAU

0

200

400

600

800

• Gradient profile has impact on selectivity and resolution

• Steeper gradient better with optimum 5 – 50% for this example

Impact of Gradient Profile on mAb Fragment Analysis AdvanceBio RP-mAb

5 to 30% 5 to 40% 5 to 50% 5 to 60%



Papain digestion

IntactFc

2 * Fab

November 16, 2016

25

Column Robustness

High Reproducibility Low Carry-over

Long Column Lifetime


Reproducibility AdvanceBio RP-mAb

November 16, 2016

26

• Injection to injection repeatability supports consistent column performance

A

B

C

Intact Reduced

Analysis of intact mAb

Average %RSD

A RT 4.5404 0.09

Area 4521.294 0.09

B RT 4.8494 0.05

Area 158.43 0.80

Analysis of reduced mAb

Average %RSD

A RT 3.234 0.06

Area 496.162 0.91

B RT 4.6574 0.04

Area 909.85 0.69

C RT 5.0118 0.02

Area 127.028 1.10



November 16, 2016

27

No Carry-Over AdvanceBio RP-mAb

• No protein carry-over between runs

Zoom view

Zoom view

• Injection of 3 production batches of intact mAb each followed by 1 blank injection, shows no carry-over

• Injection of 3 production batches of reduced mAb each followed by 1 blank injection shows no carry-over

Intact

Reduced



Long Column Lifetime AdvanceBio RP-mAb

November 16, 2016

28

Peak Compond RT_1st Injection

RT_1003 Injection

RT change (min)

RT change%

1 Ribonuclease A 0.957 0.942 0.015 1.57%2 Lysozyme 1.872 1.86 0.012 0.64%3 Cytochrome C 2.111 2.097 0.014 0.66%4 α-Lactalbumin 2.909 2.899 0.01 0.34%5 Catalase 6 Carbonic Anhydrase 3.994 3.981 0.013 0.33%

AdvanceBio RP-mAb SB-C8

• Stable over 1,000 injections =

40,000 column volumes

0

1

2

3

4

0 200 400 600 800 1000

Ret

entio

n tim

e [m

in]

# of injections

Ribonuclease A

Cytochrome C

Lysozyme

α-Lactalbumin

CarbonicAnhydrase

AdvanceBio RP-mAb SB-C8


Excellent Batch-to-Batch Reproducibility AdvanceBio RP-mAb (different column batches)

November 16, 2016

29

Samples

1. Ribonuclease A 13.7 kDa

2. Cytochrome C 12 kDa

3. Holo-Transferrin 80 kDa

4. α-Lactalbumin 14.2 kDa

5. Catalase 240 kDa

6. Carbonic Anhydrase 29 kDa

• Reproducible retention time and peak shapes over 6 different column batches

Batch 1

Batch 2

Batch 3

Batch 4

Batch 5

Batch 6

1 2 3

4 5 6



Fast Intact mAb Analysis AdvanceBio RP-mAb

November 16, 2016

30

• AdvanceBio RP-mAb C4 provides a sharp peak and resolves fine detail in less than two minutes

• AdvanceBio RP-mAb Diphenyl resolves additional fine detail - the Diphenyl phase is unique to Agilent

All phases provides good separation of intact mAb with AdvanceBio RP-mAb

Diphenyl resolves additional fine detail.

min 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

-4

-2

0

2

4

6

8

10

12

14

min 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

0

20

40

60

80

100

120

140

• AdvanceBio RP-mAb C4 • AdvanceBio RP-mAb SB-C8 • AdvanceBio RP-mAb Diphenyl


• AdvanceBio RP-mAb C4 provides better resolution than protein columns from competitors

November 16, 2016

31

Zoom view

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

0

50

100

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

0

50

100

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

0

50

100

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

0

50

100

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

-5 0 5

10

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

-5 0 5

10

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

-5 0 5

10

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

-5 0 5

10

AdvanceBio RP-mAb C4, 450Å, 3.5 μm

Brand A C4, 400Å, 3.4 μm

Brand B C4, 200Å, 3.6 μm

Brand C C4-30, 300Å, 2.6 μm

Fast Intact mAb Analysis AdvanceBio RP-mAb


High Resolution Separation of Intact mAb AdvanceBio RP-mAb

November 16, 2016

32

• AdvanceBio RP-mAb C4 provides superior peak shape at a lower pressure than a UHPLC protein column from a competitor

490 bar

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

0

50

100

min 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

mAU

0

50

100


910 bar

Brand D C4, 300Å, 1.7 μm


Fast & High Efficiency Separation of Intact mAb AdvanceBio RP-mAb

November 16, 2016

33

1 x10

0

0.4

0.8

1.2

1.6

2

2.4

2.8

3.2

3.6

min 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5


AdvanceBio RP-mAb Diphenyl, 450Å, 3.5 μm

Brand A, C4, 300Å, 3.5 μm

mAU

Width = 0.219 Width = 0.187 Width = 0.227

• AdvanceBio RP-mAb provides superior peak shape


November 16, 2016

34

0

1

2

3

4

5

6

LC

HC

m i n1 2 3 4 5 6 7 8 9

m AU

High Resolution mAb Fragments: LC/UV AdvanceBio RP-mAb

Reduced mAb

AdvanceBio RP-mAb C4, 450Å, 3.5 μm Brand A , C4, 300Å, 3.5 μm

0

2

4

6

8

0

1

2

3

4

m i n1 2 3 4 5 6 7 8 9

m AU LC HC

LCHC

mAb1 mAb2

Rs = 2.0 Rs = 0.6

Rs = 3.4

Rs = 0.8

• AdvanceBio RP-mAb provides superior peak shape and high resolution than competitor protein column


Reduction

LC HCIntact

November 16, 2016

35

High Resolution ADC Fragments: LC/UV AdvanceBio RP-mAb

Papain digested ADC

0

1

2

3

4

5

6

0

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10

Brand A , C4, 300Å, 3.5 μm

0

0.5

1

1.5

2

2.5

3

0

0.5

1

1.5

2

2.5

3

1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Reduced ADC

ADC: Lys conjugated

min

mAU

min

mAU

• Separation of different drug conjugated species


L0 L1, L2

H0, H1, H2, H3

L0 L1, L2

H0, H1, H2, H3

Fc 0 - 1

2 * Fab 0 - 2

Fc 2 * Fab


November 16, 2016

36

AdvanceBio RP-mAb Diphenyl

High Resolution ADC Fragments: LC/UV AdvanceBio RP-mAb

Reduced ADC

• Lys-conjugated ADC

• Cys-conjugated ADC Heavy chain

Light chain

Heavy chain Light chain

• Separation of different drug conjugated species


November 16, 2016

37

High Resolution mAb Fragments: LC/UV AdvanceBio RP-mAb

• mAb

• pH stressed mAb

• Oxidized mAb

Herceptin

pH stressed Herceptin

Oxidized Herceptin

Herceptin

pH stressed Herceptin

Oxidized Herceptin

AdvanceBio RP-mAb C4, 4.6 x 50 mm

Stressed variants intact

reduced

• Separation of stressed-induced antibody variants


Heavy chain Light chain

Fast, High Resolution mAb Fragment Analysis AdvanceBio RP-mAb

November 16, 2016

38

min 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

min 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

min 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

min 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200


Brand A C4, 400Å, 3.4 μm

Brand B C4, 200Å, 3.6 μm

Brand C C4-30, 300Å, 2.6 μm

• AdvanceBio RP-mAb provides superior peak shape and resolution than other columns designed for protein separations


Papain digestion

IntactFc

2 * Fab

Fab Fab/2

Fc Fab/2

Fc/2

November 16, 2016


0.5

1

1.5

2

2.5

min1 2 3 4 5 6 7 8 9 10 11 12 13 14

mAU

Fast, High Resolution mAb Fragment Analysis AdvanceBio RP-mAb


Fc/2

Fd

LC

• Separation of mAb subunits with superior peak shape and resolution

Ides digestion

IntactReduction

2*LC

2*Fc

2 * Fd

Benefits of AdvanceBio RP-mAb Columns

Pain Features and Advantages Benefit

Insufficient resolution

Superficially porous particles of smaller particles (3.5 µm) with wide pore (450Å) increase resolution for mAb but maintain compatibility with all LC instruments

Improved confidence in analysis results (accuracy)

Long analysis times

Due to Poroshell technology, analysis time of mAb characterization has been shown to decrease significantly over fully porous particles of the same size

Improved throughput - reduced costs

Short column lifetime

Column with robust Poroshell packed bed and with 2 µm frit decreases chances of bed-collapse or inlet blockage

Improved resource use - reduced costs

November 16, 2016

Agilent Confidential


November 16, 2016

41

• AdvanceBio RP-mAb columns - analyzing monoclonal antibodies for biopharma discovery, development, and QA/QC applications

Summary AdvanceBio RP-mAb


Improved accuracy: Superficially porous particles (3.5 μm) with wide pores (450Å) increase mAb resolution while maintaining compatibility with all LC instruments

Speed: Shorter analysis times compared to columns packed with fully porous particles of the same size

Lower costs: The robust Poroshell packed bed and 2 μm inlet frit extend column lifetime by helping prevent inlet blockage

Flexible method development: Range of chemistries – SB-C8, C4, and Diphenyl

November 16, 2016

42

Q & A


development of analysis methods for therapeutic … 16, 2016 1 development of analysis methods for...

Documents