Use of Solid Core Chromatography for the Analysis of Pharmaceutical Compounds

Dafydd MiltonProduct Manager, LC & LC/MS columns

Tony EdgeR&D Principal

March 2014

Introduction

• The Pharmaceutical Process• Mapping out different sectors

Wh t t f h ll f d• What types of challenges are faced• Understanding the drivers in the pharmaceutical industry

• Solid Core Chromatography• Understanding the benefits of the technology• Bar for bar greater efficiency• Bar for bar greater efficiency• How to optimise the separation

• Understanding chemistry• Optimization of the morphology

• Coupling Solid Core Chromatography to Pharmaceutical Analysis• Coupling Solid Core Chromatography to Pharmaceutical Analysis• Design of new workflows• Improving column lifetimes

2

• Improving assay robustness

The Pharmaceutical Process – Beginning

3

The Pharmaceutical Process – Further Development

4

Solid Core Material – Features and Benefits

• Features• More uniform particle sizing• More uniform particle sizing• Better packing of particles• Reduced pore depth• Reduced mass transfer effects in mobile phase

• BenefitsBenefits• More Efficient Chromatography• Allows the use of low pressure systems

• Competitive Edge• Bar for bar gives better separations than porous materialsg p p

5

Liquid Chromatography Particle Design

2.6 µm80 Å

Solid Core Particles

80 Å

2.6 µm150 Å

Conventional Fully Porous Non-Porous Solid Core

4 µm80 Å

1.x µm80 Å

Reduce Size to improve kinetics at expense of

operating pressure

Low sample capacity

Very high pressure

Small particle kinetics

Reasonable pressureVery High Sample Capacity

Lower Efficiency

Low Sample Capacity

Very High Efficiency

High Sample Capacity

High Efficiency

6

operating pressurey y g y g y

Pressure Comparison

900

1000 BAP 200

2 11

600

700

800

900

600 bar limit

pp ddL 30

230

400

500

600

ress

ure

(bar

)

HPLC pressure limit

100

200

300Pr

u = 8 7mm/s0

0 200 400 600 800 1000

Flow rate (µL/min)

Accucore RP MS 2 6µm <2µm 3µm 5µm

u = 8.7mm/s

Accucore RP-MS 2.6µm <2µm 3µm 5µm

Columns: 100 x 2.1 mmMobile phase: H2O / ACN (1:1)Temperature: 30 °C

Wide flow rate range with P < 600 bar

7

Temperature: 30 °C

The Theory … the van Deemter equation

CCBAHETP uCuCu

AHETP sm

8

A – Term Eddy Diffusion or Multiple Paths

Packing Efficiency D90/10~1.5

Porous Silica

Packing Efficiency D90/10~1.1Accucore

9

B – Term Longitudinal Diffusion

Longitudinal diffusion: (HETP = H + H + H )Longitudinal diffusion: (HETP = HA + HB + Hc)

Th l t t ti i l t th d f b d l tThe solute concentration is lower at the edges of a band; solute diffuses to the edges.

Time

High concentration C t ti t ilib iHigh concentration. Concentration at equilibrium

• The B-term depends on;

10

• Void volume of the column

C – Term Resistance To Mass Transfer

• The C term depends on;Diff i diff i th i th ili• Differences in diffusion path in the silica pores

• Differences in the radial diffusion path in the liquid• Size of molecule

11

• Significant for large molecules but not for small molecules

Efficiency Comparison – Van Deemter20.0

tical

10 0

15.0

Theo

ret

5.0

10.0

uiva

lent

Pl

ates

0.00 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0ei

ght E

q

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0He

Linear velocity of mobile phase (mm/s)Accucore RP-MS 2.6µm 5µm 3µm <2µm

Columns: 100 x 2.1 mmMobile phase: H2O / ACN (1:1)Temperature: 30 °CDetection: UV at 254 nm Flow rate range: 0.1 to 1.0 mL/min

Highest efficiency and lowest rate of efficiency loss with flow

rate for solid core

12

Flow rate range: 0.1 to 1.0 mL/minAnalyte: o-xylene rate for solid core

Van Deemter – Limitations

• Classical interpretation of how a column is performing

• 3 parameters• A – eddy diffusiony• B – longitudinal diffusion• C – Resistance to mass transfer

• Optimization of these parameter will give the best peak shape/efficiency

• However it does not take into account;• Analysis time

P t i ti t• Pressure restrictions on a system

13

Kinetic Plots

• Allows for fairer comparisons of analytical systems• Van Deemter just compares pure separation ability• Van Deemter just compares pure separation ability

• Incorporates time of analysisp y• Analysts want FASTER chromatography• Van Deemter plots do not specify the time of analysis

• Incorporates pressure limitations of systems• Van Deemter does not account for a pressure limitation p

on system

• Based on three very simple classical equations• Based on three very simple classical equations

14

Kinetic Plots – Retention Time1000010000

1000(s)

of p

eak

100

ion

time

Accucore allows optimisation of retention times

Ret

enti

Solid core produces sharper peaks in less time

101,000.00 10,000.00 100,000.00

Efficiency

p

15

Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency

Impedance

• Devised by Knox and Bristow in 1977Defines the resistance a compound has to moving• Defines the resistance a compound has to moving down a column relative to the performance of that column

• Allows for pressure to be incorporated

Often plotted with a reverse axis• Often plotted with a reverse axis• Mimics van Deemter plot• Minimum value optimum conditions 2

PtE p

• Often plotted as a dimensional form2N

E• t/N2

• t0 or tr both used

16

100,000

Kinetic Plots – Impedance100,000

20

NPtE

ce

2N

10,000

mpe

dan

Im

S lid i lSolid core requires less pressure to obtain sub 2 µm

efficiencies

1,000100.001,000.0010,000.00100,000.001,000,000.00

Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency

17

µ µ µ µ Efficiency

Resolution Equation

'11 50 k

'11

41 5.0

kkNR s

EfficiencyParticle size / packing

Selectivity(Chemistry)

RetentionFactorParticle size / packing (Chemistry) Factor

(Surface area)

18

The Impact of Selectivity on Resolution

Efficiency SelectivityRetentionEfficiency SelectivityRetention2 5

3.0

2 5

3.0

NR=k’

k’+1-14

NR=k’

k’+1k’

k’+1-1-14

2.0

2.5

(R) 2.0

2.5

(R)

k +1 4

k2

k +1k +1 4

k’221.5 N

solu

tion

(

1.5 N

solu

tion

(

=k2k’1

=k2

1 = 2

10.5

1.0kR

es

0.5

1.0kR

esSelectivity () has the greatest impact on 1.00 1.05 1.10 1.15 1.20 1.25

0.0 N

1.00 1.05 1.10 1.15 1.20 1.250.0

Nimproving resolution. 0 5000 10000 15000 20000 25000

0 5 10 15 20 25

Nk

0 5000 10000 15000 20000 25000

0 5 10 15 20 25

Nk

S19

Stationary phase, mobile phase, temperature

Stationary Phase Characterization

• Hydrophobic retention (HR)

Hydrophobic Interactions

y p ( )• k’ of neutral compound

• Hydrophobic selectivity (HS)• α two neutral compounds that have different log P

• Steric Selectivity (SS)• α sterically different moleculesα sterically different molecules

• Hydrogen bonding capacity (HBC)y g g y ( )• α molecule that hydrogen bonds and a reference• Good measure of degree of endcapping

20

• Gives indication of available surface area

Stationary Phase Characterization

• Activity towards bases (BA)

Interactions with Bases and Chelators

• Activity towards bases (BA)• k’, tailing factor (tf) of strong base• Indicator of free silanols

• Activity towards chelators (C)• k’, tailing factor (tf) of chelator• Indicator of silica metal content

21

Stationary Phase Characterization

Interactions with Acids and Ion Exchanges

• Activity towards acids (AI)• k’, tf acid• Indicator of interactions with acidic compounds• Indicator of interactions with acidic compounds

• Ion Exchange Capacity (IEX pH 7.6)g p y ( p )• α base / reference compound• Indicator of total silanol activity

• All silanols above pKa

I E h C it (IEX H 2 7)• Ion Exchange Capacity (IEX pH 2.7)• α base / reference compound• Indicator of acidic silanol (SiO-) activity

22

• Indicator of acidic silanol (SiO ) activity

Column Characterization (Visualization)

HR /10

HSAI

Accucore C18HR /10

HSAI

Accucore RP-MS

SSIEX (2.7) SSIEX (2.7)

HBC

IEX (7.6)BA

C HBC

IEX (7.6)BA

C

HR /10

HSAI

Accucore PFPHR /10

HSAI

Accucore Phenyl-Hexyl

HS

SSIEX (2.7)

AI HS

SSIEX (2.7)

AI

HBC

IEX (7.6)BA

C HBC

IEX (7.6)BA

C

23

Widest Range of Solid Core Selectivity Options

500mAU 1,2,3 curcuminoids

2 00

2.50HR /10

HSAIAccucore RP-MS

Solid Core C18

0.50

1.00

1.50

2.00 HS

SSIEX (2.7)

AI Accucore C18

Accucore 150-C18

Accucore C8

Accucore 150-C4

Accucore Polar Premium1

0.00

HBCC

Accucore aQ

Accucore Polar Premium

Accucore Phenyl-Hexyl

Accucore PFP

23

Polar Premium shows different selectivity and separates the peaks

IEX (7.6)BA

Accucore Phenyl-X

Accucore C30

0.0 1.0 2.0 3.0

0

Minutes

24

Accucore Columns – Selectivity Choices

Columns: Thermo Scientific™ Accucore™ C30 ColumnAccucore C18 ColumnKi t C181

Different selectivity for K2 isomers

350 mAUAccucore C18

Kinetex C18Dimensions: 2.6 µm, 100 x 3.0 mmMobile Phases: Methanol:buffer, 98:2

Buffer = 2 mM ammonium acetate

1

250

300 2+2’

Flow: 650 µL/minTemperature: 20 ºCInjection: 5 µLDetector: UV 250 nm

2+2’

Ki t C18

200

250

Detector: UV 250 nmPeaks: 1. Vitamin K2, 50 µg/mL

2. Vitamin K1, 50 µg/mLOther peaks formed by UV irradiation1 C30 shows better

separation for K1

Kinetex C18

100

150

2

2’

separation for K1 isomers

Accucore C3050

0

Vitamin K2 Vitamin K10.00 1.25 2.50 3.75 5.00 6.25 8.00-25

min

25

Very Fast Separations with Superb Resolution

• Separation of atorvastatini t di t (ATC AT1

Fully porous C185 μm, 250 x 4.6 mm

intermediates (ATC-AT1 with ATC-AT1-Difluro)

• All customer requirements 60 min• All customer requirements were met

• Reduction of run time from 60 min to

60 min

8 min• Resolution improved using the

Accucore PFP columnAccucore PFP

2.6 μm, 150 x 3.0 mm 8 min• Mobile phase constituents kept

similar

• HPLC compatible method (270 bar)

26

Available Databases for Column Characterisation

http://www.usp.org/app/USPNF/columnsDB.html

27

Some Basic Column Requirements

• Column Ruggedness• Stable under isocratic conditions• Stable under gradient conditions• Stable at low pH• Stable at high pHg p• Stable at elevated temperatures

28

Solid Core Column Stability – Ruggedness

Accucore RP-MS 2.6 µm 100 x 2.1 mm IDMobile Phase: 60/40 ACN/H2OFlow Rate: 400 µL/min

Accucore RP-MS 2.6 µm 100 x 2.1 mm IDMobile Phase A: Water (0.05% TFA)Mobile Phase B: Acetonitrile (0.05% TFA)W h H O (0 05% TFA)Injection Volume: 1 µL

Column Temp: 30 °CWash: H2O (0.05% TFA)Injection Volume: 1 µLColumn Temperature: 30 °C

Efficiency (o-Xylene)

Asymmetry (o-Xylene)Asymmetry (o Xylene)

4 000 + isocratic test 6 000 f t di t4,000 + isocratic test injections with no decrease in performance

6,000 + fast gradient injections with no change in retention

29

Solid Core Column Stability – Low pH

40

Column Stability at pH < 2 pH 1.8(0.1% TFA)

30

35

30,000 l

20

25

tion

Fact

or Acetaminophenp-HBAo-HBA

column volumes(5.5 days)

10

15Ret

ent

AmitriptylineNortriptylineDIPPDNPP

(5 5 y )

5

00 5000 10000 15000 20000 25000 30000 35000

Column Volumes

Solid core columns are stable at low pH

30

Solid core columns are stable at low pH

Solid Core Column Stability – High pH

pH 10.5(0.1% ammonia)

30,000 columnr column volumes(5.5 days)nt

ion

Fact

orR

eten

S lid l t bl t hi h H31

Solid core columns are stable at high pH

Solid Core Column Stability – Elevated Temperature

8

9

Column Stability at 70°C Mobile phase: MeOH/H2O (65:35)

Flow rate: 0.4 mL/min

6

7

8

r

Column temperature: 70 °C

Column: Accucore C18 50 x 2.1 mm

Run time: 5 min

4

5

6

entio

n Fa

ctor

Phenol

Butylbenzene

Run time: 5 min

2

3Ret

e o-Terphenyl

Pentylbenzene

0

1

0 2000 4000 6000 8000 10000

9,000 column volumes

(400 i j ti )0 2000 4000 6000 8000 10000

Column Volumes(400 injections)

Solid core columns are stable at high temperature

32

Solid core columns are stable at high temperature

Work Flow Solutions – Generic Methods

• Used where sample throughput is critical• Compound management• Discovery DMPK• Ability to run at high flow rates without compromising chromatographyAbility to run at high flow rates without compromising chromatography

• Require robust methods• Assays cannot afford to fall over• Many samples means long column lifetime• For bioanalytical samples need columns that are robust with plasma extractsFor bioanalytical samples need columns that are robust with plasma extracts

• Require orthogonal chemistries• Reversed phase / HILIC etc.

33

Faster than 5 and 3 µmFully porous 5 µm, 150 x 4.6 mm

Rs = 2.64

5 µL injection

∆P = 59 bar

Gradient and flow rate:• Fully porous 5 μm 150 x 4.6 mm

35–60 %B in 10.0 min1000 µL/min solvent used 17 mL1µL injection ∆P 23 b 1000 µL/min solvent used 17 mL

•Fully porous 5 μm, 100 x 2.1 mm 35–60 %B in 6.7 min210 µL/min solvent used 2.4 mL

F ll 3 100 2 1

Fully porous 5 µm,100 x 2.1 mm

Rs = 1.641µL injection ∆P = 23 bar

• Fully porous 3 μm, 100 x 2.1 mm 35–60%B in 4.0 min350 µL/min solvent used 2.4 mL

• Accucore RP-MS 2.6 μm, 100 x 2.1 mm Fully porous 3 µm, 100 x 2.1 mm

Rs =1.96 1µL injection ∆P = 97 bar

35–60 %B in 3.5 min400 µL/min solvent used 2.4 mL

ACCUCORE 2.6 µm, 100 x 2.1 mm

Rs = 2.50 1µL injection ∆P = 218 bar

Reduced analysis time and solvent costs

Minutes0 1 2 3 4 5 6 7 8 9 10

-100100 x 2.1 mm

34

Reduced analysis time and solvent costs

Shorter Columns – Faster Separations

ACCUCORE 2.6 µm, 100 x 2.1 mm Gradient and flow rate:

• Accucore RP-MS 2.6 μm, 100 x 2.1 mm

Rs = 2.50

35 –60%B in 3.5 min400 µL/min

• Accucore RP-MS 2.6μm, 50 x 2.1 mm 35–60%B in 1.8 min

mA

U

400 µL/min

Rs = 1.51

ACCUCORE 2.6 µm, 50 x 2.1 mm

Double productivity with 50 mm column

Minutes0.0 1.0 2.0 3.0 4.0

35

Double productivity with 50 mm column

Example of a HILIC Separation

Column: Accucore HILIC 2.6 μm, 150 x 3.0 mm

Flow: 0.5 mL/min

B k 290 b

Separation of decitabineand α anomer impurity

Backpressure: 290 bar

Temperature: 40 °C

Injection: 5 µL and α-anomer impurityj µ

Detection: UV @ 244 nm

Mobile phase: 5% A (20 mMammonium acetate)ammonium acetate)95% B (acetonitrile)

36

Regulatory DMPK – Bioanalysis

• Methods can be optimized• Possibilities to optimize stationary phase chemistries• Possibilities to optimize stationary phase chemistries• Analysis times still important for PK studies

• Methods will tend to form final clinical method• Sensitivity can be an issue due to efficacious nature of drug

• Injections with plasma• Injections with plasma• Columns must not block

• Can result in peak splitting• Can result in columns overpressurising• Can result in retention time shift

• Metabolism studies• Need columns with high resolution

37

• Ideally limited sample prep, so columns stable with diluted urine

Lower Pressure than Sub 2m

• Flow rate: 500 μL/min• Mobile phase: A Water; B Acetonitrile

Sub 2 µm, 100 x 2.1 mm

• Mobile phase: A–Water; B–Acetonitrile

Accucore RP-MS 2.6 µm, 100 x 2.1 mm

Accucore RP-MS 2.6 µm, 100 x 2.1 mm

Sub 2 µm, 100 x 2.1mm

Maximum pressure (bar)

171 338

Minutes0.0 0.5 1.0 1.5 2.0 2.5

Equivalent performance, lower pressure

38

(50% lower)

Accucore with TLX – Method Conditions

Sample Preparation• Acetonitrile with internal standard added to spike plasma sample• 2 (600 μL) parts acetonitrile : 1 part spiked plasma (300 μL)

Precipitated sol tion mi ed on orte mi er

Autosampler Method LC Method

• Precipitated solution mixed on vortex mixer• Sample centrifuged and supernatant transferred to 2 mL vial• 10 μL injected onto system

p• Wash Solvent A – 20% acetonitrile + 0.1% Formic Acid +

80% water• Wash Solvent B – 45%IPA + 45% acetonitrile + 10%

Acetone + 0.1% Formic Acid

• Mobile Phase A – 0.1% Formic Acid in water• Mobile Phase B – 0.1% Formic Acid in acetonitrile• Columns: Cyclone 50 x 0.5 mm (TFC), Accucore C18 50 x

2.1 mm (Analytical)• 100 μL sample loop, 100 μL syringe

39

Accucore with TLX – Retention Stability

4.4000

Rosuvastatin Retention

4.1000

4.2000

4.3000

3.9000

4.0000

Min

utes

3 6000

3.7000

3.8000

3.5000

3.6000

0 500 1000 1500 2000 2500Injection

~2,400 injections on Accucore column with TLX system – no change in retention

40

y g

Accucore with TLX – Backpressure Stability

140 Backpressure Plots

100

120

60

80

Bar

Linear (Loading Pump Pressure at t=0) Linear (Eluting Pump Pressure at t=0)

20

40

00 500 1000 1500 2000 2500

Injection

~ 2,400 injections on Accucore column with TLX system – no increase in t=0 backpressure

41

y p

Accucore with TLX–Pressure Traces

300Backpressure Traces

250

150

200

Bar

100

Injection 2 Elute

Injection 500 Elute

Injection 2395 Elute

500 50 100 150 200 250 300 350

Seconds

~ 2,400 injections on Accucore column with TLX system – backpressure traces

42

y p

Greater Peak Capacity than 5 or 3m

220

2405 µm,100 x 2.1 mm Gradient: 65–95%B in 2.1 min,

95% B for 0.4 min

3 100 2 1

Flow rate: 400 μL/min

3 m, 100 x 2.1 mm

120

140

160

180

capa

city

ACCUCORE 2.6 µm, 100 x 2.1 mm

40

60

80

100

120

mal

ised

pea

k c

0 0 0 5 1 0 1 5 2 0 2 5 3 0

0

20

40

Accucore 2.6µm 3µm 5µm

Nor

m

Minutes0.0 0.5 1.0 1.5 2.0 2.5 3.0

Higher peak capacity – more peaks can be separated per injection

43

g p p y p p p j

More Sensitive than Fully Porous 5 and 3m

Gradient and flow rate:• 5 μm, 100 x 2.1 mm

35–60 %B in 6 7 min

S/N = 169 5m, 100 x 2.1 mm

35–60 %B in 6.7 min210 μL/min

• 3μm, 100 x 2.1 mm 35–60 %B in 4.0 min350 μL/min

S/N = 368

350 μL/min•Accucore RP-MS 2.6μm, 100 x 2.1 mm

35–60 %B in 3.5 min400 μL/min

mA

U

S/N = 399

3m, 100 x 2.1 mm

S/N = 399

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

ACCUCORE 2.6m, 100x2.1mm

Higher S/N ratios – detection and quantification of low level impurities

Minutes

44

of low level impurities

Production - Quality Control Workflows

• Robust methods• Loading capacity can be an issue as looking for impurities• Loading capacity can be an issue as looking for impurities• Selectivity important• Resolution also important

• Need to be able to transfer the methods to CRO’s• Need to be aware of differences caused by different instrumentation• Need to be aware of differences caused by different instrumentation

45

Loading Capacity

Columns:

• Accucore RP-MS 2.6 μm,100 x 2.1 mm

• <2 μm,100 x 2.1 mm2,500,000

1 2

Effect of Loading - Accucore

R² = 0.9998

R² = 0.99932,000,000

0.8

1

1.2

d Va

lue

A

R² = 0.9721

1,000,000

1,500,000

eak

area

0.2

0.4

0.6

Nor

mal

ise As

N

Tr

As

NTr500,000

Pe

00 5 10 15 20 25

Load on Column (µg)

00 0.5 1 1.5 2 2.5

Load on column (µg)<2µm Accucore 2 6μm Competitor

No loss in performance with 2 μg loaded on a

<2µm Accucore 2.6μm Competitor

46

2.1 mm ID Solid core column

Method Transfer and Optimisation

5 μm, 150 x 4.6 mmMethod Transfer Calculator:

www.thermoscientific.com/crcRs = 2.645 µL injection

Gradient and flow rate:• 5 μm, 150 x 4.6 mm

35 60 %B in 10 0 minRs = 2 50 35–60 %B in 10.0 min1000 μL/min

• Accucore RP-MS 2.6 μm, 100 x 2.1 mm 35–60 %B in 3.5 min

Rs = 2.50

400 μL/min• Accucore RP-MS 2.6 μm, 50 x 2.1 mm

35–60 %B in 1.8 minutes400 μL/min

ACCUCORE 2.6 μm, 100 x 2.1 mm

Rs = 1.51

1µL injection

400 μL/min

ACCUCORE 2.6 μm, 50 2 11µL injection

Minutes0 1 2 3 4 5 6 7 8 9 10

050 x 2.1 mm

Scalable from fully porous 5 μm columns

µ j

47

Scalable from fully porous 5 μm columns

System Considerations• Column: Accucore RP-MS 2.6 μm, 100 x 2.1 mm

• Gradient: 65–95 % B in 2.1 min

Dwell volume: 100 µL

95 % B for 0.4 min

• Flow rate: 400 µL/minAccela 1250

Dwell volume: 800 L

Surveyor Accela Surveyor Agilent

800 µL

Minutes0.00 1.00 2.00 3.00 4.00

Accela1250

Surveyor Agilent 1100

Run time (min)

2.5 3.0 3.5Dwell volume: 1000 µL

min0 0 5 1 1 5 2 2 5 3 3 5

Agilent 1100 AveragePW (1/2 Height)

0.02 0.02 0.04

min0 0.5 1 1.5 2 2.5 3 3.5

Solid core can deliver performance on a b f diff t t

48

number of different systems

System Considerations

• Minimise volume dispersion

Always Optimize System Configuration

• Tubing–short L, narrow ID• Low injection volume• Low volume flow cell• Low volume flow cell

• Optimise detector sampling rate Need enough points to define

peak (minimum of 10, >20 for quantitation)

5 pts

Fast scanning MS

• Low dwell volume pump for fast45 pts

9 pts

Low dwell volume pump for fast gradients

49

Analyzing Biomolecules

• Move to produce bigger molecules• Difficult to copy• Difficult to copy• Greater success rates

• Chromatography requirements• Need less retentive phase• Need wide pores to cope with larger molecules• Need wide pores to cope with larger molecules

50

Peptides – Resolution & Peak ShapeRT: 0 11 15 03RT: 0.11 - 15.03

1.12

8.743.76 7.09 13.468.9111 20

Accucore 150-C18

11.2013.63

1.01 C ti l S lid C C181.018.52

6.983.6013.13

15.0310.84

Conventional Solid Core C18

100000 10.20< 2 µm Wide Pore Fully Porous C18

0

50000

uAU

8.491.5914.4912.035.25 14.31

y

2 4 6 8 10 12 14Ti ( i )

-50000

51

Time (min)

Proteins – Excellent Resolution vs < 2 µm Wide Pore

• Sharper and higher peaks80000

100000 Accucore 150-C4Backpressure: 185 bar

higher peaks than < 2 µm Wide Pore Fully Porous C4

40000

60000

uAU

Porous C4• Better resolution

and sensitivity100000

0

20000

• Significantly lower backpressure

60000

80000

100000 < 2 µm Wide Pore Fully Porous C4Backpressure: 320 bar

p

40000

60000

0 1 2 3 4 5 6 7 8 9 10Ti ( i )

0

20000

52

Time (min)

Conclusions

• The Pharmaceutical Process• Mapping out different sectors

Wh t t f h ll f d• What types of challenges are faced• Understanding the drivers in the pharmaceutical industry

• Solid Core Chromatography• Understanding the benefits of the technology• Bar for bar greater efficiency• Bar for bar greater efficiency• How to optimize the separation

• Understanding chemistry• Optimization of the morphology

• Coupling Solid Core Chromatography to Pharmaceutical Analysis• Coupling Solid Core Chromatography to Pharmaceutical Analysis• Design of new workflows• Improving column lifetimes

53

• Improving assay robustness