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How can Analytical HPLC Techniques

using Agilent Infinity Series UHPLC

Quaternary 1290 & 1260 BioInert Reduce

the Risk of NBE Candidate Failure?

May 22, 2013 1

BioPharm Application Examples using

Buffer Advisor & Blend Assist

Patrick Cronan

Applications Scientist

Agilent Technologies

Boston, MA

Drug Development Lifecycle

2013 BioPharm Seminar Series NBE

Characterization

2

Primary customer Secondary Ta

HPLC methods to confirm target

protein identity and impurity

profiles

Methods will be mainly UV based

as will be used to define

specification

Methods determine final product

QC release criteria

HPLC is used to obtain complete

characterisation of target protein

identity and form.

Methods will be MS based to

maximise the information

obtained from each analysis

Methods developed are used for

process optimization

HPLC Methods

will be fully

validated

Carried out in

accordance with

SOP

Methods must be

robust and

transferable

HPLC is utilised from discovery through process development to

support manufacturing and QC

International Conference for Harmonization (ICH) Documents related to biopharmaceuticals


Characterization

3

• ICH Q6B: Specifications: test procedures and acceptance criteria

for biotechnological/biological products. Guidance on setting and

justification of specifications for proteins and polypeptide from

recombinant or non-recombinant cell cultures. Limited to well-

characterized biotechnology products

• ICH Q5C: Quality of biotechnological products: stability testing of

biotechnological/biological products

• ICH Q5D: Quality of biotechnological products; derivation and

characterization of cell substrates used for production of

biotechnological/biological products

Characterization Profile for NBEs


Characterization

4

Physicochemical Parameters determination of primary structure, composition, and physicochemical properties

Approach: peptide mapping, C- and N-Terminal amino acid sequencing

Higher structural order: Spectroscopy circular dichroism and NMR

isoform characterization: UV, Electrophoresis, HPLC LC/MS

Biological Activity to determine drug efficacy

Approach animal or cell assays, biochemical assays

Immunochemical Properties immunological properties

Approach: binding assays to purified antigens and defined regions of antigens

Quantity

Approach on purified protein: UV spectroscopy - absorbance to quantify

Impurities product related or derived from the process

Approach Process related: DNA and proteins determination of host cell and cell media

Approach Product related: Isomerisation, oxidation and post-translational modifications

(deamidation, truncation and aggregation) (IEX, SEC, CE)

Protein Structures


Characterization

5

Amino acids sequence in the polypeptide

Spatial arrangement of polypeptide chain folding (helices or beta-sheets via H-bonding).

Conformation of the polypeptide caused by H-bonding and hydrophobic interaction

Spatial arrangement of more than a single polypeptide

Different Methods to analyze primary sequence and the structure

Typical Structure of a Monoclonal Antibody (mAb)


Characterization

6

Light Chain

Fc

Fab

Antigen

binding

Hinge

Glycosylation

site Truncation

(lysine)

Disulfide

shuffling

Pyroglutamate

Deamidation/oxidation

Heavy Chain

Potential locations for

product/process

related alterations

influencing drug safety

and efficacy

Monoclonal Antibodies: Impurities


Characterization

7

• Impurities are product- or process related compounds such

as aggregates or deamidated, isomerized, and oxidized

forms (charge heterogeneity) of the mAB

• arise during cloning, fermentation and purification processes

• can affect the mAbs’ tertiary structure and antigen-binding

properties

• Glycosylation pattern can be critical for the function of

mAbs

• Changes in structure and form can alter the efficacy or

safety of the drugs

Interactions during Analysis: Analysis of Charge Variants

• high salt or pH changes (incompatibility/corrosion on standard (metal)

LC equipment

• unspecific interaction of molecule with surfaces

• metal ions eluting from instrument can cause disturbance of

chromatography or column poisoning

• Long runs

8

min0 5 10 15 20 25 30 35

mAU

0

100

200

300

400

0.8561.360

1.688

1.969

2.100

2.243

2.649

2.937

3.211

4.782

6.906

14.049

15.901

16.565

17.745

23.144

min0 5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

1.431

1.559

2.725

24.828

26.560

33.902

MAb-major

Right- MAb-subspeciesMAb-subspecies-left

Agilent Bio MAb, NP10

ProPac WCX-10

15-30% B in 30 min

05-30% B in 30min

Resolved basic and acidic variants on a bio-

inert LC

Massive fronting and tailing makes quantitation impossible


Characterization

1260 Infinity Bio-inert LC: Features at a Glance


Characterization

Titanium based quaternary pump with integrated

degasser and active seal wash (600 bar) up to 10 mL/

min

Thermostatted Column Compartment with two

Bio-inert heat exchangers (option #019)

Wellplate sampler with ceramic needle and PEEK

tubing for metal free sample flow path.

Carryover 40 ppm

Universal Bio DAD Detector, 80 Hz, (G1315C)

with Bio-inert flow cell

Bio-inert Fraction Collector, Chiller, Manual Injector,

Valves, Fluorescence Detector and MWD available

New Capillary Design Ensures Bio-inertness


Characterization

10

Capillaries

Metal cladded PEEK capillary

Newest fitting design: Hybrid technique

New capillary technolgy enables

600 bar AND is completely metal free !!

min 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

500

600

0 %

MeO

H

10 %

MeO

H

50 %

MeO

H

70 %

MeO

H

90 %

MeO

H

11


Characterization

1260 Infinity Stainless Steel LC

System

Solvents:

10 mM Ammoniumacetate + 0 – 90 % Methanol

Reducing Sample-System Interactions Model Compound: ATP

Reducing Sample-System Interactions Model Compound: ATP

min 0 1 2 3 4 5 6

mAU

0

100

200

300

400

500

600

700

0 %

MeO

H

10 %

MeO

H

50 %

MeO

H

70 %

MeO

H

90 %

MeO

H

12


Characterization

Chromatographic conditions

Flow: 0.5 mL/min

Injection volume: 0.2 μL

Column temperature: 40 °C

DAD: 254 nm, Bandwidth 4

Peak width: <0.1 min (2.5 Hz)

Flow cell: bio-inert, 10 mm (1260 Infinity),

bio-inert 1260 Infinity LC

No Column used

Chromatographic Techniques for mAb Analysis


Characterization

13

SIZE CHARGE HYDROPHOBICITY RECOGNITION

Size Exclusion Ion Exchange Reversed Phase Affinity

2-

1-

+

Application Technique Agilent Columns Notes

Primary Structure

Analysis

Reversed phase

separations

ZORBAX 300Å

Poroshell 300Å

ZORBAX 300Extend-C18

PLRP-S

RP separations require (or cause) denaturing of the

protein to get detailed info

Aggregation

analysis

Molecular Size

Size exclusion

separations

Bio SEC-3

Bio SEC-5

ProSEC 300S

ZORBAX GF Series

Protein aggregates are a major concern in drug product.

They influence the final formulation and can potentially

cause immunogenic responses.

Chemical

Modification

Charge Variant

Ion-exchange

separations

Agilent Bio IEX

Agilent Bio MAb

PL-SAX

PL-SCX

Protein analysis using ion-exchange requires buffered salt

gradients or pH gradients.

Aggregation Studies: Regulations


Characterization

14

ICH (Q6B):

• „The category of aggregates include dimers and higher multiples of

the desired product. These are generally resolved from the desired

product and product-related substances, and quantitated by appropriate

analytical procedures (e.g., size exclusion chromatography, capillary

electrophoresis)” (ICH Q6B)

• “Multimers and aggregates should also be appropriately characterized

using a combination of methods. The formation of aggregates, sub-visible

and visible particulates in the drug product is important and should be

investigated and closely monitored on batch release and during stability

studies.

Size Exclusion Chromatography What to Consider in mAb SEC?


Characterization

15

• Size in solution is related to retention time (large size: early elution, small

size: late elution), acts as molecular sieve

• Particle size and pore size determine resolution

• Small injection volume is critical (< 1% of column volume)

• non-specific interactions between the protein and the stationary

phase (column material) must be minimized

by : selection of a stationary phase with low unspecific interaction

by : additives to mobile phase (organic modifier, n-propanol, or salt)

• Quantitation: Usually UV detection (determination of the ratio of

aggregates/monomer), select 220 nm and 280 nm

Monitoring Aggregation and Impurities by SEC


Characterization

16

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Column: Agilent Bio SEC-3 300Å, 7.8x300mm

Mobile phase: 150 mM Phosphate, pH 7

Flow rate: 1.0 mL/min

Temperature: Ambient

Sample: Monoclonal antibody (10 L, 5 mg/mL)

Buffer/Excipients

MAb

Dimer MAb

Aggregates

Low Molecular

Weight Impurities

Detection: UV (diode array detector)

Aggregation Analysis with Increased Sensitivity Accurate and Precise Quantitation of Aggregates by Light

Scattering


Characterization

17

• Refractive Index

• Viscometer

• Dual Angle Light Scattering detector

Agilent 390-MDS

Agilent 1260 Infinity Bio-inert Quaternary LC System

Enables identification of (mAb)n for aggregate composition

Provides increased sensitivity to monitor effect of process variables and

molecular weight information to determine aggregate form – dimer, trimer etc

Light Scattering for higher order aggregates Gamma Globulin (MW 150kD), Agilent Bio SEC-5 500Å, 7.8 x 300 mm


Characterization

18

Monomer

Dimer High MW

aggregates

RI imbalance

(fresh eluent) UV, 254nm

LS 90

RI

Charge Variant Analysis


Characterization

19

Light Chain

Fc

Fab

Antigen

binding

Hinge

Glycosylation

site Truncation

(lysine)

Pyroglutamate Heavy Chain

Disulfide

shuffling

Deamidation/ Oxidation

Charge Variants (charge heterogeneity) are modifications

The alterations can be triggered by biological processes

(e.g. enzymes, mutations or through chemical reactions)

Sites where charge variants can occur

Incomplete C-terminal

processing of Lys and Asn

Deamidation of Asn and Gln

and isomerization

Oxidation of Met, Trp, Cys,

His

Sialylation of terminal glycans

Disulfide mediated

conformational change

Glu cyclization =

pyroglutamate

Adduct formation

Regulatory Aspects


Characterization

20

“4.4.2. Purity and impurities

• As noted in the characterization section, monoclonal antibodies may display

a complex purity/impurity profile that should be assessed by a

combination of orthogonal methods,

• and for which individual and/or collective acceptance criteria should be

established for relevant product-related variants.

• For example, separation methods based on charge heterogeneity should be

considered to quantitatively and qualitatively monitor charge variants.”

From: GUIDELINE ON DEVELOPMENT,

PRODUCTION, CHARACTERISATION

AND SPECIFICATIONS FOR

MONOCLONAL ANTIBODIES AND

RELATED PRODUCTS

Monoclonal Antibody Heterogeneity


Characterization

21

T. Xiang et al. / J. Chromatogr. B 858 (2007) 254–262

Carboxypeptidase B cleavage site

Carboxypeptidase B Cleavage

of C-terminal Lysine.

Samples: CHO-cell derived, humanized monoclonal antibody, IgG1

Lysine (lys or K)

H3N-C-C-O-

+ H O

I II

I

CH2(CH2)3-NH2

Basic

Polar

The addition of lysine to one or

both heavy chains

Charge Variants Charged Isoform Analysis of Monoclonal Antibodies


Characterization

22

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

Minutes

0.00 2.00 4.00 6.00 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

Agilent 1260 Bio –inert LC, Agilent Bio MAb, NP5, 4.6mm x 250mm Buffer A: 10 mM Sodium Phosphate, pH 7.50 Buffer B: A + 100 mM NaCl, pH 7.50 Gradient: 15-95% B in 60 min Flow rate: 0.8 mL/min. Sample: 5 ul 5 mg/mL, mAb PEEK column hardware

Acidic

Isoforms

Basic Isoforms

HPLC Analysis of Charged Isoforms


Characterization

23

Column: Agilent Bio MAb 5um, 4.6x250mm PEEK

Eluent A: 10 mM Na phosphate buffer, pH 5.5

Eluent B: A + 0.5 M NaCl

Flow rate: 0.85 ml/min

Gradient: 5 – 30%B in 25 minutes

Volume: 5 ul of 1mg/ml

Detection: UV 225nm

Instrument: Agilent 1260 Infinity Bio-inert system

min 5 7.5 10 12.5 15 17.5 20 22.5 25

mAU

0

5

10

15

20

25

Lys-1

Lys-2

Lys-0 Basic Charged Variants

K K K

Pro

tein

Net

Charg

e

+

_

pH 4 6 8 10 12

Iso

ele

ctirc

Po

int

(pI)

+

- - - - - -

- - - - - - - -

- -

pH lower than

the protein pI

pH greater than

the protein pI

Protein Isoelectric Point - Charge

April 2012, Seminar

24

Cation

Exchange

Particle

Ion Exchange: Salt Gradient

May 22, 2013

Confidentiality Label

25

Positively charged salt Ions compete

and displace positively charged proteins

Experimental Situation

Chromatographic Conditions

Flow Rate: 1.5mL/min

Gradient: 0 min – 200mM NaCl

2 min – 200mM NaCl




Injection Volume: 20uL

Temperature TCC: 35˚C

DAD: 280nm/4nm

REF: Off

peak width: >0.013 min

(0.25s resp time – 20Hz)

Column Agilent Bio Mab, PEEK,

4.6 x 250mm, 5um

Solvents Buffer A: H20

Buffer B: NaCl 3M

Buffer C: MES (2-(N-morpholino

ethanesulfonic acid monohydrate) 60mM

Buffer D: MES-Na (2-(N-morpholino

Ethanesulfonic acidsodium salt) 35mM

Sample BioRad Cation Exchange Standard:

Myoglobin: 17,699 pI 7.4, 6.9

Cytochrome C: 12,384 Da pI 10.7, 9

Ribonuclease A: 13,700 Da pI 8.88

Buffer Advisor Software Workflow

Buffer Advisor Software Timetable import into Chemstation

29

30

Buffer Advisor Software pH scouting

pH 5.4

pH 5.8

pH 6.0

pH 6.4

pH 6.8

pH 7.0

M

M

R

R

C

C

Myoglobin: pI 6.9

Cytochrome C: pI 10.7, 9

Ribonuclease A: pI 8.88

31

Buffer Advisor Software stable pH

Primary Structure Analysis


Characterization

32

When to use Reversed-phase (RP) separations?

• Intact Proteins

• Reduction and alkylation to separate heavy and light chains

• Enzyme digest of proteins, peptide maps

– may need more than one enzyme to get >95% coverage (large

proteins)

- looking for changes in the peptide fingerprint due to amino acid

substitution, omission, modification (single point mutation)

- provides resolution at the single amino acid level

• Amino acid analysis

Characterization of Primary Structure


Characterization

33

Levels of Characterization

Improve

accuracy

and

resolution

Intact mAb

Reduction/Alkylation

Light and Heavy Chain

Enzymatic Digestion

Peptide Map


Characterization

34

Intact and reduced mABs by reversed phase

separation using small particle column technology

Analysis of a CHO cell derived

intact mAb with an Agilent

RRHD 300 SB C8 , 2.1mm x

50 mm, 1.8 um particle

column on a 1290 Infinity

binary LC

Analysis of a CHO cell derived

reduced mAB with an Agilent

RRHD 300 SB C8 , 2.1mm x

100 mm, 1.8 um particle

column on a 1290 Infinity

binary LC

1290 Infinity Quaternary Pump - BlendAssist

Simple tool for online-dilution of modifiers and gradient set-up

You need different concentrations of modifiers in your analysis, would like to have just one stock-

solution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a

simple tool – BlendAssist!

Water Water

1% TFA

ACN

ACN

1% TFA

Desired method conditions - example:

1. 5 to 95% gradient of ACN with 0.1% TFA in

Water and 0.08% TFA in ACN

2. 20 – 80% gradient of ACN with 0.5% TFA in

Water and 0.4% TFA in ACN

Without BlendAssist you need to either pre-mix the

required solvents or by using stock-solutions of

TFA in Water and ACN to program complex

gradients (%A, B, C, D).

With BlendAssist: just program your binary

organic/aqueous gradient and define the dilution

factor!

35

1290 Infinity Quaternary Pump - BlendAssist

Simple tool for online-dilution of modifiers and gradient set-up

36

min 1 2 3 4

Norm.

0

5

10

15

20

25

30

35

40

6 dynamically mixed mobile phases

6 premixed mobile phases (same user)

min 1 2 3 4

Norm.

0

5

10

15

20

25

30

Analysis of Glucocorticoids - Comparing dynamically mixed mobile phase vs. user influence

37

A:B=70:30

A:B=70:30

Decrease the Diffusion Distance!

1. Rapid Resolution High Definition (RRHD) 300 Family (C3, C8, C18, diphenyl)

Developed very small (totally porous) particles (<2 um)

RRHD1.7um, UHPLC

Superior efficiency

Very narrow bands

Ultra high resolution

Fast flows

Short column lengths (pressure

constraint)

2. Poroshell 300 and AdvanceBio120

Limit diffusion distance into a particle (shell particle)

HPLC

Superior efficiency

Very narrow bands

Ultra high resolution

Fast flows

Long column lengths (no pressure

constraint)

5um Poroshell 300

2.6um AdvanceBio 120

38

2. 5um Poroshell 300 and 2.7um AdvanceBio 120

5.0 um 4.5 um

0.25 um

Poroshell 300 AdvanceBio 120

Use AdvanceBio120 for peptide maps containing small proteins and peptides

Peptide digests of proteins or monoclonal antibodies

Use for LC/MS applications with formic acid

Use Poroshell 300 for intact protein analysis and large peptide fragments

Does the sample contain all polypeptides or larger?

Poroshell 300 can be used with very large proteins/monoclonal antibodies, complex biologicals

39

Premixed versus Blend Assist;

0.05%TFA-Water/0.04%TFA-ACN

41

Premixed

Blend Assist

Note Fine Structure

Triplicate Runs Blend Assist: Stock 1%TFA;

0.05%TFA Water/0.04%TFA ACN

42

Comparison of Three Columns

Tryptic Digest

43

Zorbax 1.8 micron, 2.1mm x 100mm

PoroShell 300

PoroShell 120

Maximum Peaks Plotted for Three Columns

44

LC-based N-Glycan Analysis confirm glycan pattern consistency in QA/QC of the NBE


Characterization

45

• Suitable approaches: • Release of glycans(PNGase F), Labeling with 2AB, LC separation by

HILIC, detection: Fluorescence

• Release of glycans (PNGase F), High Performance Anion Exchange

Chromatography with pulsed amperometric detection (HPAED)

• Glycan release, separation and detection by HPLC/Chip-MS using the

mAB glyco-chip and the Agilent Q-TOF

min 0 10 20 30 40 50

LU

0

5

10

15

20

25

30

35

40

Glycan ladder

analyzed by

HILIC with 1290

Infinity LC

Glycan analysis

benefits from large

power range of 1290

for highest resolution

and smallest peak

shape

46


Characterization

Glycan Pattern by UHPLC and Fluorescence

Detection

(1,6) G1F

(1,3) G1F

G2F G2FS1 G2FS2

G0F

G0

Man5

2100 0A 0G

1463.55

2000 0A 0G

1317.49

2110 0A 0G

1625.60

2120 0A 0G

1787.6574 Man5

1235.44

2121 1A 0G

2197.814

2122 2A 0G

2488.910

min 15 20 25 30 35 40 45

LU

0

2

4

6

8

Gradient glycoproteins

Flow rate 0.5 mL/min

Gradient 0-6 min – 85% B

10 min – 80%B

60 min – 64% B

65 min – 50% B

67 – Flow 0.25 mL/min

68 min – 0% B

73 – 0% B

75 min – 85% B

80 min – Flow 0.5 mL/min

Injection

volume

1 μL

Column T 60 °C

FLD

(wavelength) Ex. 260 nm/ Em. 430 nm

Quality by Design Analytical Methods and Biologic Production


Characterization

47

Concept of QbD: develop robust methods which are easily transferable in the development

lifecycle and across multiple sites

HPLC methods are suitable for providing feedback to development and manufacturing for

achieving optimum expression and yield from the purification.

Early characterization in early discovery will provide insight for characterization at later stages

and will reduce cost and decrease the chance of failures later in the development process.

Traditionally the life cycle

was a series of discrete steps

discovery…development

Discovery

Development

Discovery

Process

Research

Development

Minimize risk for the candidate

before Scale-UP

Biologics Production


Characterization

48

Bio-Reactor

producing biologic Purification Step 1 Purification Step 2

Pure Biologic

Formulation

At each step in the production process HPLC

will be used to test monitor target molecule

concentration and/or form

If profile differs from expected

then LC/MS verification of

profile changes is carried out

In-Process Testing


Characterization

49

• Analytics are required at each stage of the purification process to determine

efficiency of the purification step

• For example a purification step designed to remove aggregates will be monitored

using SEC to quantify the efficiency of aggregate removal and to ensure that the

required purity has been achieved before moving on to the next step

• The HPLC method must be robust and is most likely to be UV based rather than

using MS of LS

• Where abnormality is seen then fractionation and characterisation may be

instigated to determine process corrective action

Reducing Risk of Failure


Characterization

50

Form Function HPLC Techniques

Biophysical Screening Structure RP/IEX/SEC

Thermal Stability RP/SEC

Aggregation SEC

Physico-chemical

Screening

Peptide Maps RP

Charge variation RP/IEX

Glycosylation HILIC

Oxidation RP/IEX

With 1260 Infinity Bio-inert

LC, Agilent can provide most

comprehensive solution for

these challenges

Agilent (U)HPLC Instruments and Bio Applications

Application 1260 Bio-inert

LC

1290

Quaternary

LC

Comments

Ion-exchange

(charge variants) X For salt and pH gradients

Size exclusion

(aggregation) X If salt is added

HIC X High salt application

Hilic (Glycan) X For highest throughput and

resolution

Reversed phase

(peptide mapping) X For highest throughput and

resolution

Glycan analysis

(Hilic and FLD) X For highest throughput and

resolution

Glycan Analysis

(HPAED) X

Universal System

for any NBE assay X If salt gradients and RP is used

on a single system


Characterization

51


Characterization

52

Thank You