acquity uplc®: an analytical platform for biopharmaceuticals · 2012-08-13 · ©2011 waters...

©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation

ACQUITY UPLC®: An Analytical Platform for Biopharmaceuticals

John Barackman

Waters Technical Support Specialist

©2011 Waters Corporation 2

Topics

Components of LC Resolution

— Physical

— Chemical

Ultra-Performance Liquid Chromatography

— What is Required; How does it differ from HPLC

Our Waters ACQUITY UPLC Family

Overview ACQUITY UPLC Configurations for

Biopharmaceutical analysis

Tools for Methods Transfer


The Components of Resolution

Efficiency Selectivity Retentivity

Physical Van Deemter Equation

— Column length

— Particle size, shape, packing

— Mobile phase flow rate (linear velocity)

System induced Bandspread

Chemical

Dependent upon

— Analyte properties

— Stationary phase properties

— Mobile phase properties



Physical


The Driving Force for UPLC

The “Van Deemter Equation” published 1956, correlates diffusion,

resistance, particle size and shape with mass transfer leading to

non ideal behaviour in chromatography

H = a(dp) + b(Dm) + c(dp2)(µ) µ(dp) Dm


The “Van Deemter Equation” published 1956, correlates diffusion,

resistance, particle size and shape with mass transfer leading to

non ideal behaviour in chromatography

H = a(dp) + b(Dm) + c(dp2)(µ) µ(dp) Dm

Resolu

tion

In

creases

as p

artic

les g

et s

malle

r

Two Important Points Regarding the Van Deemter Plot


Two Important Points Regarding the Van Deemter Plot

For particle sizes below approx, 2 µm, the optimum flow rate profiles flattens out. Achieve high throughput by pushing flow rate without significant loss of resolution.

At each particle size range, there is an optimum linear velocity (flow rate) that achieves maximum resolution for that particle size.


The Driving Force for UPLC

The Van Deemter Equation is valid for large molecules.

— Diffusion is slower so ideal flow rates are slower

0

0.0005

0.001

0.0015

0.002

0.0025

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

u (mm/sec)

H (c

m)

3.5 micron particles peptides

1.7 micron particles peptides

Van Deemter Plot For a 1500 Dalton Peptide

3.5 mm

1.7 mm


Smaller, Rounder, Narrower Distributions, Better Packing

Particle size, shape, and packing effect diffusivity and the efficiency of mass transfer which, in turn, impacts resolution.


10 min

1980’s - 2000

5 – 2.5µ spherical micro-porous

1500-4000 psi

50,000 - 80,000 plates/meter

3.9 x 150mm

Late 1970’s

10µ irregular micro-porous

1000-2500 psi

25,000 plates/meter

3.9 x 300mm

10 min

Evolution of Chromatographic Efficiency

Early 1970’s

40µ pellicular non-porous coated

100-500 psi

1000 plates/meter

1m columns

10 min

Note: Since peaks are narrower for the same mass injected, you gain sensitivity by using smaller particles.


Why Not Go Smaller?


Chromatographic Resolution: Impacting Efficiency


Two components of efficiency:

• From the Column Packing

• Everywhere Else (“Extra-Column”)


Loss of Resolution Due To Extra-Column Bandspread

In this region, both analytes (blue and red) are not separated [a partial co-elution –

shown as a “purple” band]

System with MORE

Band Spreading

System with LESS

Band Spreading

Better separation More concentrated “Bands”

Higher Sensitivity

Extra-column bandspread is “non-ideal” behavior (loss of resolution) caused by the LC system outside of the column packing and is

“on top of” the column contribution.


Sources of Bandspread

Band Spreading will occur:

1. Along the flow path from the injector (“sample band”)

2. Into, through and out of the column (“analyte bands”)

3. Into the detector

3

2

1


Sources of Bandspread

Band Spreading will occur:

1. Along the flow path from the injector (“sample band”)

2. Into, through and out of the column (“analyte bands”)

3. Into the detector

3

2

1

• UPLC instruments must be designed specifically to take advantage of the sub-2µm particles

Pumps

Injectors

Tubing

Fittings

Column Heaters

Detectors, flow cells, nebulizers

→ Must minimize extra-column bandspread and increase data

rate

• Using HPLC components of any kind would perform poorly with UPLC columns

• However: HPLC columns work fine on UPLC systems!


Putting it All Together UltraPerformance LC®

A Class of Liquid Chromatograph Based On:

— Chromatography columns with very small, pressure-

tolerant particles, narrow size distributions, efficiently

packed

— Instruments designed to take advantage of the small

particles

o Pumps, Injectors, Tubing, Fittings, Column

Heaters, Detectors all designed to withstand the

pressures required, minimize extra-column

bandspread, and utilize the high data rates

required

If the Entire System is Holistically Designed for UPLC

Then:

— Significant improvements in resolution, sensitivity

AND sample throughput are realized


402 pesticide residues detected in a 8-minute UPLC run on TQD

Benefits of UPLC

• Resolution • Speed • Sensitivity


Benefits of UPLC Maximize Throughput


Benefits of UPLC Maximize Resolution

(3.5 µm)

(1.7 µm)

C18 RP Peptide maps at constant L/dp and gradient time.




Chemical • Focus on the

stationary phase


ACQUITY UPLC Column Chemistries

Requirements for UPLC Columns:

— Sub–2 µm particles (1.7 µm typical)

— Pressure tolerant (≥ 15,000 psi)

— Efficiently packed (low dispersion)

— Narrow size distribution

Additional Desirable Characteristics:

— Very wide pH stability (pH 1-8 and pH 1-11 typical)

— High lot-to-lot reproducibility

— Wide range of selectivity, retentivity, porosity options

— Scalable from UPLC HPLC PREP

Waters manufactures most of its own particles and columns


Ever-Expanding ACQUITY UPLC® Column Offering

BEH C18

BEH C8

BEH Phenyl

BEH Shield RP18

BEH HILIC

BEH Amide

CSH C18

CSH Fluoro-Phenyl

HSS T3

HSS C18

HSS C18 SB

Five particle substrates

• 130Å, 200Å and 300Å BEH [Ethylene Bridged Hybrid], HSS

[High Strength Silica] and CSH [Charged Surface Hybrid]

Wide and growing selection of column chemistries

• 15 stationary phases

• BEH 130Å C18, C8, Shield RP18, Phenyl, HILIC and Amide

• BEH 300Å C18 and C4

• BEH 200Å SEC

• HSS C18, T3, C18 SB

• CSH C18, Fluoro-Phenyl and Phenyl-Hexyl

Proven application-based solutions

• AAA, OST, PST, PrST and Glycan

Transferability between HPLC and UPLC

XBridge HPLC and ACQUITY UPLC BEH columns

HSS HPLC and ACQUITY UPLC HSS columns

XSelect HPLC and ACQUITY CSH columns

VanGuard Pre-columns

eCord Technology

Nano, UPLC, HPLC, Semi-PREP, PREP Scale (most chemistries) CSH Phenyl-Hexyl

©2010 Waters Corporation | COMPANY CONFIDENTIAL 23

Column Chemistries For Biomolecules

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ACQUITY BEH300 C4, 1.7 µm



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Waters Protein-PakTM Hi Res IEX



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ACQUITY BEH200 SEC, 1.7 µm

For Protein Analysis



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ACQUITY BEH200 SEC, 1.7 µm

For Protein Analysis


For Peptide Analysis


ACQUITY BEH Amide, 1.7 µm For Oligosaccharide Analysis



ACQUITY BEH Amide, 1.7 µm

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ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm

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ACQUITY BEH Glycan, 1.7 µm

For N-Linked Glycan Analysis



ACQUITY BEH Amide, 1.7 µm

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ACQUITY BEH Glycan, 1.7 µm

ACQUITY OST C18, 1.7 µm

For Oligonucleotide Analysis



Waters Corporation

What Waters Has To Offer

In 2004, Waters Corporation introduced the first true UPLC: the ACQUITY UPLC


Classic ACQUITY

High Throughput

Clinical/IVD

MS Inlet

H-Class

H-Class Bio

General Purpose

Methods Transfer

Bio-Analysis Nano ACQUITY

PATrol System


New Members of the ACQUITY Family

ACQUITY UPLC I-Class

- SFC at UPLC Scale - Complementary to RP, NP, GC

- Flexible Architecture - Lowest System

Dispersion of ANY UPLC - Ultimate MS Inlet


An Overview of Selected ACQUITY Systems

ACQUITY System Details

Introduction to the various pump and injector design paradigms, sample organizer, column heaters, and

detector types that make up our family of UPLC systems


High Pressure mixing, dual pump

—Two solvents at a time

—Smaller system volume/mixing volume

The BSM: A High-Pressure Mixing System

A high pressure mixer is capable of very efficient gradient formation.

A smaller dwell volume improves system throughput.

Smaller System Volume = Smaller Dwell volume

Multiple Pumps

ACQUITY UPLC

Detector Injector Column

Pump A

Pump B

Mixer


Standard ACQUITY UPLC System

• Binary Solvent Manager (BSM), High Pressure Mixing

• Sample Manager – Rheodyne Injector (SM)

• Column Heater, Column Manager (CM)

• System Characteristics

o <120 µL dwell volume

o <10 µL bandspread

• Supports all current ACQUITY UPLC detectors

o PDA/PDAeλ, TUV, FLR, ELSD

o SQD, TQD, XEVO MS Platforms

o Empower and MassLynx

• Optimum High-Throughput and MS Inlet BSM


Rheodyne Based Injector

SAMPLE

PVDD

SAMPLE /

METERING

SYRINGE

FROM

PUMP

TO

COLUMN

INJECT VALVE

INJECT POSITION

SAMPLE

LOOP

3 2

14

5 6

Needle Overfill IIStep 1: Sample Aspiration

Weak Wash Solvent

Air Gap

Buffer Volume

Sample

Mobile Phase

SOLVENT TYPE

Volumes Represented

are not to Scale

Internal Valve Passage

V = 0.100uLA rheodyne based injector minimized dwell volume and allows for a range of injection modes to meet your particular requirements.

The ACQUITY UPLC SM is a Rheodyne Base

Injector Design


Standard ACQUITY UPLC System

• Binary Solvent Manager (BSM), High Pressure Mixing

• Sample Manager – Rheodyne Injector (SM)

• Column Heater, Column Manager (CM)


o <120 µL dwell volume

o <10 µL bandspread

• Supports all current ACQUITY UPLC detectors

o PDA/PDAeλ, TUV, FLR, ELSD

o SQD, TQD, XEVO MS Platforms

o Empower and MassLynx

• Optimum High-Throughput and MS Inlet

SM


Rheodyne Loop Injection Loading the Sample into the Loop

Injection Modes: Partial, PLNO, Full Loop


Rheodyne Loop Injection Pushing the Sample onto the Column

Sample Loop contains Sample, air gaps and weak wash

40


ACQUITY Column Heaters

Standard Configuration: • Single Column • Temperature range: 5°C above ambient to 90°C • Up to 4.6 mm diameter, up to 150 mm length columns including filter and guard column Optional Configurations: • Four column capacity Column Manager with column select valves • 30-cm Column Heater/Cooler available for legacy methods


High Throughput System

Optional Sample Organizer • Up to 22 microtiter and 10

2mL vial tray capacity

• Up to 8448 Samples

• 15 second plate cycle time

• 4 to 40⁰C temperature

controlled


ACQUITY UPLC – High Throughput 0.86 min

> 1670 injections per day.

ACQUITY UPLC – High Throughput


Intro to the NanoACQUITY UPLC

Flow

Sensor

Modules

Heating/Trapping

Module

Sample

Manager

Binary Solvent

Manager

Auxiliary Solvent

Manager

• Binary high pressure blending without splitting

• 200 nL/min to 100 µL/min

• Low dispersion design, UPLC pressure capable (10,000 psi)

• Tunable UV/Vis, PDA, MS and MS/MS

• Wide range of column selectivity's available

• 75 – 150 µm diameters

• 100 – 250 mm length

• 1.7 – 3.5 µm particle sizes


1D Separation – Reverse Phase C18

2000

1500

1000

500

0

Cou

nts

10080604020

Retention Time (min)

7 - 40% ACN Gradient 250 ng Protein Digest Load

250 nL/min 1.7µm BEH C-18, 75µm x 25cm nanoACQUITY with Synapt HDMS


NanoACQUITY UPLC 2D System Schematic

Analytical Column Trap

Needle

Trap

Valve 6

5 4

3

2 1

Inject Valve

6

5 4

3

2 1

RP 1

Syringe

nanoBSM

nanoBSM

Waste

XBridge C18

300µm x 5cm

pH 2 Sample

pH 10 Symmetry C18

180µm x 2cm

BEH C18

75µm x 15cm

Plug

Plug

High organic and pH are reduced before trapping


2D RP/RP of Rat Brain Digest

11.1% ACN

20.8% ACN

17.4% ACN

14.5% ACN

45% ACN


The QSM: A Low-Pressure Mixing System

Low Pressure mixing, single pump

— Solvent is mixed before it is passed

through the pump

Gradient Proportioning Valve

Detector Injector A B

C D

Column Pump

Single Pump Design

ACQUITY UPLC H-Class

Low pressure mixing simplifies the pump design and expands the range of solvents that can be blended. Common GPV/Pump design to many legacy systems.


ACQUITY UPLC H-Class/H-Class Bio

• Quaternary Solvent Manager (QSM)

• Sample Manager – Flow Through Needle (SM-FTN)

• Column Heater with Active pre-heating


— <400 µL dwell volume

— <10 µL bandspread

— 2 ml/min max flow rate

— 15,000 psi max pressure

• Reproduce Common Legacy Methods

• Supports all current ACQUITY UPLC

detectors

• H-Class Bio: All wet-able surfaces made

from biocompatible materials QSM


The QSM: A Low-Pressure Mixing System

Low Pressure mixing, single pump

— Solvent is mixed before it is passed

through the pump

Gradient Proportioning Valve

Detector Injector A B

C D

Column Pump

Single Pump Design


Low pressure mixing simplifies the pump design and expands the range of solvents that can be blended. Common GPV/Pump design to many legacy systems.

An Optional 6-Way Solvent Select Valve can be fitted into the QSM to

aid methods development. (A, B, C, D1->D6)


Another Benefit of Auto•Blend Rapid Method Scouting

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0.05% TFA – 5% D

0.1% TFA – 10% D

0.025% TFA – 2.5% D

©2011 Waters Corporation 52 *Available THF/Hexane Kit Installed

Using Auto•Blend for Complex Gradient Delivery

Time

(min)

%

Water

%

Acetonitr

ile

%

THF*

%

Methano

l

0.0 64 29 4 3

5.0 56 34 7 3

9.0 41 38 7 14

Aldehydes and Ketones as DNPH Derivatives


Auto•Blend Plus™ Technology AQUITY H-Class/H-Class Bio

Program methods directly in units of pH and Molarity

Calculation of required proportions from physical constants

— pH is calculated using Henderson-Hasselbalch equation with

pKa provided

— Typically use pKa corrected for salt concentration

OR:

— Empirical calibration table covering operating range of buffer

and salts selected

o Can produce a near true linear pH gradients as required

Independent gradients for pH and salt concentration


Instrument Control Method Auto•Blend™ to Auto•Blend Plus™

Click to convert the IM to Auto•Blend PlusTM


Typical Gradient Table Auto•Blend™ Plus

QSM percent table is converted to units of pH and Salt concentration.

The allowed pH and [salt] ranges are based on the respective buffer

system selected


Benefit of Auto●Blend Plus™ Rapid Methods Development

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pH 6.1

pH 7.6

pH 7.1

pH 7.0

pH 6.9

A mixture of proteins was separated using cation exchange chromatography- alpha-Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) –

A

A

A+B

A

A

B

B

B

B

C

C

C

C

C


Flow-Through Needle Based Injectors (FTN)

Wash Solvent

Purge Solvent


ACQUITY UPLC H-Class/H-Class Bio Direct Inject Sampling with the FTN

Sample Manager – Flow Through Needle (SM-FTN)

No re-configuration necessary across the entire UPLC injection

volume range

— Up to 10uL injection standard

— Up to 1000uL with extensions

No wasted sample and low carryover

— Needle and loop are part of flow path

o Low carryover performance

Good precision, linearity, and accuracy

Full range of ANSI plates and vials

H-Class Bio: Wet-able surfaces are

made of biocompatible materials

A flow-through injector design, while adding some dwell volume provides a single, simple injection model and provides a wide linear range, with good accuracy and high precision. Common injection model to many legacy systems.

SM-FTN


Sample Manager –FTN Sample Loading

Inject Valve Load Position


Sample Manager –FTN Sample Injection


SM-FTN Wide Linearity Range

Benzocaine Tetracaine Procaine R2=0.999991 R2=0.999997 R2=0.999989


SM-FTN Repeatability

Method: ACQUITY UPLC BEH200 1.7 µm 4.6x150mm 50mM KPO4, 100mM KCl, pH 6.8 40⁰C, 0.5 mL/min flow, 220 nm detection


3.5 AU Full Scale

Stress Injection @ 2mg/mL 1st Blank Injection

Carryover = 0.0002%

3.5 AU Full Scale

SM–FTN Low Carryover


ACQUITY UPLC H-Class Bio What’s different from the H-Class?

Many Components Re-Engineered for Bio-Compatibility

— Primary & accumulator pump heads

— Check-valves

— UPLC Valves (Vent, Injector, CM, etc..) & SSV

— Mixer housing, mixer manifold, sinkers

— SM Needle Assembly and Injector Port

— CM-A Active Pre-Heater (APH) Assembly

— UPLC tubing assemblies

— PDA & TUV Flow Cells

Bio-Compatible Materials:

— Minimize corrosion (particularly to halides)

— Improve sample recovery (particularly for bio-molecules)

— Minimize iron adducts (IEX and LCMS)

— Limit oxidation

— High strength, durable

Wet-Able Surfaces are made principally from Titanium and Nickel-

Cobalt Alloys


ACQUITY UPLC I-Class System Optimized for Low Dispersion

Binary Solvent Manager (BSM)

— 18,000 psi capable

Sample Manager (Rheodyne or FTN)

— Fixed-Loop (FL) Sample Manager

— Flow-Through-Needle (FTN) Sample Manager

Column Management (2 options)

— Single Column Heater

— Dual Column Manager

o Max 2 columns

o Optional 2D Technology feature

Detection

— MS

— TUV or PDA only – new lower dispersion flow cells

All Existing ACQUITY UPLC Chemistries

— Plus optimum platform for 1 mm ID columns


Instrument Contribution to Extra-Column Effects

Engineering developments have specifically improved dispersion in every part of the system flow path to allow the I-Class to minimize system induced band spread:

— Injector, fittings, flow path, sealing surfaces, reduced tubing volumes, improved flow cell dispersion

— ~ 95 uL dwell volume, < 7 uL band-spread

22

det

2

,det

2

,

2

,

2

,

2

,

2

, Fectorectorvpostcolumnvcolumnvprecolumnvinjectorvtotalv

Injection volume

+ injector band-

spreading

Tubing between injector

and column

Column volume

+ frits

Tubing between column

and detector

Band- spreading inside the detector

cell +

tubing

Time-based Band-

spreading in the

Detector (Sampling Rate; Time Constant)


ACQUITY UPLC Systems with 2D Technology Solves Key Challenges

Increase Selectivity & Sensitivity

Eliminate Unwanted

Interferences

Mobile Phase Flexibility

Characterize the Most Complex Samples


2D Capability Available on All ACQUITY UPLC System (New Systems and Upgrades)


System with 2D

Technology

ACQUITY UPLC I-

Class System with

2D Technology

ACQUITY UPLC

System with 2D

Technology


Flexibility of ACQUITY UPLC Systems with 2D Technology

Typical Experiments that can be Performed with these Systems

— Trapping

— Heart Cutting

— Parallel Column Regeneration

— At-Column Dilution (ACD)


ACQUITY UPLC Systems with 2D Technology Example Flow Path (Trapping Config Shown)

©2011 Waters Corporation 71 Time

3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40

%

0

100

MRM of 6 Channels ES+

327 > 269.9

1.46e6

4.31

250 uL Clozapine 1 ppb 2D – 3 min gradient


Peak distortion Volume Overload


Increase Sample Loading and Sensitivity with Trap and Back-Transfer Configuration


HPLC to UPLC Methods Transfer

Tools to Help Make Methods Transfer Easier


Column Selector and Calculator Calculator Tools

Transfer Existing HPLC Methods to the H-Class/H-Class Bio Redevelop Existing HPLC Method to UPLC Methods

Using our Column Calculator and Column Selector Tools (Free! www.waters.com)

Quickly scale/translate between column selectivity, column size, particle diameters, injection volumes, gradient times and system dwell volumes


Transfer Methods Between UPLC and

HPLC with Ease

Utilize existing Assets

Run HPLC methods on ACQUITY UPLC H-Class

Future-proof your lab

Transfer from HPLC-to-UPLC


Transferring HPLC Methods Across

Multiple HPLC and UPLC Systems


Questions?

acquity uplc®: an analytical platform for biopharmaceuticals · 2012-08-13 · ©2011 waters...

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