purification process speed-and efficiency-hayward-etal

Utilizing LC & SFC separations with Utilizing LC & SFC separations with UV, ELSD & MS detection for UV, ELSD & MS detection for

purification in drug discovery:purification in drug discovery:

Driving toward capacity, quality, Driving toward capacity, quality, efficiency, and rapid turnaroundefficiency, and rapid turnaround

Xu Zhang, David P. Budac, QingPing Han, and Mark J. Hayward

Lundbeck Research USAParamus, NJ

Lundbeck Research USA Chemistry – Analysis and Purification

The need for processThe need for processefficiency and qualityefficiency and quality

� Med chemists, on average, spend ≥50% of their time on purification, even when state of the art tools are provided.

� 20 medicinal chemists means ≥≥≥≥10 FTE effort in purification.� If 1-2 experts could do all the purification, a huge human

efficiency increase in medicinal chemistry can be realized.� Time is of the essence in drug discovery (LO cycle time).

So, turnaround time must be fast (high need: hand crafted cmpds).

� Losing compounds costs a lot of Med chemist time. Thus, quality (minimized losses) has crucial impact on efficiency.�Med Chem compounds cost ≈≈≈≈2k$ ea. (total FTE cost /

average # of compounds).�You can’t afford not to make every effort not to lose

them (Aim for highest success rate or at least 6 sigma).�Don’t be penny-wise (on instrument, solvent, or salary)

and then pound foolish on the big $ = compounds.

�Quality and speed are crucial in every respect!1


Purification Purification –– needs and goalsneeds and goals� Early Drug Discovery �� Full coverage (Med Chem definition)

� Hit to Lead Parallel Synthesis – 40+ mg, approximately 20-100 compound/batch, every few days

� Lead Optimization – hand crafted for in vivo – 100 - 500 mg, some requiring ultra high purity, approximately 20+ compound/batch daily

� Development Candidate Candidates – Up to 50 g with at least 10 g at ultra high purity, 2 day turnaround on 20 g, 20 - 25 compounds/yr

� Variety of sample quantities – 10s of mg to 10s of grams

� Variety of sample qualities� Sample purities range from 5 to 95% prior to purification.� Impurities may or may not be baseline resolved according to OA-LCMS

(levers needed).*� Dissolution remains an ongoing challenge.*

� Above needs define scale or capacity!� Success requires following capabilities:

�100 mg per injection� 20 – 120 compounds/day� ≥≥≥≥120 injections & collections/day� ≥≥≥≥0.5 g/hr in full gradient mode

2

*Experts are crucial in addressing these challenges


Given the needs and goals there are some significant challenges

• Operational philosophy must be simple and streamlined yet diverse enough to cover most chemical space and keep quality high– Integrate Med Chem, analysis, purification, drying, and Compound

Management into the way of working– Trusted partnership throughout the process is crucial for efficiency

• Mass per injection is 5 fold higher than norm (LC/MS based)– Adapt off the shelf components to increase capacity

• Cycle times are 3 fold faster than norm (LC/MS based)– Apply fast LC techniques

• FTE load must be low to achieve desired gains in efficiency– Automate everywhere possible for tasks and transparency of data

• Med Chemists already have their own instrumentation. Why should they come to you?– Must be able to do it much better / faster than Med Chemists– Must gain the trust of the Med Chemists

3


Purification Operational PhilosophyPurification Operational Philosophy� Tried and true technique: get a quality separation at

analytical scale, then scale up (50 fold in volume).� Required to achieve high success rate.� Nevertheless, must be able to adapt at prep scale

� 2 x 2 x 2 matrix of gradient fast analytical LC methods adapted to the preparative scale (needs more levers):� 2 columns – C18 and C8.� 2 gradients – C18 gradients favor moderate LogP to polar

compounds and C8 moderate to high LogP� 2 pHs – 4 and 6.5 (extremes rarely needed).� Crucial efficiency component: align with OA-LC/MS and

achieve high Med Chemist competence (simplicity)

� Routine 100 mg per injection & Gaussian peaks.� Success rate as close to 100% as possible (measure

and collect waste, if needed).� Automated with optimized conditions built into

predictable, calibrated methods.4


Operational Philosophy:Operational Philosophy:Why use analytical data and scale up?Why use analytical data and scale up?

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

2.0e-2

4.0e-2

6.0e-2

8.0e-2

1.0e-1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

18:38:1305-Jun-2006

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

2.0e-2

4.0e-2

6.0e-2

8.0e-2

1.0e-1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

HNJ_19813-127-002_03 2487 TUVAn2

1.99e63.38

1.86

0.58 1.47

2.16

2.952.64

4.314.18

HNJ_19813-127-002_03 2: Diode Array 254

Range: 1.007e-13.38

229.9

1.85238.9 2.47

228.92.15227.9

2.93209.9

4.32228.9

4.18228.9

Waste stream after fraction collector when collection works perfectly

Stream prior to fraction collector

In many cases, only data from the actual sample allows accurate threshold prediction due to earlier eluting peaks and baseline rise from low level impurities.

Losing compounds is not a viable option for achieving increased speed. Background levels at ~15% peak height (UV & MS)

Collected peak

5


Column choice(Polarity)

Gradient choice (Polarity)

pH choice(Ionization/Polarity)

Neutral-pH~7

8 LC/MS method choices: 2 x 2 x 2 polarity matrix (3 binary choices = simplicity)

Highly polar(H2O soluble)

not as commonsome intermediates

C8

High OrganicC8 only

C18

Acid-pH~4

Low OrganicC18 only

Moderate OrganicC8 & C18

Most likely:mod LogP &

polarity,neutral pH

(for CNS cmpds)

Chemical space:

pH dimension (polarity) is truly orthogonal to column/ gradient (see Thu talk)

Column/gradient choices allow access to extremes & optimization in mid region

6

Low polarity& high LogPMore typical


2x2x2 approach: Optimize pH & polarity

Goal: sharp peaks in the middle 60% of the chromatogram

(0.4min < RT < 1.6min)

Elutes too earlySolutions:

1. C8 �� C18 Column2. Less organic gradient3. Change pH

Broad Peak(s)Solution:

1. Change pH, thenre-evaluate RT / polarity

Elutes too lateSolutions:

1. C18 �� C8 Column2. More organic gradient3. Change pH

Simple technique to achieve optimal separation:•Send molecules through column un-ionized (adjust pH of sample and mobile phase).•Select column/gradient combo in order to achieve near 1.0 minute retention time.

7


Operational philosophy: Med Chemists perform the pre-analysis used for purification

• Why make Med Chemists do all that work?– Our Med Chemists perform OA-LC/MS 11 times per day (avg)

• Following reaction progress (>90%) and final products (<10%)

– They want to do it. They already do it = efficiency

• How will Med Chemists know which method to use?– We train them

– Med Chemists can become highly competent in the use of OA-LC/MSs choosing column, gradient, & pH (2 x 2 x 2)

– Binary choices often sufficient to allow expert results without being expert

• Med Chemist entry point for purification– OA-LC/MS data where one can say “I want that peak or those peaks

in bottle(s)” and structure of compound

– Highly interactive process with purification expert

– Trusted partnership throughout the process is crucial for efficiency

– Making excellent progress toward same approach with SFC8


Challenges of 100 mg/injectionChallenges of 100 mg/injection

� Compared to analytical scale, injection mass increased 105 fold but mobile phase volume for separation can increase only about 50 fold� Maintaining speed and resolution requires compensation in the

chromatographic system.� Injection process must be adapted for high load.� Adsorption and buffering capacity must be adjusted for high load.

� Collection volume and separation time can limit number of compounds collected�High separation efficiency (analytical like) must be routine to keep

collection volumes reasonable.�High separation efficiency (fast analytical like) must be routine to

keep cycle times reasonable.

� Additional processes must be automated� Adapted injection process must be automated.� Extra adsorption / buffering capacity must be on-line.� Automatic column switching & regeneration.� Solvent and waste handling must be streamlined

9


O

N

N

100 mg

25 mg

50 mg

Challenges of apparent massChallenges of apparent mass--overloadoverload

Conventional Wisdom:Higher mass loading kills chromatographic performance!

Thus one injects lower mass (10-20 mg: common LC/MS scale).

Doxylamine(pKa 8.7): 25-100 mg injection. Mobile phase temperature: 45°°°°C. Buffer: 0.2% formate. Mobile phase pH = 6.5

10

Un-ionized form

Ionized form


Achieve 100 mg/injectionAchieve 100 mg/injection� Injection via “at column dilution”

� Deliver fully-dissolved sample to column – improve mass and volume loading

� Completely separate sub-system (pump, valves), enabling sample-dependent injection method selection from software

�High separation capacity� Larger diameter column (30 mm) provides enough stationary

phase surface area to retain compounds� Higher temperature improves adsorption kinetics� Higher buffer concentration enhances buffer capacity � Completely separate buffer mixing sub-system enables buffer

capacity selection within the separation methods software

�High separation efficiency � Higher temperature allows for lower back pressure and faster,

higher velocity separations� Use all other known techniques, i.e. minimize extra-column

volume, small particles (3 µm)� On-line back-flush maintains column condition (>>2000

injections) and eliminates delay time for column re-equilibrium

11


RP-LC Purification System SchematicAll components under full software control (MassLynx V4.1) [except automatic heaters]

12

Concentrated buffers at 1-4 M: NH4COOH,

NH4COOCH3, CH3COOH, NH3, H2O etc…

Acetonitrile

heater

Waste barrels

Waste level sensor

and auto switcher

MilliQ Gradient water

purification and auto-

delivery system

Waste UV

Concentrated buffer pump

mixer

Dilution solvent selections (ACN,

50/50 ACN water mixture, etc..)

At columndilution pump

Photo diode array

MS ELSD

Injection

port

Fraction

collector

Back-flushregenerating pump

heater

heater

6-pos. column selectors

choose up to 6 column

chemistries

Column

water bathMakeup pump

Inertsil

C18

Inertsil

C8

XBridge

C18

SunFire

C18

splitter

Binary pump

B A

Degassers

Back-flush solvent selections

(ACN, 5% acetic acid and DMF)


Waste barrels

Waste UV (2487)

mixer

Photo diode Array-2996

MS-ZQ ELSD-2420

Injection

port

Fraction

collector

Back –flush/regenerationP50 CO2 + P50 Modifier

(awaiting software)

heater

heater

6-pos. column selectors

choose up to 6 column

chemistries

OJ

AS

OD

AD

Splitter

Degassers

G700 with Bulk Tank

P-200

CO2

P-50

modifier

515

PR 40 PSI

Waste level sensor and auto switcher

Concentrated buffer pump (515)

(front panel control)

At column dilution pump (Thar analytical FDM)

heater

CO2 ventSIIIGLS

heater Dilution solvent selections

(alcohols, 50/50 alcohols and CO2

mixture, etc..)

Diethylamine, triethylamine, isopropylamine,

ammonium formate, formic acid etc in alcohols

Alcohols

Make up pump

515

NP-SFC Purification System SchematicMost components under full software control (MassLynx V4.1)

heater

Columns 5-6:

Ethyl pyridine – 5

Variety of columns - 6

Much of our SFC design philosophy comes from our established approach toward RP-LC/MS based purification

13


LC System Photo: overall pictureLC System Photo: overall picture

14


LC System Photo: pumps (5) pictureLC System Photo: pumps (5) picture

15


LC System Photo: injector/collector (LC System Photo: injector/collector (combocombo), ), columns, UV detectors (2) picturecolumns, UV detectors (2) picture

16


LC System Photo: columns & detectorsLC System Photo: columns & detectors[MS, ELSD & UV (2)] picture[MS, ELSD & UV (2)] picture

17


LC/MS & SFC/MS System Photos:LC/MS & SFC/MS System Photos:note on vacuum pump ergonomicsnote on vacuum pump ergonomics

18

There are noise abatement solutions that work.(required in a Danish lab)


SFC Instrument photo (viewed from right)

19


Instrument photo (viewed from front)

20


““At Column DilutionAt Column Dilution””Approach for Sample InjectionApproach for Sample Injection

� Nature of injection �� instantaneous >10x dilution!� Choice of dilution solvent can have big impact on keeping samples in

solution as injection mass increases and can help chromatographic performance

� Goal: Deliver sample to stationary phase as individual molecules in solution (best way: separate pump)

� Flow parameters are important� Flow rate must be sufficiently high to deliver sample without introducing

band-broadening (7.5 mL/min into 100 mL/min).

� Diverting at column dilution solvent after injection process can be helpful to eliminate the effects of injection solvent on the separation (divert at 0.3-0.5 min).

� Dilution solvent composition is an untapped resource for scaling up injection mass� 100% B not always universal best choice

� 50/50 clearly much better in about half the cases

� Binary choice (50%/50% A/B & 100%B) covers small scale (100mg inj)

� Further refinement of %B and buffering can be worthwhile for larger scales where ≥200mg injections are desired (it’s all about solubility)

21


At Column Dilution: RPAt Column Dilution: RP--LCLCFlow Rate & DivertFlow Rate & Divert

�Elevated flow rate to avoid wasting time and minimize band-broadening before column.

�Target: injector sweep time 15 s max, 8 s typical.

�Elevated flow rate also helps prevent sample loss.

� Diverting “at column dilution” flow also can improve separation by allowing dissimilar injection and separation solvents.

�Improved solubility by lowering pH eliminates injection precipitation

1 mL/min

2 mL/min8 mL/min

Example: 200 mg imipramine

ACN

50:50:H2O/ACN 1% acetic Acid

N

N

22


Time1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

2.5e-1

5.0e-1

7.5e-1

1.0

1.25

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

2.5e-1

5.0e-1

7.5e-1

1.0

1.25

60742-048-010-boc-t79 2: Diode Array 250

Range: 1.3792.24

2.67

60742-048-010-boc-t94 2: Diode Array 250

Range: 1.3152.54

3.62

100% B injection

50/50 CO2/MeOH injection

(best choice in about half of cases)

At column dilution: all the same applies to SFCExample: challenging chiral resolution 100 mg injection

(all aspects mirror RP-LC, i.e. goal is solubility)

Already purified by RP-LC: we know that only 2 enantiomers present

Classic sign of precipitation(in addition to pressure spike):Second peak for same analyte

23


Separation capacity and efficiency (LC & SFC)Separation capacity and efficiency (LC & SFC)� Column particle size and diameter:

� Smaller particles (3-5 µm) enhance surface area, adsorption capacity, separation efficiency, and speed.� We have tested 3 µm particles at prep scale and they work as well as

they do in analytical scale� However, we have not found suppliers that pack 3 cm columns with 3 µm

particles consistently (but we would like to)� Thus, we use 5 µm particles for all prep scale work

� 30 mm diameter consistently provides enough capacity for 100 mg injections under “infinite diameter” conditions (1 mL injections typical, 2 mL max).

� Column length:� Column length is an expensive and slow way to gain resolution

� Column cost approximately proportional to length� Separation time approximately proportional to length

� Stationary phase (SFC) and eluent choices (RP-LC & SFC) are the most time effective way to achieve resolution

� We find 50 mm length optimum for RP-LC� Adjusting eluent conditions can be done much faster to achieve resolution at this

length (total time starts to increase at shorter lengths) More in buffering section

� We find 100 mm length optimum for SFC� We haven’t found shorter chiral columns to be available (but we would

like to)24


Separation capacity and efficiencySeparation capacity and efficiency� Temperature:

� A crucial parameter that affects adsorption / desorptionrate, and thus must be properly controlled:

� Four independent heaters used:� Mobile phase heater (up to 400 Watts applied, J-KEM Sci.)

� Column heater (water bath kept at temp of mobile phase)

� Dilution solvent heater (up to 20 Watts applied, Sererity).

� Back-flush solvent heater (up to 80 Watts applied, J-KEM Sci)

� Benefits of temperature control:� Improved peak shapes due to faster adsorption kinetics �

fronting = missed adsorption opportunities and tailing = delayeddesorption

� Significant selectivity changes also possible for SFC.

� Maintain highly concentrated samples in solution. This can be especially crucial during the injection process.

� Reduces back pressure allows higher flow rates and faster runs � combination of solvent choice / temp = speed for RP-LC!

25


0.5

25oC35oC

45oC55oC

NH

N

O

O

O O

O

O

O

O

O

RPRP--LC Temperature Effect on Peak ShapeLC Temperature Effect on Peak ShapeReserpineReserpine

26

More heat!= more mass transfer= less fronting


•• To achieve resolution, one generally must have retentionTo achieve resolution, one generally must have retention

–– No retention = no separationNo retention = no separation

•• elutes in void volumeelutes in void volume

–– Must sweep multiple column volumes (kMust sweep multiple column volumes (k’’))

•• kk’’ must be greater than 2 must be greater than 2

•• 5 < k5 < k’’ < 15 very often optimal & < 15 very often optimal & ∆∆%B / k%B / k’’ should be <5 (LSS)should be <5 (LSS)

•• To get resolution fast one must sweep column volumes quicklyTo get resolution fast one must sweep column volumes quickly(k(k’’/min /min ≥≥≥≥≥≥≥≥ 3 whereas typically k3 whereas typically k’’/min /min ≤≤≤≤≤≤≤≤ 1 prep scale)1 prep scale)

–– Maximize/increase Maximize/increase velocityvelocity (flow) while maintaining mod (flow) while maintaining mod ∆∆%B / k%B / k’’

–– Tune temperature to match Tune temperature to match velocityvelocity

–– Make good choices Make good choices (required to achieve first 2)(required to achieve first 2)::

•• Column type Column type –– polymer vs. silica/BEHpolymer vs. silica/BEH

•• Mode of operationMode of operation –– isocratic (static) vs. isocratic (static) vs. gradientgradient (linear sweep from A (linear sweep from A to B)to B)

•• SolventSolvent (B) (B) –– acetonitrileacetonitrile (ACN) vs. methanol (MeOH)(ACN) vs. methanol (MeOH)

RPRP--LC: Temperature Effect on SpeedLC: Temperature Effect on Speed

27


RPRP--LC: Velocity vs. temperature for:LC: Velocity vs. temperature for:elution mode and column choiceelution mode and column choice

Optimal Velocity vs. Temperature

10

35

60

85

110

135

160

0 5 10 15 20 25 30

Optimum Eluent Velocity (mm/s)

Sep

ara

tio

n T

em

pera

ture

(C

)

Fit gradient ACN - silica and BEH

Observed gradient ACN - silica and BEH

Isocratic ACN silica - Guiochon etal

Iso (& grad) MeOH silica - Guiochon etal

Isocratic (& grad) ACN polymer - Carr etal

Range where bothanalyte and silicastability are well

established

BEH or silica with reduced H2O content only(not polymer)

BEH or polymer only(particle stability)

Limited stability for silica

MeOH Gradient ACN

HighVelocity

That’sWhyACN!

28


•• Temperature offers much greater ability than other Temperature offers much greater ability than other

techniques to achieve higher velocitiestechniques to achieve higher velocities

•• Gradients with acetonitrile are truly fast!Gradients with acetonitrile are truly fast!–– Optimum velocity with gradient ACN is much more Optimum velocity with gradient ACN is much more

responsive to temperature elevation than any other mode responsive to temperature elevation than any other mode

of operationof operation

–– Isocratic operation is at least 3 fold slower than gradient Isocratic operation is at least 3 fold slower than gradient

(ACN)(ACN)

–– Other solvents (alcohols) behave like isocratic operation Other solvents (alcohols) behave like isocratic operation

even in gradient mode even in gradient mode (still >3 fold slower)(still >3 fold slower)

–– ACN gradients appear to be uniquely crucial to ACN gradients appear to be uniquely crucial to

achieving high productivity!achieving high productivity!

RPRP--LC: Scientific case forLC: Scientific case forgradient ACN operationgradient ACN operation

29


RP-LC: Acetonitrile (ACN) usage – Why?• We use gradient ACN for purification

Let’s compare methanol use with ACN– One third velocity or 3 fold more time (35 vs. 100mL/min @50°C)

– Half the solubility (loading) or double the number of injections

– Total 6 fold loss in productivity! (add instruments and FTEs!)

– Double injections = double solvent = 12k$/mo (vs. 20k$/mo for ACN) (also doubles waste volume)

– Lower organic strength = lower column lifetime (more column cost) & less reliability

– Overnight runs = lower reliability– Lower reliability = lower quality & lost compounds– Must also change OA-LC/MS to MeOH (Med Chem disruptive)

• Conclusion: the expense of 6 fold loss in productivity and lower quality would seem to thoroughly outweigh the potential 8k$/mo in savings on solvent

30


Time2.10 2.15 2.20 2.25 2.30 2.35 2.40 2.45 2.50

AU

1.0

2.0

3.0

4.0

5.0

carbamazepine-4 2: Diode Array 230

Range: 5.5152.25

SFC: Effect of separation temperature on carbamazpine peaks

Peak shape doesn’t change much with increasing temperature compared with RP HPLC condition (note opposite effect on retention). However, temperature can still be very helpful with selectivity (see next).

30 oC40 oC

50 oC

31


Temperature can dramatically enhance selectivity, sometimes in unexpected ways

Mixture of endo/exo isomers/enantiomers at (A) 30, (B) 40, (C) 50, and (D) 60°°°°C

SFC: Temperature tuning the separation

D

C

B

A

32


RPRP--LC Mobile Phase Buffer CapacityLC Mobile Phase Buffer Capacity� Background:

� Goal: send desired compound through column un-ionized.� Buffer must be more concentrated than analytical scale because sample

is more concentrated.� Don’t over rely on aqueous pKa and Henderson–Hasselbach equation to

know ionization state – Use peak shape! (see talk on Thu)� KEY MESSAGE: must view un-ionized state loosely, as pKa shift or

anion complex with buffer can be sufficiently un-ionized for good separation peak shape for basic drugs where pH < pKa (pKa - pH = 2-4 is OK!).

� i.e. more buffer goes a long way toward reducing analyte charge.

� Practical Approach:� Mix buffer on-line like “at column dilution:”

� Flow rate proportional to buffer concentration.� Valve makes it easy to have 6 buffers on-line & method selectable.

� Target high pH and buffer concentration for high pKa compounds:� pH 4 and 6.5 can cover a very full range of drug-like compounds.� Lower buffer concentration (0.2%) usually works well for bases when

pH > pKa. Also, more quickly removed during drying process.� Higher buffer concentration crucial for high mass loading.� Higher buffer concentration (1%) at pH 7.5 usually works well with

high loading of stronger base intermediates when pH ≤ pKa. 33


O

N

N

[formate] = 260 mM (1%)

[formate] = 130 mM (0.5%)

[formate] = 52 mM (0.2%)

RPRP--LC Mobile Phase Buffer CapacityLC Mobile Phase Buffer CapacityEffect on peak shape & peak capacityEffect on peak shape & peak capacity

DoxylaminepKa = 8.7100 mg injection. Mobile phase pH = 7.5Mobile phase temperature 45°°°°C

Buffer needs to reach 10x peak concentration to correct peak shape

34


RPRP--LC Mobile Phase Buffer CapacityLC Mobile Phase Buffer CapacityDirect effect on loading and relative to pH shiftDirect effect on loading and relative to pH shift

Amitriptyline @ pH = 7.5 (pKa = 9.2)

0.1

0.4

0.7

0.0 0.5 1.0Buffer concentration (%)

Peak w

idth

@

base (

min

)

100 mg

50 mg

25 mg

100 mg Amitriptyline (pKa = 9.2)

0.1

0.4

0.7

1.0

0.0 0.5 1.0Buffer concentration (%)

Peak w

idth

@

base (

min

)

pH 6.5

pH 7.5

Buffer capacity (buffer concentration) has a large impact on column loading (peak width).

The effect on loading from buffer concentration can be considerably larger than that of the pH effect.

35


Noscapine at 25 and 100 mg loading at different pH conditions. (Aqueous pKa = 7.8)

RP-LC peak shape / loading with pH: 3 distinct states

36

3333rdrdrdrd pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:Distinctive peak shape, loading pattern and analytestate. Retention increases with loading (anti-Langmuir behavior). Analyte is likely to be at least partially protonated, but buffer anions may form neutrally charged complex when present in sufficient concentration. Rapid equilibrium seems to result in behavior like non-ionized complex (not protonated base) even at the effective pKa ≈ 4.5 in eluent.

2222ndndndnd pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:Basic compound primarily exists as protonatedbase, buffer anions provide charge shielding (nonlinear Langmuir behavior).

1111stststst pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:pH range with distinct chromatographic behavior:Basic compound exists as free base despite buffer pH being at or below the aqueous pKa. Buffer concentration does not affect chromatographic behavior (linear Langmuir behavior). Acetonitrilelikely mitigates protonation. Peak shape and loading still suggest linear Langmuir behavior.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

1.12

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-11.53

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

2.20

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

3.07

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

3.12

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

3.15

pH = 3.5

pH = 5.5

pH = 4.5

pH = 4.0

pH = 7.5

pH = 6.5

Big transition from a small change in pH.

Time0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

2.78pH = 5.0

Small transition from a big change in pH.

20% ACN 60% ACN

Frequently provides

maximum selectivity


Another practical aspect of getting the right buffer concentration

Chromatograms of a compound synthesized in-house. (a) 60 mg loading with 48 mM of formate (b) 80 mg loading with 96 mM of formate.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

1.0e-1

2.0e-1

3.0e-1

4.0e-1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

1.0e-1

2.0e-1

3.0e-1

4.0e-1

1#1,2:21

1#1,1:101

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

1.0e-1

2.0e-1

3.0e-1

4.0e-1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

1.0e-1

2.0e-1

3.0e-1

4.0e-1

FEB2007_317 2: Diode Array 254

Range: 5.028e-10.55

1.53

1.782.12

FEB2007_321 2: Diode Array 254

Range: 5.28e-11.52

1.22

0.982.17

1.82

(a)

(b)

Un-retained desired compound

37


(a)

(b)2.43

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

2.38

1.53

0.75

3.622.72

2.23

1.57

0.87

3.632.75

Chromatograms of a compound synthesized in-house. (a) 40 mg loading with 48 mM of formate (b) 55 mg loading with 96 mM of formate. Mobile phase pH: 6.5

Another practical aspect of getting the right buffer concentration (selectivity)

Impurity resolved

38


1 0 0 m g , 5 % t o 2 0 % M E O H + 0 .2 % D E A , 1 0 0 G /M IN , b p 1 2 0 , s p 3 0 0 ,4 0 o C

T im e1 .6 0 1 .8 0 2 .0 0 2 .2 0 2 .4 0 2 .6 0 2 .8 0 3 .0 0 3 .2 0 3 .4 0 3 .6 0 3 .8 0 4 .0 0

AU

0 .0

2 .5 e - 2

5 .0 e - 2

7 .5 e - 2

1 .0 e - 1

1 .2 5 e - 1

1 .5 e - 1

1 .7 5 e - 1

2 .0 e - 1

2 .2 5 e - 1

2 .5 e - 1

2 .7 5 e - 1

im p r a m in e -8 2 : D io d e A r r a y 3 2 0

R a n g e : 3 .7 3 6 e - 12 .9 1

Buffering can help a lot with peak shape under high loading conditions

SFC peak shape becomes much better with adding 0.2%DEA in MeOH.

No additive

0.2% DEA

Imipramine100 mg injection

While we don’t understand the chemistry as well as we do RP-LC (yet), buffering

also helps SFC

39


Other Automation Features:Other Automation Features:Column BackColumn Back--flush Regeneration flush Regeneration –– a simple but crucial component for successa simple but crucial component for success

� Without back-flushing, columns show increased peak width in as few as 50 injections

� Benefits of back-flushing column� Prolongs lifetime of columns; >4000 injections (> 400 g)

without loss of performance (increased peak width).� Prevents carryover and pressure gain.� Much more consistent performance.� Allows for re-equilibration of column prior to starting next cycle,

i.e. no time lost.

� Back-flushing technique� Gradient back-flush repeated 3 times over duration of run� Flow rate of 20 mL/min sufficient (1/5th of prep flow)� Acidic buffer (5% acetic acid in water removes bases well) and

organic (ACN) removes lipophilic compounds.� Resolution difference between columns (in pair) may be ideal

way to evaluate condition (significant difference = dead column)

� DMF is a quick way to dislodge nitrogen containing tar40


• ELSD Characteristics

�Mass based detection (not concentration)

�Fairly analyteindependent

�+/- 20% accuracy readily achievable

�Automated inclusion in FractionLynx report

Other Automation Features (LC & SFC):Other Automation Features (LC & SFC):ELSD collected mass estimationELSD collected mass estimation

41


• FractionLynx (FL) Reports captured by NuGenesis SDMS

� Our pipeline the the Med Chemist ELN

� Automated by printing FL browser reports to SDMS.

� Report includes waste UV chromatogram to show compound was collected (not lost). This (and ELSD mass) builds data driven trust with Med Chemists

� Convincing nature of data presentation minimizes need for post purification QC for ordinary compounds (95% purity threshold cmpds).

Other Automation Features (LC & SFC):Other Automation Features (LC & SFC):Immediate access to data by Med ChemistsImmediate access to data by Med Chemists

42


• Benefits of making buffers on-line

� Greater selection (6)

� Far less labor: people handle only small volumes of concentrated buffer

� Software select buffer concentration

� Achieves best water quality direct from Millipore Gradient

� Water circulates in ceiling & loop is tapped at point of use

RPRP--LC LC �� Other Automation Features:Other Automation Features:Water plumbed directly to point sourceWater plumbed directly to point source

43


SFC has analogous set up of “A” solventCO2 source photos

44


• Waste set up:� 30 gal. drum (110 liter)� Keeping drums in ventilated

cabinet achieves best safety & aesthetics.

� Simple industrial level sensor detects full & switches to stand-by drum.

� Simple industrial level sensor detects full & switches on blue light to indicate need to replace drum.

� Entire waste handling process can be maintained by anyone.

� SFC process is same but waste package volume reduced to 20 liters and is easily placed under lab bench (smaller sensor used)

LC LC -- Other Automation Features:Other Automation Features:Waste collected in drums w/ automatic switchingWaste collected in drums w/ automatic switching

45


• Waste set up:

� 110 liter (30 gal) drum is best balance between capacity & move-ability (55 gal. drums would work in same set up).

� 30 gal. capacity allows a full (24 hr) day of operation before drum must be replaced (2nd drum full). Thus, waste management workflow is decoupled from LC/MS workflow.

� Use of DOT approved containers & labels allowed us to shift drum removal & replacement to night time cleaning staff.

LC LC -- Other Automation Features:Other Automation Features:Waste collected in drums w/ automatic switchingWaste collected in drums w/ automatic switching

46


LC & SFC drying fractions

• Med Chemists perform drying and downstream aspects through delivery to compound management

• Med Chemists and Analytical own the process• We have used lean 6σ approach to streamline

our way of working• We have found a simple, time efficient way to

remove buffers and water without extra heating• We have 3 collection packages and workflows

depending on mass purified that rapidly move compounds to transfer to compound management (CM)

• Automated SD file generation for registration

47


RP-LC: Drying fractions - volatile buffer removal

• Can be washed, but that is a manual approach = laborious

• Can use more heat and time (roto-vap, Biotage V-10, or Genevac), but that may not be good for compound and we want faster not slower (8-12 hr Genevac)

• Alternative: dry down to viscous goo at 35°C achieving approximately 95% volume reduction (2-3 hr in Genevac), then re-dissolve in pure acetonitrile– High organic content drives off buffer and water first

• Dry again (4-5 hr in Genevac)– Extra step for Genevac but less total time

– Result: no formate, acetate, or water (by NMR) and 1-2% residual acetonitrile

– Easily achieved / automated with V-10 using acetonitrile as wash solvent or by adding pure acetonitrile tube at end of batch

48

*volatile buffers are easily removed with a single pass dry for SFC fractions because there is no water


LC & SFC collection packaging: operating with downstream process in mind

• Libraries (≤50 mg): collect directly into pre-tared, bar-coded 16 x 100 mm tubes accepted by CM in Genevac racks (Genevacto dry = done)

• Singletons (≤2 g approx): collect into EPA tubes w/ one tube for each 100-300 mg injection � V-10 xfer & dry to done into one or two pre-tared, bar-coded 4 mLtubes accepted by CM

• Multi-gram: (≥10 g) collect into 500 mL jars� rotovap

49


LC & SFC drying tools: 3 needed• Genevac for libraries

– 100% next day

• Biotage V-10 for singletons (≤1 g)– Like Genevac with

automatic pipetting and serial work flow

– Acetonitrile tube added to drive off buffer / H2O at low temp (35°C)

– Multiple tubes transferred to one or two 4 mL pre-tared CM tube

– >75% same day

• Roto-vap for multi-gram– Mostly next day

50


Purification performedusing a neutral pH method and C18 columnTemperatures: 40 o C

RPRP--LC purification example I: LC purification example I: Crude Synthesized ProductCrude Synthesized Product

Analytical result

submitted sample

Analytical result

purified sample

Prep LC

purification

51


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

11:47:0623-Aug-2006

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

MAJG_42700-018-001_03 2487 TUVAn2

2.28e62.88

2.45

2.27

3.84

MAJG_42700-018-001_03 2: Diode Array 254

Range: 7.557e-12.88250.9

2.68209.9

2.48209.9

Waste stream after fraction collector

Stream prior to fraction collector

Background levels at >20% peak height (UV & MS)

Collected peak

RPRP--LC purification example II: LC purification example II: Closely Eluting Species with High BackgroundClosely Eluting Species with High Background

Purities of >95% are routinely achieved for samples such as these.

52


LC purification example III: LC purification example III: 200 mg Injection of Development Candidate200 mg Injection of Development Candidate

Typical injection for purification of gram quantities of material. In this case 8 grams were purified in 4 hours for ultra high purity (no visible impurities for toxicology study). Level of recovery was >90%.

Full Scale Same Data

2% Scale

Concentrations

of fractions

10-20mg/mL.

On cooling

crystals fall

out of solution.

53


SFC/MS: cycle time 5 min60 mg/injection.

AD-H column - 3x15 cm, 5umUV & MS detection – one peak

desired, one tube collection Resulting ee is 100%

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

NP-HPLC: cycle time 23min40 mg/injection

IA column - 2x25 cm, 5 umUV detection – must fish out

relevant tubesResulting ee is 90%

SFC example 1: Chiral purification comparison of enantiomeric mixture by prep SFC/MS and NP-LC

(in house compound)

Comparison: SFC is 7 fold more productive, far less laborious, and delivers higher quality!

54


SFC example 2: Chiral resolution of flurbiprofen(well known chiral example, comparison of isocratic with gradient)

Top and middle: 100 mg and 50 mg/injection with 5 to 40% MeOH in 5 min gradient; Bottom: 50mg/injection with 15% MeOH isocratic

100g/min and 30x150 mm AD-H column, BP 120 bar

Gradient often gives better separation / loading and does not cost time!

Message: USE GRADIENT!

100 m g, 5 to 30 % M EOH , 100 G/M IN, bp120 sp 280,40oC

Tim e0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

AU

0.0

2.0

4.0

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

AU

0.0

2.0

4.0

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

AU

0.0

2.0

4.0

flurbiprofen-t7 2: D iode Array 250

Range: 5.5153.362.71


Range: 5.5123.462.76


Range: 5.3411.97 2.57

55


Prep SFC/MS fully separates desired product (m/z = 364)from starting material (m/z = 288)

m/z = 288

Prep LC/MS: desired product and starting material are partially co-eluted

m/z = 364

m/z = 288

m/z = 364

SFC example 3: Purification comparison of reaction mixture by prep LC/MS and SFC/MS (in house compound)

We couldn’t find LC separation. SFC/MS was straight forward.56


04-Mar-201015:44:12

Time0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

3.5e-1

4.0e-1

4.5e-1

AF28962-1c Sm (Mn, 3x4) 2: Diode Array 230

Range: 5.078e-1Area%55.2744.73

Area72270.7158490.75

Height503336204468

Time3.246.49

AD-H, 3x15cm, 30:70 IPA/CO2, 100g/min, 280 nmA mix of isomers (meta/para 55:45) separated by SFC – 250 mg loading

Complete co-elution with RP HPLC

SFC example 4: Purification of achiral product isomers by prep SFC/MS with chiral column (in house compound)

We couldn’t find RP-LC separation. SFC/MS was straight forward.57


RPRP--LC summaryLC summary� Achieved analytical quality for 100+ mg injections

� Good RT / threshold correlation with OA-LC/MS.� Gaussian peaks routinely.

� High throughput� 1-3 g/hr full gradient routine purification rate OR >20 g day (single cmpd).� High velocity separations – 4 mm/s (UPLC = 5 mm/s).� 5 min run time, k’ = 20 separations, 6 min cycle time (early terminate library gradients).� Column switching eliminates need for column wash or equilibration time.� Theoretical >200 compounds purified / day (actual peak demand 400 / week).

� Versatile� Range of buffers and columns selected (C8 and C18) cover a wide range of

compound purification applications.� Equipped with additional column selection to allow purification at high pH, and

if desired, HILIC or reverse phase chiral purification.

� High Reliability/Success Rate� Back-flushing prolongs column life (>4000 injections per or >400

grams purified on each column).� 2 full systems means “always on.” (2 work flows, no down time).� >99.99% success rates with >99% same day turnaround.

� Quality = Human Efficiency = Saves You $� One expert purifies > 95% of all compounds (>150g / month).� >10 to 1 increase in human efficiency (Med Chemist time reduction).� Even-though all out quality would seem to cost ≈≈≈≈20% more, it

saves a lot of Med Chem effort worth many, many fold more.58


SFC summary and overall thoughtsSFC summary and overall thoughts� We are doing essentially the same thing with SFC that we do

with RP-LC (100 mL/min, 3 cm columns, most hardware same) and we achieve essentially the same results in the same time� 1-3 g/hr full gradient routine purification rate� Reliability and success rates are the same� Same day turnaround on achiral separations

� However, there are some exceptions� Chiral column / solvent screening takes longer (turnaround 1-3 days chiral)� Sometimes chiral strategy requires resolution of intermediates� We don’t understand buffering as well, so we can’t yet exercise the same

degree of control on resolution using this lever� RP-LC came first, so we tend to go there first� Med Chemists just starting to learn OA-SFC/MS

� It is important not to be religious about one technique� Many want to force fit into one or the other �� there is no such thing as a

universal technique� Instead, one should play to the strengths of multiple techniques� RP-LC is best suited for separating based on the sum of the lipophilic

parts of molecules� NP-SFC is best suited for separating based on the specific polar

functional groups and shapes of the molecules in specific regions� There is considerable overlap between RP-LC & NP-SFC in the kinds of

molecules that can be separated �� Use this to enhance capacity!59


RP-LC Purification ParametersFlow Rates: 100 mL/min total with 1- 5 mL/min buffer and 7.5 mL/min from the dilution pump (for the first 0.3-0.5 minutes if dissimilar to eluent).

Temperatures: 45-55°C for mobile phase, columns (water bath), and back-flushing. 45-75°C for the dilution heater.

Work horse columns: C18 Inertsil ODS-3, 30x50mm, 3 µµµµm particles.

C8 Inertsil C8-3, 30x50mm, 3 µµµµm particles.

Tubing ID: 0.03” prior to column, 0.02” after column.

Splitter: 1/10000 split with 1 mL/min MeOH / 0.1% formic acid as makeup solvent.

Mobile Phase: (A) Water purified by Millipore Milli-Q Gradient system (B) ACN UV grade from B&J (important).

Buffers: Neutral, 0.2-1.0% ammonium formate (high purity);

Acidic, 0.2-1.0 % acetic acid in ACN/water (high purity).

Dilution Solvents: Varies but predominantly 1:1 ACN/water or 100% ACN.

Back-flush Solvent: 3 gradient sweeps (A) 5% acetic 1% ACN buffer (B) ACN (sometimes DMF plug).

Pumps / Injector / Detectors / Collectors / Software: Waters

Heaters: J-KEM Scientific, LAUDA and Selerity Technologies 60


Highlight of new application in SFC:Open Access (OA) SFC/UV/ELSD/MS

• To gain efficiency, complementary capabilities, and greater capacity, we have deployed OA-SFC/UV/ELSD/MS

– True orthogonal separation option for Med Chem support (TLC with awesome detectors)

– Still has broad overlap with RP-LC/UV/ELSD/MS for Med Chemsupport, thereby providing added capacity for routine reaction monitoring

– Also opens up chiral method development and ee measurement to “everyone”

– 3 achiral column choices & 7 for chiral (6 modifier / buffer options)

• Using new detector interfacing techniques and recent software releases, SFC/UV/ELSD/MS is ready for prime time in providing immediate gratification in the above applications

61


Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

100

60744-024-003 1: Scan ES+ 251

2.67e60.61

251.0

60744-024-003 1: Scan ES+ 213

9.67e60.69

213.0

Orthogonal SFC separations can be highly complementary to the frequently used RP-LC

• Truly orthogonal SFC approach can separate starting material and products that RP-LC can’t

• These SFC methods also are aligned with preparative scale methods allowing immediate purification

• MS used in this application due to lack of chromaphore

LC/MS

SFC/MS

60744-024-003, diol

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

60744-024-003b 1: Scan ES+ 251

6.89e72.21

60744-024-003b 1: Scan ES+ 213

6.13e70.82

MH=251

MH=213

MH=251

MH=213

Normal phase separation gives TLC-like outcome for polar intermediates

SFC/MS

62


Time0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

LS

U

0.000

200.000

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

AU

0.0

5.0e-1

1.0

OA-SFC/UV/ELSD/MS can provide similar information as OA-LC/UV/ELSD/MS

• Chromatograms showing starting material and product (reaction progress)

• Essentially same data with either approach except reverse elution order (TLC-like)

• Note the improved quality of ELSD with SFC!

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

92

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

28

0.65

0.41

1.24

2.032.001.930.69

0.39

0.07

0.200.11 0.33

0.440.660.57

1.851.761.66

LC/MS

SFC/MS

UV

UV

ELSD

ELSD

63


Chiral screening of many methods on a single sample login (MassLynx / OpenLynx SCN 798)

Method set for achiralanalysis

First set of methods for chiralcolumn screening

Second set of methods for chiral column screening if first set doesn’t work

New software makes method screening easy!

64


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

0.0

5.0e+1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

0.0

5.0e+1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.000.0

5.0e+1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

0.0

5.0e+1

chiral-sample1-9 3: Waters 2998 Range: 7.103e+11.48

chiral-sample1-10 3: Waters 2998 Range: 5.639e+12.672.54

0.63



3.48

Screening chiral conditions for preparative method development (4 x 3)

AD-H

OD-H

AS-H

OJ-H

IPA

MeOH AD-H

OD-H

AS-H

OJ-H

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.0

5.0e+1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.0

5.0e+1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.0

5.0e+1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.0

5.0e+1




2.53


0.663.20

EtOH

OD-H

AS-H

OJ-H

AD-H

Screening 4 columns and 3 solvent gradients showed AD-H with IPA gives a useful separation

Scaled preparative version of same method was immediately used to resolve 10g on same day

OA-SFC/UV/ELSD/MS is a viable screening approach for preparative workTime

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.0

5.0e+1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

2.0e+1

4.0e+1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.0

5.0e+1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.000.0

5.0e+1

chiral-sample1-1 3: Waters 2998 Range: 5.865e+13.72 4.29




1.03

IPA

OD-H

AS-H

OJ-H

AD-H

65

purification process speed-and efficiency-hayward-etal

Technology

quality med chemists

high logp

med chem compounds

high purity

high success rate

process efficiency

fast high need

lot of med chemist time