optimizing quantification of mab and adc aggregates · detecting and quantifying mab aggregation...

AdvanceBio SEC Optimizing Quantification of mAb and ADC Aggregates: Speed versus Resolution

February 16, 2016 Agilent Technologies 1

Optimizing Quantification of mAb and ADC

Aggregates: Speed versus Resolution


What is size exclusion chromatography?

Why is resolution important?

How much resolution is needed?

How to speed up the analysis?

What is the benefit?

Dr Linda Lloyd, BioColumns Product Manager

Dr Andrew Coffey, Senior Applications Chemist

Aggregation


Aggregates can be tangled clusters of denatured mAbs

• Irreversibly formed during production

• Expression, downstream processing

• Storage as drug substance or drug product

Aggregates can be ordered structures of native mAbs

• May be reversible or irreversibly

• Interaction may be hydophobic and/or electrostatic

• Dimer produced from association of two monomers

Aggregation is impacted by

• Biochemical and biophysical properties of the mAb

• Physicochemical environment

Aggregation impacts

• Process yield and economics

• Product immunogenicity

When does aggregation matter?


• candidate

selection

• protein

cloning

Discovery

• clone

selection

• stability

studies

• QbD

Development

• process

monitoring

• Continuous

improvements

• QC

Manufacture

Characterization

SEC for soluble aggregates

Aggregation is critical quality attribute - size exclusion chromatography is required technique to quantify aggregation

100s of samples for analysis

Clone selection/development

• Bio-Monolith Protein

A/Protein G

How much is

expressed

• AdvanceBio SEC How much is

aggregated

• AdvanceBio Glycan

Mapping

What is the

glycan form

Formulation

Drug product (DP) requires

formulations that meet the target

product profile (TPP)

Require analytical methods needed

to determine stability and set shelf

life

Requires high throughput assays to

monitor multiple formulations as a

function of time


Small number of samples

Candidate Characterization

• Multiple

characterization

techniques

What is the

candidate

• Aggregation What are the

critical quality

attributes

• Is the level of

aggregation likely to

cause late stage failure

Is it suitable to

move forward

Quality control

Batches of drug product (DP) must

meet the defined impurity profile

Require analytical methods with

sensitivity and resolution to ensure

batch complies with specification

Require robust analytical methods

that deliver accurate data for every

sample


What does that mean for SEC


• Columns must deliver accurate, precise quantitation for mAbs and next generation biologics

• High resolution for more accurate quantitation

• Faster analysis speeds for delivery to deadlines

• No change to sample integrity

• Sensitivity for quantitation at low levels - 1 to 5%

• Methods must be easily transferred to other locations

AdvanceBio SEC columns improve lab productivity by providing robust, reliable methods

that eliminate sample re-analysis and increased sample throughput.

Size Exclusion Chromatography

• Size Exclusion Chromatography (SEC) allows the separation of

molecules based on the size in solution.

• Larger molecules are excluded from the pores of the stationary

phase and elute first.

• Smaller molecules diffuse into the pores and elute later.

• There is no interaction between the analyte and the stationary

phase.

• It is the method of choice for quantifying the amount of

aggregates, dimers and larger oligomers, of therapeutic

proteins such as monoclonal antibodies (mAbs).


SEC animation


Detecting and quantifying mAb aggregation




# Time Area Area

%

1 4.66 2.8 0.06

2 5.18 10.9 0.22

3 5.72 32.3 0.66

4 6.58 4849.0 98.40

5 8.13 20.4 0.41

6 8.80 14.2 0.29

1 2

3

4

5 6

Some definitions

• Column volume

• Exclusion limit / Void volume

• Interstitial volume

• Pore volume

• Total permeation

• Non-specific interaction


Column Dimensions: 7.8 x 300 mm

Column Volume = 14.3 mL

What are these inside the column?

• Interstitial volume

• Pore volume


Interstitial or inter-

particle volume

Pore Volume or intra-

particle volume

Silica particles

min2 4 6 8 10 12 14

Norm.

0

50

100

150

200

250

300

What are these regions on a chromatogram?


Colu

mn V

olu

me

Pore

Volume

Interstitial

Volume

Exc

lusi

on

Lim

it /

Void

volu

me

Tota

l P

erm

eati

on

Non

-speci

fic

inte

ract

ion?

Rule Number #1

• Choose the right pore size

– It is essential to select a column that has pores

sufficiently large to allow your molecule to

permeate into the pore structure of the stationary

phase and not be excluded.

– It is also essential to choose a pore size that is not

too large.


For monoclonal antibodies the optimum pore size is around 300Å …

Agilent SEC pore size selection


SEC Chromatogram of proteins

min2 4 6 8 10 12 14

mAU

0

50

100

150

200

250

300

350


AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7 µm

1.0 mL/min, 150 mM Sodium Phosphate pH 7.0

UV, 220 nm

1 Thyroglobulin aggregates

2 Thyroglobulin

3 γ-Globulin (IgG) dimer

4 γ-Globulin (IgG)

5 Ovalbumin dimer

6 Ovalbumin

7 Myoglobin

8 Uridine

9 Vitamin B12 1

2

3

4

5

6 7

8

9

Creating a calibration curve

Thyroglobulin

dimer Thyroglobulin

IgG dimer

IgG Ovalbumin

dimer Ovalbumin

Myoglobin

Aprotinin

Neurotensin Angiotensin II

Uridine

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Log(M

W)

Retention Time (min)

AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7µm Protein or Peptide MW Log(MW) RT (min)

Thyro Aggs 1340000 6.13 4.64

Thyroglobulin 670000 5.83 5.13

IgG Dimer 300000 5.48 5.65

γ-Globulin 150000 5.18 6.53

Oval Dimer 88600 4.95 7.25

Ovalbumin 44300 4.65 8.14

Myoglobin 16950 4.25 8.78

Aprotinin 6511 3.81 9.21

Neurotensin 1672 3.22 10.36

Angiotensin-II 1040 3.02 10.64

Uridine 244 2.39 11.22


What impacts resolution

• Resolution at half height


Rt - Rt2 1

W1/2

𝑅𝑠 = 2 (𝑅𝑡2 − 𝑅𝑡1)

1.7 (𝑊0.5 1 + 𝑊0.5 2)

We have maximised Pore Volume in

order to give greatest retention time

difference …

… we have optimised Particle Size

and column loading in order to give

sharper peaks (while retaining the

ability to run on 400 bar instruments).

Mobile phase selection


min2 4 6 8 10 12 14

mAU

0

50

100

150

200

250

300

min2 4 6 8 10 12 14

mAU

0

50

100

150

200

250

4.6 x 300 mm

150 mM Sodium Phosphate, pH 7.0

0.35 mL/min

4.6 x 300 mm

20 mM Tris, 90mM NaCl, pH 8.0

0.35 mL/min

AdvanceBio SEC columns

• Optimized pore size – 300Å for larger biotherapeutic proteins, mAbs and ADCs

– 130Å covering a wide range of smaller proteins and polypeptides

• Optimized pore volume – Highest pore volume for maximum resolution

• Optimized particle size – 2.7 µm totally porous particles for high efficiency and applicability

from 400 bar to UHPLC

• Optimized column loading – Multiple column dimensions for speed and resolution with

maximum lifetime

• Optimized surface chemistry – Proprietary hydrophilic coating to reduce non-specific interactions


Characterization and QC

Need more

resolution

Slower flow

rates

Multiple

columns


Need more resolution

• Resolution at half height


Rt - Rt2 1

W1/2

𝑅𝑠 = 2 (𝑅𝑡2 − 𝑅𝑡1)

1.7 (𝑊0.5 1 + 𝑊0.5 2)

Column Performance Uridine (small molecule)

120000

125000

130000

135000

140000

145000

150000

155000

160000

165000

170000

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Pla

tes/

m

Flow Rate (mL/min)

Plates/m


Column Performance Vitamin B12

100000

110000

120000

130000

140000

150000

160000

170000

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Pla

tes/

m

Flow Rate (mL/min)

Plates/m


Column Performance Resolution (BioRad and IgG I9640)

1.30

1.40

1.50

1.60

1.70

1.80

1.90

2.00

2.10

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Reso

luti

on F

act

or

Flow Rate (mL/min)

Rs IgA/IgG

Rs Ova/Myo

Rs I9640


Increased sample throughput


Need more

speed

Shorter 15cm

columns

Faster flow

rates

Initial Conditions

min2 4 6 8 10 12 14 16 18

mAU

0

10

20

30

40

50

6.9

90

8.0

00

8.7

57

10.1

80

11.6

64

15.2

20


5µm Diol-type SEC column

7.8 x 300 mm

Flow rate 0.8 mL/min

Run time 20 min

Resolution monomer / dimer 1.77

Same flow rate, 15cm column Same resolution, much shorter run time

min1 2 3 4 5 6 7 8

mAU

0

20

40

60

80

100

120

140

2.9

12

3.3

18

3.5

77

4.0

93

4.8

77

7.1

64


AdvanceBio SEC 300

7.8 x 150 mm


Run time 9 min


Highest flow rate tested, 15cm column Resolution less than 1.5, but sufficient for rapid screening

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

20

40

60

80

100

120

1.5

58

1.9

13

2.1

91

2.6

09

3.8

19


AdvanceBio SEC 300

7.8 x 150 mm


Run time 5 min


Flow rate impact on sample throughput

0

50

100

150

200

250

300

350

400

450

0.0

5.0

10.0

15.0

20.0

25.0

0.8 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

Sam

ple

Thro

ughput

*

Run T

ime (

min

s)

Flow Rate (mL/min)

Throughput (samples/24h) Run Time


Increase speed of analysis

min2 4 6 8 10 12 14

Norm.

0

20

40

60

80

100

120

140


0.5 mL/min

1.0 mL/min

1.5 mL/min

Signals normalised and aligned.

Run time reduced from 12 to 4

minutes

3X sample throughput

Sufficient resolution

AdvanceBio SEC 300Å 7.8 x 150 mm

Reduce laboratory running costs

More to

increase

productivity

Longer column

lifetime

Fewer

columns,

reduced

downtime


Extended Lifetime

min2 4 6 8 10 12 14

mAU

0

10

20

30

40


Every 200 injections of IgG I9640 during

extended lifetime experiment (14 days; up to

1200 injections)

Economic Value


250Å Diol-type, 5µm 7.8 x 300 mm

Economic value calculation …


250Å Diol-type, 5µm

Multiple sample types

mAbs and next generation biologics

Need more

generic

methods

Reduced

secondary

interactions

Fewer

methods,

better peak

shape


Difficult Samples Problematical mAbs …


290Å 3µm

250Å Diol-type, 5µm AdvanceBio SEC 300Å

300Å 3µm

Difficult Samples Antibody Drug Conjugates (ADCs)


290Å 3µm

250Å Diol-type, 5µm

200Å 1.7µm

AdvanceBio SEC 300Å

Recap


Need more speed Shorter 15cm columns Faster flow rates

Need more resolution Slower flow rates Multiple columns

Need more generic

methods

Reduced secondary

interactions

Fewer methods, better

peak shape

Column choice - resolution or speed

- requires different column formats


0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

Flow Rate (mL/min)

Two 7.8 x 300 mm

7.8 x 300 mm

7.8 x 150 mm

Speed Resolution

Separation of protein standards and IgG

using different Agilent HPLC systems

min0 2 4 6 8 10 12 14

Norm.

0

25

50

75

100

125

150

175

200

Agilent 1100 LC System

min2 4 6 8 10 12 14

Norm.

0

25

50

75

100

125

150

175

200

Agilent 1260 Bio-inert LC System

min2 4 6 8 10 12 14

Norm.

0

25

50

75

100

125

150

175

Agilent 1290 Infinity LC System

Conditions

Eluent 150 mM Sodium phosphate buffer, pH 7.0

Flow rate 1.0 mL/min (7.8 mm ID columns) or 0.35 mL/min (4.6 mm ID columns)

Temperature Ambient

Sample 1 mg/mL protein concentration

Instrument Agilent 1100 HPLC instrument (legacy, 400 bar)

1260 Infinity Quaternary Bio-inert (600 bar)

1290 Infinity Binary LC with G1315D DAD and Bio-inert flow cell (1200 bar)

Wavelength 220 nm


Agilent AdvanceBio SEC Ensuring reliability


min0 2 4 6 8 10 12 14 16

mAU

0

10

20

30

40

50

60

70

Injection #1

Injection #1200

Injection #800

Injection #400

Injection Rs

#1 2.16 (after 0 days)

#400 2.16 (after 4 days)

#800 2.20 (after 8 days)

#1200 2.19 (after 12 days)

Effect of analysis temperature of on monomer /

dimer / aggregate content of γ-Globulin


0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

14.0%

16.0%

18.0%

20.0%

65.0%

67.0%

69.0%

71.0%

73.0%

75.0%

77.0%

79.0%

81.0%

83.0%

85.0%

15°C 20°C 25°C 30°C 35°C 40°C 45°C 50°C 55°C

Aggs

and D

imer

Are

a %

Monom

er

Are

a %

Injection Number

Monomer% Aggs% Dimer%

Control of temperature


min4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

mAU

0

25

50

75

100

125

150

175

200

225

15 °C 167.5 bar

Rs 1.758

Monomer Area% 74.8%

25 °C 140.6 bar

Rs 1.825

Monomer Area% 73.9%

Column dimensions


4.6 or 7.8 x 300 mm 4.6 or 7.8 x 150 mm

Two 4.6 or 7.8 x 300 mm

min5 10 15 20 25

mAU

0

10

20

30

40

50

60

70

min5 10 15 20 25

mAU

0

10

20

30

40

50

min2 4 6 8 10 12 14

mAU

0

25

50

75

100

125

150

175

min2 4 6 8 10 12 14

mAU

0

5

10

15

20

25

30

35

40

min2 4 6 8 10 12 14

mAU

0

25

50

75

100

125

150

175

min2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

60

70

80

min1 2 3 4 5 6 7

mAU

0

50

100

150

200

250

min1 2 3 4 5 6 7

mAU

0

20

40

60

80

100

120

Robustness or sensitivity


min2 4 6 8 10 12 14

mAU

0

25

50

75

100

125

150

175

200

min2 4 6 8 10 12 14

mAU

0

25

50

75

100

125

150

175

200

4.6 mm ID (1260 Bio-inert)


Inj vol 2 µL

7.8 mm ID (1260 Bio-inert)


Inj vol 6 µL

Pressure 174.4 bar

Rs 1.91

Rs 2.23

Pressure 154.1 bar

Rs 1.82

Rs 2.12

Resolution 150 mm vs 300 mm vs 2 x 300 mm

min5 10 15 20 25

mAU

0

20

40

60

80

100

120

min5 10 15 20 25

mAU

0

5

10

15

20

25

30

35

40

min5 10 15 20 25

mAU

0

10

20

30

40

50


7.8 x 150 mm

Pressure 102.3 bar

Rs 1.63

7.8 x 300 mm

Pressure 188.7 bar

Rs 2.06

2 x 7.8 x 300 mm

Pressure 330.1 bar

Rs 2.53

• For resolution use longer column lengths 300 mm

– Reduce flow rate

• For speed use shorter columns 150 mm

– Increase flow rate

• Generic methods for wide range of sample types

– Use an innovative SEC column that has minimal

non-specific interactions

Summary


The AdvanceBio SEC columns

provide solutions for all the above

Thank you !


optimizing quantification of mab and adc aggregates · detecting and quantifying mab aggregation...

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