The development and manufacture of biopharmaceuticals is a dynamic and rapidly growing industry. By the nature of their production, biopharmaceuticals are highly complex heterogeneous mixtures that require many analytical techniques for characterization and routine testing. As a result, many manufacturers incorporate life cycle management into their respective workflows to take advantage of newer technologies and methodologies to ensure efficacy and patient safety. In this presentation, we will address the range of chromatographic categories – HPLC, UHPLC, and UPLC – and define the characteristics associated with each. The discussion will continue with several examples of methods transferred from legacy HPLC instrumentation to modern UHPLC and UPLC instruments. We will compare qualitative and quantitative data across each chromatographic class. Resolution, sensitivity, and overall run time will be used as metrics to assess the success of the method transfer to the respective LC platform, to ensure the transferred methods are in line with current acceptance criteria. Learn: - The importance of selecting the correct instrumentation to meet user needs. - Which parameters influence method transfer from one LC platform to another. - How workflows can benefit from features such as Multi-flow path technology and Gradient SmartStart when transitioning methods. Interested in more detail? Watch the related on-demand webinar: http://view6.workcast.net/register?pak=3479247014905635

1. ©2015 Waters Corporation 1
Brooke Koshel, Ph.D.
Senior Scientist
Waters Corporation
Life Cycle Management of Chromatographic
Methods for Biopharmaceuticals

2. ©2015 Waters Corporation 2
Outline
 Global trend towards new technologies
through life cycle management
 Defining LC platforms for life cycle management
of analytical procedures
 Life cycle management of analytical procedures
using UHPLC and UPLC systems
– Method transfer of existing HPLC methods
on the ACQUITY ARC UHPLC System
– Method transfer of existing HPLC methods
on the ACQUITY UPLC H-Class Bio System

3. ©2015 Waters Corporation 3
Current Global Environment
Biopharmaceutical Industry
 Highly competitive, regulated business environment
– Lower costs without compromising product quality
– Maintain regulatory and compliance requirements
 Challenges to increase profitability:
– Increasing regulatory pressures
– Increased quality expectations and competitive pressures
– Need for Efficiency : “Lean” laboratory operation
 Need to deliver a sustainable competitive advantage
– Invest in core competencies
– Invest in technologies to achieve business objectives
 Manufacturers are incorporating Life Cycle
Management
– Take advantage of newer technology and methodologies
for increased ROI and quality standards

4. ©2015 Waters Corporation 4
ICH Q10 Incorporates Life Cycle
Management into the Quality System
ICH Q10 definition of Innovation :
“The introduction of new technologies
or methodologies”

5. ©2015 Waters Corporation 5
ICH Q6B Recommends New Technologies
for Biologics

6. ©2015 Waters Corporation 6
US FDA
Emphasis on New Technologies
http://www.fda.gov/downloads/drugs/guidancecomplianceregula
toryinformation/guidances/ucm386366.pdf
Over the life cycle of a product, new
information (e.g., a better
understanding of product CQAs or
awareness of a new impurity) may
warrant the development and
validation of a new or alternative
analytical method.
“New technologies may allow for
greater understanding and/or
confidence when ensuring product
quality. Applicants should periodically
evaluate the appropriateness of a
product’s analytical methods and
consider new or alternative methods.”
VIII. LIFE CYCLE
MANAGEMENT OF
ANALYTICAL PROCEDURES

7. ©2015 Waters Corporation 7
Life Cycle Management of Analytical
Procedures with New Technologies
 New technologies allow for greater understanding when ensuring product
quality.
– Reduces exposure to unnecessary compliance risk
– Reduces validation costs
– Patients get quicker and safer access to drugs
 Gives regulators the confidence that industry can be responsible for greater
self-management of improvements and changes
– Companies with good quality management systems
– Well controlled processes and products
– Appropriate technologies are being used for product safety
 Increases ROI by decreasing equipment down time, overheads, solvent usage,
etc.
Proper management of the analytical equipment lifecycle
is required to meet business and regulatory requirements

8. ©2015 Waters Corporation 8
Considerations for Life Cycle Management
of LC-Technology
Performance
Confidence in technology
Ease of Use
Reduces training
Avoidance of Human Error
Method Transfer
Ease of transfer
Current vs. new methods
Revalidation/refiling
Flexibility
Interface with other labs
CRO/CMO/R&D, Dev./QC
etc.
Future-Proofing
Incorporate
technology advances while
maintaining workflow
Robustness
Long term reliability of
methods
Informatics
Ease of data processing
Degree of compliance

9. ©2015 Waters Corporation 9
Outline
 Global trend towards new technologies
through life cycle management
 Defining LC platforms for life cycle management
of analytical procedures
 Life cycle management of analytical procedures
using UHPLC and UPLC systems
– Method transfer of existing HPLC methods
on the ACQUITY ARC UHPLC System
– Method transfer of existing HPLC methods
on the ACQUITY UPLC H-Class Bio System

10. ©2015 Waters Corporation 10
Platforms for Life Cycle Management
LC Separation Categories
ACQUITY ArcAlliance® HPLC ACQUITY UPLC
H-Class Bio
Chromatographic Resolution Increases
Overall Run Time Decreases
Method Sensitivity Increases

11. ©2015 Waters Corporation 11
Platforms for Life Cycle Management
LC Separation Categories
Chromatographic Resolution Increases
Overall Run Time Decreases
Method Sensitivity Increases
AU(x10-3)
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Retention Time (min)
15 20 25 30
AU(x10-3)
0.0
1.0
2.0
3.0
4.0
5.0
1.0 1.5 2.0 2.5
Retention Time (min)
HPLC UPLC
*
*

12. ©2015 Waters Corporation 12
What is at the Root of the Performance
Differences Across the LC Categories?
 Dispersion – n. Broadening of an analyte band due to both
on-column effects (diffusion and mass transfer kinetics which
are both dependent on particle size and linear velocity) and
system effects (tubing internal diameter (I.D.) and length,
connections, detector flow cell volumes, etc.)
 True separation performance is governed by the system
dispersion paired with a flow rate range that yields the
highest possible efficiency for a given analytical column
Read more at :
https://www.waters.com/waters/Chromatographic-Bands,-Peaks-and-Band-
Spreading/nav.htm?cid=134803614

13. ©2015 Waters Corporation 13
Defining the LC Categories:
Power Range vs. Dispersion
 LC systems trying to cover a wide
“power range” (flow rate / pressure
envelope) end up compromising
extra-column dispersion, and
therefore performance, in efforts to
accommodate both sub 2 µm and
traditional column technologies.
 Flow rate range and available
pressure alone have little bearing on
the actual separation power of the
system and do not provide an
appropriate measurement of
system performance.
The difference between UHPLC and UPLC
True separation performance is governed by system dispersion
mAU 0.00
200.00
400.00
AU
0.00
0.20
0.40
Minutes
1.10 1.20 1.30 1.40 1.50 1.60
Rs = 0.52
Rs = 1.53
Rs = 1.44
Rs = 2.84
Vendor A UHPLC
with Higher
‘Power Range’
ACQUITY
UPLC H-Class
with Lower
‘Power Range’

14. ©2015 Waters Corporation 14
Defining the LC Categories by
Dispersion
Dispersion > 30 µL
Columns accepted:
• 3.0 – 4.6 mm ID
• 3 - 10 µm particles
Optimal:
• 4.6 mm ID, 5 µm
Typical operating pressure:
• < 6,000 PSI
Dispersion 12 - 30 µL
Columns accepted:
• 2.1 - 4.6 mm ID
• 1.7 - 5 µm particles
Optimal:
• 3.0 mm ID, 2.x µm
Typical operating pressure:
• 6,000 – 15,000 PSI
Dispersion < 12 µL
Columns accepted:
• 1.0 - 4.6 mm ID
• 1.6 - 5 µm particles
Optimal column:
• 2.1 mm ID, 1.7 µm
Typical operating pressure:
• 9,000 – 15,000 PSI
HPLC UHPLC UPLC
Alliance HPLC 34 μL
Shimadzu
Prominence 35 μL
ACQUITY Arc 25 µL
Agilent 1260 SL 25 µL
ACQUITY UPLC 10 μL
H-Class CH-A 7 μL
I-Class CH-A 5 μL
All dispersion values measured at 5 σ

15. ©2015 Waters Corporation 15
Bridging The Performance Gap
Between HPLC and UPLC Technology
HPLC UPLC
UHPLC
Extends the ACQUITY family into laboratories
requiring method compatibility with HPLC and
UHPLC (2.x µm) separations
Reasons For Slow Adoption
•Not yet evaluated UPLC technology
•Do not require a UPLC level of performance
•Budget
•Training

16. ©2015 Waters Corporation 16
Outline
 Global trend towards new technologies
through life cycle management
 Defining LC platforms for life cycle management
of analytical procedures
 Life cycle management of analytical procedures
using UHPLC and UPLC systems
– Method transfer of existing HPLC methods
on the ACQUITY ARC UHPLC System
– Method transfer of existing HPLC methods
on the ACQUITY UPLC H-Class Bio System

17. ©2015 Waters Corporation 17
Lifecycle Management of an Analytical
Procedure using UHPLC
HPLC UHPLC
HPLC Methods
or
Updated
UHPLC
Methods
Isocratic Methods
OR
Gradient Methods

18. ©2015 Waters Corporation 18
ACQUITY Arc System
Comprehensive
detector portfolio
-UV/Vis
-Photodiode Array
-Fluorescence
-Refractive Index
-Evaporative Light Scattering
-Mass Detection
Negligible carryover
Flow-through-needle design with
user definable wash settings
Thermal management options
-Heating or heating/cooling
-Supports columns up to 300 mm
-Optional column switching
Auto•Blend Plus™ Technology
Automated online solvent blending at
specific pH and ionic strength that
supports reversed phase, SEC, and IEX
Quaternary solvent
management
Precise and accurate blending of up to 4
solvents with automated solvent
compressibility. Optional 6 solvent
select valve expands flexibility
Gradient SmartStart
Counteract system dwell volume
differences without altering the
gradient table. Minimize cycle times
by managing gradient start and pre-
injection steps in parallel
Arc Multi-flow path™
Technology
Plug-and-play method
compatibility with HPLC or UHPLC.
Replicate methods by selecting the
most appropriate flow path.

19. ©2015 Waters Corporation 19
Transfer HPLC Method to ACQUITY Arc
Arc Multi-flow path Technology
For UHPLC Separations
Lower System Volume
To injector and column
For HPLC Separations
Higher System Volume
Choose the path that best
fits your application
Path 1: 1100uL
Path 2:
700uL

20. ©2015 Waters Corporation 20
ACQUITY Arc System:
Quaternary Solvent Manager-R
Pressure transducers
Gradient proportioning valve Solvent degasser
Passive
check
valves
Arc Multi-flow path
Technology
Seal wash
pump
Optional
solvent
select
valve

21. ©2015 Waters Corporation 21
Transfer HPLC Method to H-Class
Transferring Isocratic Methods
From HPLC to UHPLC
Application Example
Size Exclusion Chromatography (SEC)
Scenarios
Method Equivalency
Method Improvement

22. ©2015 Waters Corporation 22
Transfer HPLC Method to ACQUITY Arc
Isocratic: SEC
Instrument
HPLC
(Quaternary)
UHPLC
ACQUITY Arc
(Quaternary)
Column Chemistry
TOSOH Biosciences G3000SWXL, 5 um
(250 Å pore size)
No Change
Dimensions 7.8 mm ID x 300 mm No Change
Mobile Phase
0.02 M sodium phosphate,
0.3 M sodium chloride, pH 6.8
No Change
Flow Rate 500 ml min-1 No Change
Temperature 30 oC No Change
Injection Volume 30 ml No Change
Run Time 35 min No Change
CDS Empower® Empower
Sample: Rituximab

23. ©2015 Waters Corporation 23
AU
0.00
0.10
0.20
0.30
AU
0.00
0.10
0.20
0.30
Retention Time (min)
15 30
Sample: Rituximab
Acquity Waters
Waters
Waters
Acquity
Acquity
Waters
System Mode Rs
Relative Peak Area
(%)
HMW
Species
Monomer
x̅ σ x̅ σ
Arc HPLC 1.5 1.29 0.01 98.65 0.01
Agilent
1100
HPLC 1.5 1.28 0.01 98.63 0.01
HMWPeak
Dimer
Monomer
AU
-0.001
0.000
0.001
0.002
0.003
0.004
Minutes
10.00 15.00 20.00 25.00 30.00
AU
-0.001
0.000
0.001
0.002
0.003
0.004
Minutes
10.00 15.00 20.00 25.00 30.00
HMWPeak
Dimer
Monomer
Transfer HPLC Method to ACQUITY Arc
Isocratic: SEC

24. ©2015 Waters Corporation 24
Injection Area HMW Species
1 80304
2 81069
3 81890
4 81153
5 82012
Mean 81285.6
Std. Dev. 693.00
%RSD 0.85
Inj Rs
Monomer-
Dimer
1 1.51
2 1.54
3 1.52
4 1.58
5 1.55
AU
-0.001
0.000
0.001
0.002
0.003
0.004
Minutes
10.00 15.00 20.00 25.00 30.00
5 Injections
AU
0.00
0.05
0.10
0.15
0.20
Retention Time (min)
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
HMWPeak
Dimer
Monomer
Transfer HPLC Method to ACQUITY Arc
Isocratic: SEC

25. ©2015 Waters Corporation 25
Transfer HPLC Method to H-Class
Transferring Isocratic Methods
From HPLC to UHPLC
Application Example
Size Exclusion Chromatography (SEC)
Scenarios
Method Equivalency
Method Improvement

26. ©2015 Waters Corporation 26
Update HPLC Method to UHPLC
Improvements in Separations
Dimer Monomer
Method Scaled for new column dimensions
AU
-0.001
0.000
0.001
0.002
0.003
0.004
Minutes
6.00 8.00 10.00 12.00 14.00
AU
-0.001
0.000
0.001
0.002
0.003
0.004
Minutes
12.00 15.00 18.00 21.00
HPLC UHPLC
Resolution
95.0
95.5
96.0
96.5
97.0
97.5
98.0
98.5
99.0
99.5
100.0
Arc
(HPLC)
Agilent
1100
Arc
(UHPLC)
PeakArea(%)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Arc
(HPLC)
Agilent
1100
Arc
(UHPLC)
PeakArea(%)
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
Arc (HPLC) Agilent
1100
Arc
(UHPLC)
Sample: Rituximab
Column
TOSOH Biosciences
G3000SWXL
7.8 mm ID x 300 mm
Column
Waters XBridge® Protein
BEH
7.8 mm ID x 300 mm
3.5 mm5 mm

27. ©2015 Waters Corporation 27
Transfer HPLC Method to H-Class
Transferring Gradient Methods
From HPLC to UHPLC
Application Examples
Example 1: Ion Exchange
Chromatography(IEC)
Example 2: Peptide Mapping
Scenarios
Method Equivalency
Method Improvement

28. ©2015 Waters Corporation 28
Transfer HPLC Method to ACQUITY Arc
Gradient: Ion Exchange
Instrument
HPLC
(Quaternary)
UHPLC
ACQUITY Arc (Quaternary)
Column
Chemistry
Dionex ProPac WCX-10, 10 um No Change
Dimensions 4 mm ID x 250 mm No Change
Mobile Phase
0.2 M MES in water, pH 6.0
0.02 MES 0.4 M NaCl in water, pH 6.0
No Change
Flow Rate 700 ml min-1 No Change
Temperature 30 ºC No Change
Run Time 115 min No Change
Injection Volume 40 ml No Change
CDS Empower Empower
Sample: Rituximab
Arc Multi-flow path Technology: HPLC Flow Path 1

29. ©2015 Waters Corporation 29
Arc Multi-flow path Technology
 Flow Path 1: For HPLC Separation (Larger Dwell Volume)
– Selectable dwell volume that emulates both system volume and mixing
behavior
 Does not impact the gradient table
– Falls within USP <621> guidelines on transferring gradient methods
between different chromatographic systems
System Emulation with Arc
Multi-flow path Technology
Select Path 1

30. ©2015 Waters Corporation 30
AU
0.000
0.002
0.004
0.006
0.008
0.010
AU
0.000
0.002
0.004
0.006
0.008
0.010
Retention Time (min)
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00
Acquity Waters
Waters
Waters
Acquity
Acquity
Waters
Transfer HPLC Method to ACQUITY Arc
Gradient: Ion Exchange
System Mode Rs
Relative Peak Area
(%)
K1 K0
x̅ σ x̅ σ
Arc HPLC 2.1 4.78 0.04 70.30 0.11
Agilent 1100 HPLC 1.8 5.14 0.05 69.67 0.28
Sample: Rituximab
K1
K0
K1
K0

31. ©2015 Waters Corporation 31
Transfer HPLC Method to ACQUITY Arc
Ion Exchange: High repeatability of results
K0
AU
0.00
0.05
0.10
Retention Time (min)
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
6 Injections
AU
-0.0025
0.0000
0.0025
0.0050
0.0075
0.0100
Minutes
30.00 40.00 50.00 60.00
Inj Rs
K0-K1
1 2.11
2 2.10
3 2.08
4 2.11
5 2.15
6 2.14
Injection Area K1
1 269505
2 272294
3 267541
4 266328
5 268459
6 265468
Mean 268265.8
Std. Dev. 2445.71
%RSD 0.91
K1
Sample: Rituximab
K1
K0

32. ©2015 Waters Corporation 32
Transfer HPLC Method to H-Class
Transferring Gradient Methods
From HPLC to UHPLC
Application Examples
Ion Exchange Chromatography(IEC)
Peptide Mapping
Scenarios
Method Equivalency
Method Improvement

33. ©2015 Waters Corporation 33
Transfer HPLC Method to ACQUITY Arc
Gradient: Peptide Mapping
Instrument
HPLC
(Quaternary)
UHPLC
ACQUITY Arc (Quaternary)
Column
Chemistry
XBridge BEH C18 130Å, 3.5 mm No Change
Dimensions 4.6 mm x 100 mm No Change
Mobile Phase
H2O with 0.1% (v/v) TFA
Acetonitrile with 0.1% (v/v) TFA
No Change
Flow Rate 500 ml min-1 No Change
Temperature 40 ºC No Change
Run Time 60 min No Change
Injection Volume 75 ml No Change
CDS Empower Empower
Sample: Waters MassPREP™ Peptide Mixture (Infliximab)
Arc Multi-flow path Technology: HPLC Flow Path 1 with Gradient Offset

34. ©2015 Waters Corporation 34
Transfer HPLC Method to ACQUITY Arc
Gradient: Peptide Mapping
Retention Time (min)
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
No Gradient Offset
ACQUITY Arc
Agilent 1100 Series
Sample:
Waters MassPREP™ Peptide Mixture
Dwell volume
differences between
instruments results
in retention time
differences

35. ©2015 Waters Corporation 35
Arc Multi-flow path Technology
 Flow Path 1: for HPLC
Separations
(Larger dwell volume)
 Compensates for transferring
methods from LC systems with
variable volume
 Adjust when the gradient
starts relative to the injection
sequence
 No impact to gradient table
System Emulation with Arc Multi-flow path
Technology and Gradient SmartStart
Select Path 1
Gradient
SmartStart

36. ©2015 Waters Corporation 36
Accounting for Dwell Volume
 Gradient dwell volume VD is the total volume of the system from where the
mobile phase mixing occurs to the analytical column
 Differences in dwell volume can lead to retention, selectivity and resolution
differences
t1/2 (1) t1/2 (2)
50%
100%
tG
UHPLC
HPLC
𝑡 𝐷 = 𝑡1/2 −
1
2
𝑡 𝐺
𝑉𝐷 = 𝑡 𝐷 𝐹
Programmed
gradient
Gradient
delay UHPLC Gradient
delay HPLC

37. ©2015 Waters Corporation 37
Accounting for Dwell Volume
Gradient SmartStart
ACQUITY UPLC
Quaternary
Solvent
Manager
Gradient Start :
“After injection”
OR, enter volume
Differences

38. ©2015 Waters Corporation 38
Transfer HPLC Method to ACQUITY Arc
Gradient offset aligns chromatograms
Retention Time (min)
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
No Gradient Offset
ACQUITY Arc
Agilent 1100 Series
Retention Time (min)
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
Agilent 1100 Series
ACQUITY Arc
Programmed Gradient Offset
Sample: Waters MassPREP Peptide Mixture

39. ©2015 Waters Corporation 39
AU
0.00
0.20
0.40
AU
0.00
0.20
0.40
Retention Time (min)
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 36.00 38.00 40.00
AcquityWaters
Waters
Waters
Acquity
Acquity
Waters
Transfer HPLC Method to ACQUITY Arc
Gradient: Peptide Mapping
Sample: Infliximab

40. ©2015 Waters Corporation 40
Transfer HPLC Method to H-Class
Transferring Gradient Methods
From HPLC to UHPLC
Application Examples
Ion Exchange Chromatography(IEC)
Peptide Mapping
Scenarios
Method Equivalency
Method Improvement

41. ©2015 Waters Corporation 41
Update HPLC Method to UHPLC
Gradient: Peptide Mapping
Instrument
HPLC
(Quaternary)
UHPLC
ACQUITY Arc (Quaternary)
Column
Chemistry
XBridge BEH C18 130 Å, 3.5 mm XBridge BEH C18 130 Å, 2.5 mm
Dimensions 4.6 mm x 100 mm No Change
Mobile Phase
H2O with 0.1% (v/v) TFA
Acetonitrile with 0.1% (v/v) TFA
No Change
Flow Rate 500 ml min-1 No Change
Temperature 40 ºC No Change
Run Time 60 min No Change
Injection Volume 75 ml No Change
CDS Empower Empower
Sample: Infliximab
Arc Multi-flow path Technology: UHPLC Flow Path 2

42. ©2015 Waters Corporation 42
Arc Multi-flow path Technology
Flow Path 2
For UHPLC Separations
(Lower Dwell volume)
Update HPLC Method to UHPLC
UHPLC Flow Path 2
Select Path 2

43. ©2015 Waters Corporation 43
ACQUITY QDa® Mass Detector for Mass
Confirmation
2998 PDA
New Low dispersion
analytical flow cell
2489 UV/Vis
New Low dispersion
analytical flow cell
2414 RI
2475 FLR
New Low dispersion
analytical flow cell
2424 ELS
ACQUITY QDa
Additional High Performance Detection Options

44. ©2015 Waters Corporation 44
Sample: Tryptic digest of Infliximab
Update HPLC Method to UHPLC
Peptide Mapping: Improvements in Separations
AU
0.00
0.10
0.20
0.30
AU
0.00
0.10
0.20
0.30
0.40
0.50
Intensity(x10)6
1.0
2.0
3.0
4.0
5.0
6.0
Retention Time (min)
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 36.00 38.00 40.00
Agilent 1100 Series HPLC System
3.5 µm
ACQUITY Arc System, UV/Vis
2.5 µm
ACQUITY Arc System, QDa
2.5 µm

45. ©2015 Waters Corporation 45
 UHPLC separations enables
better chromatographic
resolution of peptide
variants over HPLC
 ACQUITY QDa provides the
specificity and sensitivity for
relative quantification of
peptides
 Mass confirmation for
increased confidence
5.7%
94.3%SIR
TUV
TIC
AU
0.05
0.10
0.15
0.20
0.25
Intensity
1x106
2x106
3x106
4x106
5x106
6x106
7x106
8x106
Intensity
0.0
5.0x105
1.0x106
1.5x106
2.0x106
Retention Time (min)
15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00
Sample: Tryptic digest of Infliximab
Native
Peptide
Oxidized
Peptide
Native
Oxidized
Update HPLC Method to UHPLC
Quantification using ACQUITY QDa

46. ©2015 Waters Corporation 46
HPLC or UHPLC Methods on
UHPLC Platform
Benefits:
– Replicate established HPLC assays
without compromise
o System-to-system transfer
o “Method Transfer”
– Improve productivity with modern
UHPLC column technology
o “Method Improvement”
– Increase confidence with ACQUITY QDa
mass detection
HPLC Methods
or
Improved
UHPLC
Methods
Replicate HPLC assays or improve
methods regardless of the LC platform
used for method development

47. ©2015 Waters Corporation 47
Outline
 Global trend towards new technologies
through life cycle management
 Defining LC platforms for life cycle management
of analytical procedures
 Life cycle management of analytical procedures
using UHPLC and UPLC systems
– Method transfer of existing HPLC methods
on the ACQUITY ARC UHPLC System
– Method transfer of existing HPLC methods
on the ACQUITY UPLC H-Class Bio System

48. ©2015 Waters Corporation 48
Lifecycle Management of an Analytical
Procedure using UPLC
HPLC UPLC
ACQUITY UPLC
H-CLASS Bio
HPLC Methods
or
Updated UPLC
Methods
Isocratic Methods
OR
Gradient Methods

49. ©2015 Waters Corporation 49
ACQUITY UPLC H-CLASS BIO:
HPLC Simplicity UPLC Performance
Reproduce established
HPLC &UHPLC methods
Enables seamless
transfer to UPLC
The system of choice for
method development
Flexibility for
HPLC , UHPLC &
UPLC Methods
Biocompatible
system for analysis
in high salt mobile
phases
The system of choice for
method Transfer
Low dispersion
True UPLC
performance with band
spread of less than 10μL
for highest
chromatographic
resolution
Auto-Blend Plus
Automated online
solvent blending at
specific pH and ionic
strength that
supports reversed
phase, SEC, and IEX

50. ©2015 Waters Corporation 50
Transfer HPLC Method to H-Class
Transferring Isocratic Methods
From HPLC to UPLC
Application Example
Size Exclusion Chromatography (SEC)
Scenarios
Method Equivalency
Method Improvement
ACQUITY UPLC
H-CLASS Bio

51. ©2015 Waters Corporation 51
Transfer HPLC Method to H-Class
Isocratic: SEC
Instrument
HPLC
(Quaternary)
ACQUITY UPLC
H-Class Bio
(Quaternary)
Column Chemistry Waters BioSuite™ SEC 250Å, 10 mm No Change
Dimensions 7.5 mm x 300 mm No Change
Mobile Phase
20 mM Phosphate, 200 mM NaCl,
pH 6.8
No Change
Flow Rate 400 ml min-1 No Change
Temperature Ambient No Change
Injection Volume 20 ml No Change
Run Time 35 min No Change
CDS Empower Empower
Sample: Infliximab

52. ©2015 Waters Corporation 52
AU
0.00
0.05
0.10
0.15
0.20
AU
0.00
0.10
0.20
0.30
0.40
Retention Time (min)
10 20 30
AU(x10-3)
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
AU(x10-3)
-1.0
0.0
1.0
2.0
3.0
4.0
Retention Time (min)
15 20 25 30
Retention Time (min)
15 20 25 30
Transfer HPLC Method to H-Class
Resolution Maintained
Sample: Infliximab
System Mode Rs
Relative Peak Area
(%)
Dimer Monomer
x̅ σ x̅ σ
HPLC HPLC 1.72 0.48 0.01 99.5 0.01
H-Class Bio HPLC 1.77 0.47 0.00 99.5 0.00

53. ©2015 Waters Corporation 53
Transfer HPLC Method to H-Class
Transferring Isocratic Methods
From HPLC to UPLC
Application Examples
Size Exclusion Chromatography (SEC)
Scenarios
Method Equivalency
Method Improvement
ACQUITY UPLC
H-CLASS Bio

54. ©2015 Waters Corporation 54
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Rs(m,d)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
PeakArea(%)
99.0
99.1
99.2
99.3
99.4
99.5
99.6
99.7
99.8
99.9
100.0
PeakArea(%)
Update HPLC Method to UPLC
Improvements in Separations
AU(x10-3)
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Retention Time (min)
15 20 25 30
AU(x10-3)
0.0
1.0
2.0
3.0
4.0
5.0
1.0 1.5 2.0 2.5
Retention Time (min)
HPLC UPLC
RsDimer Monomer
*
*
* Increased resolution of low level clipsMethod Scaled for new column dimensions
Column
ACQUITY UPLC Protein
BEH SEC 200Å
4.6 mm x 150 mm
Column
Waters BioSuite
SEC 250Å
7.5 mm x 300 mm
1.7 mm10 mm

55. ©2015 Waters Corporation 55
Transfer HPLC Method to H-Class
Transferring Gradient Methods
From HPLC to UPLC
Application Example
Peptide Mapping
Scenarios
Method Equivalency
ACQUITY UPLC
H-CLASS Bio

56. ©2015 Waters Corporation 56
Transfer HPLC Method to H-Class
Gradient: Peptide Mapping
Instrument
HPLC
(Quaternary)
ACQUITY UPLC
H-Class Bio (Quaternary)
Column
Chemistry
XBridge BEH C18 130Å, 3.5 mm No Change
Dimensions 4.6 mm x 100 mm No Change
Mobile Phase
H2O with 0.1% (v/v) TFA
Acetonitrile with 0.1% (v/v) TFA
No Change
Flow Rate 500 ml min-1 No Change
Temperature 40 ºC No Change
Run Time 60 min No Change
Injection Volume 20 ml No Change
CDS Empower Empower

57. ©2015 Waters Corporation 57
VD1 – VD2
(360 ml)
Accounting for Dwell Volume
Gradient SmartStart
ACQUITY
Quaternary
Solvent
Manager
Gradient Start :
“After injection”
Enter volumetric
Differences

58. ©2015 Waters Corporation 58
Transfer HPLC Method to H-Class
Gradient offset aligns chromatograms
*
Retention Time (min)
10 15 20 25 30 35 40 45
Retention Time (min)
10 15 20 25 30 35 40 45
No Gradient Offset
HPLC
H-Class Bio
Sample: Waters MassPREP Peptide Mixture
*
Programmed Gradient Offset
HPLC
H-Class Bio
* is a system peak observed on the instrument

59. ©2015 Waters Corporation 59
0.5
1.0
1.5
2.0
2.5
3.0
1 5 9 14 18 22 26 30 34 39 44 48 52 63
RRT
Peak Number
Transfer HPLC Method to H-Class
Gradient: Peptide Mapping
A total of 56 peptide peaks were
selected for monitoring
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1819 20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54 55
56
AU
0.00
0.05
0.10
0.15
AU
0.02
0.04
0.06
0.08
0.10
0.12
Retention Time (min)
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
1
2
3
4
5 6
7
8
9
10
13
14
15
16
17
18
19 20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
3738
39
40
41
42
43
44
4546
47
48
49
50
51
52
53
54 55
56
11
12
Sample: Infliximab
H-Class Bio
HPLC

60. ©2015 Waters Corporation 60
HPLC or UPLC methods on UPLC
Platform
Benefits:
 Flexibility
– Run both legacy HPLC methods and UPLC
methods
 Future-proofing your laboratory
– Taking advantage of sub 2 µm particle
technology for separation efficiency
 Improve critical performance parameters
– Resolution and sensitivity
– Reduce analysis time
ACQUITY UPLC
H-CLASS Bio
HPLC Methods
or
Improved
UPLC Methods

61. ©2015 Waters Corporation 61
Summary
 Global trend towards new technologies through life cycle management
– Driven by industry and regulators
– Increased ROI and quality standards
 Three tiers of LC categories : HPLC, UHPLC, UPLC
– Increased resolution, sensitivity and reduced run time
– Dispersion (not pressure/flow envelope )characteristics of instruments govern
performance
 UPLC provides largest scientific and business benefits
 UPLC and UHPLC can be seamlessly incorporated into life cycle management
strategy
 Waters has solutions at every LC category for reliable robust and reproducible
solutions
Versatility without
Compromise
Performance that
enhances your
laboratory
Dependability
HPLC
Methods HPLC/UHPLC
Methods
HPLC/UHPLC/UPLC
Methods

62. ©2015 Waters Corporation 62
ACQUITY ARC System
www.waters.com/arc

63. ©2015 Waters Corporation 63