Economical Chiral Purification of Racemate using a “Catch &

Release” Like Technique

Background

• Analytical SFC methods developed on Chiralpak AY

• Modifiers of methanol, ethanol and isopropanol showed baseline separation of the enantiomers

• Attempt to scale up using pSFC with AY column up to 70% ethanol failed

• Desired enantiomer did not elute

2

Preparative SFC trace using AY column

At 70% ethanol, desired enantiomer did not elute even after 30 minutes.

Switched to 70% methanol (very broad desired enantiomer eluted).

3

Column: AY-H, 30 X 250 mm, 5µ

Isocratic: 70% Ethanol

Flow: 110 mL/min

Loading: 200 mg

Undesired enantiomer

“Catch and Release” like purification technique

• Possibility of “catch and release” like technique using flash chromatography ensued due to retention behavior of enantiomers on AY column and ethanol as the mobile phase

• Due to time constraint and unavailability of a larger preparative AY column, “catch and release” like technique on preparative SFC instrument was not feasible

4

Interactions - “Catch & Release” using SPE

vs. “Catch & Release” using CSP 5

• “Catch and release” is widely used in Ion Exchange

separation with SPE. The sorbent (anion or cation exchange)

traps the compound of interest via electrostatic interaction

and eluted with an acidic or basic modifier

•The “catch and release” technique using CSP may be

hydrophilic and hydrophobic interactions depending on which

sorbent is used

Catch and Release using SPE

Step 1 – Condition SPE with

solvent A

Step 2 – Add sample

Step 3 – Wash with solvent A to

elute unwanted compound

Step 4 – Elute compound of

interest with solvent B

6

Catch & Release using CSP

Step 1 – Condition CSP with

solvent A

Step 2 – Add racemate

Step 3 – Wash with solvent A to

elute enantiomer 1

Step 4 – Wash with solvent B to

elute enantiomer 2

Step by step “Catch & Release” using SPE

vs. “Catch & Release” using CSP

vs

mi0 2 4 6 8 10

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mi0 2 4 6 8 10

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mi0 2 4 6 8 10

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mi0 10 20 30 40 50

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LC method development using

analytical AY-H column 7

IpOHDesired

UndesiredMeOH

Desired

Undesired

EtOH

Desired

Undesired MeCN

Desired

Undesired

MeCN selected to release desired enantiomer based on elution profile and peak shape

Isopropanol (IpOH) and Methanol (MeOH): Almost baseline resolution, desired peak very broad (> 3 minute from baseline to baseline) Acetonitrile (MeCN): Partial resolution between enantiomers. Desired peak eluted in less than 2 minutes (k’ = 1) ( < 2 minutes from baseline to baseline) Ethanol (EtOH): Desired enantiomer eluted 40 minutes after the undesired enantiomer ( = 11.4)

Testing the concept -

Application of LC method to “catch and release”

the enantiomers using Chiral Stationary Phase (CSP)

• 200 gram of 20 µ Chiralpak AY CSP

is slurry packed with ethanol into a

500 mL vacuum vessel

• 5 g of crude racemate is dissolved

in ethanol and manually loaded

onto bed

• Step 1 - CSP (column) washed

with 12 column volumes (7

fractions) of ethanol applying low

vacuum

• Step 2 - Column was washed with

12 column volume (11 fractions) of acetonitrile while applying low vacuum

8

The set up

mi0 1 2 3 4 5 6 7 8 9

mA

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Step 1 – HPLC traces of fractions

collected using ethanol 9

F1F7

• Desired enantiomer was not observed (7 fractions collected)

• Fraction 7 (F7)showed only a trace of the undesired enantiomer

Collected fractions profile

Fraction monitoring conditionsColumn: Chiralpak AY-H 150x4.6 Mobile Phase: MeCN (neat)

Flow Rate: 1.5 mL/min Sample: 5 uL of each fraction collected

mi1 2 3 4 5 6 7 8 9

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QC of fractions collected from step 1 by HPLC 10

min 1 2 3 4 5 6 7 8 9

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mAU

Racemate

Undesired

(pooled F2-F6)

Column: Chiralpak IF, 150x4.6, 5µ

Mobile Phase: 50% CO2(l) / 50%

MeOH containing 20mM Ammonia

Flow Rate: 4 ml/min

BPR: 160 Bar

Column Temp: Ambient

Pooled fractions recovery

• Fraction 1 – ~0.3 g

• (>98% e.e., 93% chemical purity)

• Fractions 2 – 7: ~0.9 g

• (>98% e.e., 97% chemical purity)

• Total recovery F1-F7 = 89%*

*adjusted for the %purity of crude (54%)

* Elution order is reversed using IF column (Column switched to IF because

the analytical AY was being used to monitor the fractions and no back up

column available)

Desired

Undesired

mi2.5 5 7.5 10 12.5 15 17.5

mA

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11

Fractions monitoring conditionsColumn: Chiralpak AY-H 150x4.6 Mobile Phase: MeCN (neat)

Flow Rate: 1.5 mL/min Sample: 5 uL of each fraction collected

Solid Blue Line – F1

Dashed Purple Line – F11

Impurity

Desired

• Undesired enantiomer was not observed (11 fractions collected)

• Fraction 11 showed no trace of the desired enantiomer

Step 2 – HPLC traces of fractions

collected using acetonitrile

Collected fractions profile

12QC of fractions collected from step 2 by HPLC

min 1 2 3 4 5 6 7 8 9

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1400 mAU

Desired

(pooled F5-F9)

Column: Chiralpak IF, 150x4.6, 5uM

Mobile Phase: 50% CO2(l) / 50% MeOH containing 20mM Ammonia

Flow Rate: 4 ml/min

BPR: 160 Bar

Column Temp: Ambient

Pooled fractions recovery• Fractions 1-5 – ~0.9 g

• (>98% e.e., 60% chemical purity)

• Fractions 6–9: ~0.6 g

• (>99% e.e., 97% chemical purity)

• Total recovery F1-F9 = 84%** Adjusted for the %purity of crude (54%)

* Elution order is reversed using IF column

mi1 2 3 4 5 6 7 8 9

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1400 Racemate

Desired

Undesired

Efficiency and throughput - Preparative SFC

vs. “Catch and Release” Technique 13

Method Loading

(g)

Grams

processed

/Hr.

Solvent required KKD

pSFC 0.70 7.6 0.56 liters/gram 1.0*

“ Catch and

release”

Technique

5 10 0.20 liters/gram ~1.2