regis technologies, inc. - waters corporationmilligram scale purification one sfc 350 unit for...

Regis Technologies, Inc.

SFC Purification of Achiral Molecules Using Chiral/Achiral

Stationary Phases

Zahid AliSenior Separation Scientist

Serving the Scientific Community Since 1956

Outline

• Introduction• Method development and process

overview• Recent examples of purification of

achiral compound using chiral/achiral stationary phases

• Conclusion

Introduction

• Regis’ contract purification lab receives 100’s of chiral & achiral samples per year, of which ~70% are potential candidates for prep purification.

• Regis uses its expertise in purification technology to provide cost-effective, high purity, and fast purifications of target molecules for the Biotech, Pharmaceutical, and Diagnostics industries. Regis has proven that the use of SFC technology allows us to perform separations and purifications much faster than traditional Prep-LC methods.

Typical Project Life CycleFree Screening

Analytical Screening– Screen a number of achiral/chiral stationary and mobile

phase conditions and choose the stationary/mobile phase combination that gives optimized resolution.

– Achiral Stationary PhasesCyano , Diol, Amino, Ethyl Pyridine, Pyridyl Amide, DEAP (diethyl amino propyl)

If no separation, then screen the sample on Chiral columns

– Chiral stationary PhasesRegisCell®, RegisPack®, Whelk-O 1® , OJ®, AS®, DACH, ULMO,

Leucine, Phenylglycine, alpha-Burke 2, beta-GEM

Normal or Reversed Phase

The high cohesion and hydrogen-bond acidity of water dictates the separation characteristics in RPLC

The competition between the sample and mobile phase for polar interactions with the stationary phase,dictates separation characteristics in NPLC

Typical Project Life CycleFree Screening

Small Scale Project (1-10 grams)Analytical Methods Developed for Release

Large Scale Project (100g to Multi kg)HPLC, NMR, Residual Solvent……….

Medium Scale Project (10-100 grams)Solubility & Loading Estimation

Separation/Purification Optimization

Regis SFC Lab

Regis currently operates 2 analytical units for: Method development Free chiral/achiral screening Solubility and loading studies Milligram scale purification

One SFC 350 unit forPreparative scale purification from:

10gm to Multi-kilogram separations under GMP and non-GMP conditions

One SFC 80 unit for:Intermediate scale purification milligram-10gm scale

CO2 Delivery System

• Bulk Tank is vertical by design so we can take advantage of the head pressure created by the CO2in the tank

• 28,000 lb Capacity

CO2 Booster Pump• Booster pump can

deliver up to 1.3 kg of CO2 per min to the SFC lab at 1,400 psi

Case Study 1Achiral Purification

Case Study 1 – Proprietary achiral API, of which the compound of

interest is 98% pure by RPLC – 99%+ purity with high yield – Large scale API isolation by RP-Prep LC has

proven to be expensive and time consuming– Impurity needs to be identified and removed– Only Methanol can be used as Co-Solvent– Projected quantities of 10-15kg per month

What are the goals for this project?

Develop an SFC method that isolates the impurities from the drug substance using methanol as co-solvent

Run a reversed phase HPLC method to quantitate related substance impurities in drug substance after SFC purification

The methods differ with respect to separation mode and therefore provide orthogonal information concerning quantitation of impurity (HPLC) and the isolation of impurity and API by (SFC).

Achiral SFC Screening Result

Column: Diol (250X4.6)(ES Industries)Mobile phase: CO2:MeOH (85:15) Flow rate 4mL/min


Column: Cyano (250X4.6) (ES Industries)Mobile phase: CO2:MeOH (85:15)Flow rate 4mL/min


Column: Pyridyl Amide (250X4.6)Mobile phase: CO2:MeOH (85:15)Flow rate: 4mL/min

Chiral SFC Screening Result

APIImpurity

Column: RegisCell (250X4.6mm)Mobile Phase: CO2:MeOH(85:15)

Flow Rate: 4mL/min

ImpurityAPI

Comparison HPLC/SFC Result

SFC Screening ChromatogramColumn: RegisCell (250X4.6mm)

Impurity

Reversed Phase HPLC Column: Waters C18Inpurities

Case Study #1: Scale Up

Column: RegisCell, 5 micron, 250mm x 3cm: Mobile Phase: CO2:MeOH (80:20), 175 gm/min; Production Rate: 5gm/hr

STACKED INJECTION

Single injection

Overlay chromatogram before and after SFC purification

The project was successfully completed with a chemical purity of 99.6%

Case Study # 2Achiral Purification

What goals does the Regis SFC lab have for this job?

• Challenge:– Proprietary achiral API, of which:– The compound of interest is 89% pure by reversed

phase HPLC, and needs to be purified > 98.5% withno single impurities > 0.5%

– Large scale API isolation by RPLC is expensive and time consuming, therefore an alternative less expensive and fast purification technique under (cGMP) is needed

– Screen the sample by achiral selected stationary phases: Diol, Amino, Pyridine Amine, DEAP and Ethyl pyridine:→No separation by SFC→

– Screen the sample by chiral columns


(S,S)-Whelk-O 1, 5 micronMobile Phase: CO2:MeOH (75:25)

HPLC/SFC Result

Analytical SFC chromatogramColumn: (S,S)-Whelk-O 1, (250X4.6mm),

Mobile Phase: CO2: MeOH (75:25)

RPLCColumn: C18,(150X4.6mm), 5mic

Case Study # 2 : Scale Up

Column: Whelk-O 1, 10 micron, 250X 50mm : Mobile Phase: 30% MeOH, 300 gm/min; Production Rate: 4gm/hr

Single injection

Stack injection

Overlay chromatogram before and after SFC purification

Pre-Purification Chemical Purity 89% by HPLC

Post-Purification Chemical Purity 98.7% by HPLC

Overlay chromatogram

Impurities

Case Study 3:

Case Study 3:

• Challenge:– Separation of 2-diastereomeric mixtures of

enantiomers – The goal for this project develop a method that

separates all four stereoisomer at a purity > 99%DE

– Initially 150gm of material to be purified – Projected future project of 1kg

(R,S) stereoisomer (S,R) stereoisomer

(S,S) stereoisomer (R,R) stereoisomer

diastereomers diastereomers

enantiomers

enantiomers

Possible stereoisomeric combinations

Achiral SFC Screening

Diol(ES Industries)

DEAP (diethyl amino propyl)(ES Industries)

Ethyl Pyridine(ES Industries) Cyano

(ES Industries)

Mobile Phase CO2:EtOH (90:10), Column: (250X4.6mm), 5mic


Column: AS-H (250X4.6mm)Mobile Phase: CO2:EtOH (90:10)Flow Rate 4mL/min

Column: RegisPack (250X4.6mm), Flow Rate 4mL/min, Mobile Phase: CO2:EtOH (90:10)

Separation of Disteromeric Mixture By Multiphase Approach

A multicolumn approach was developed to separated 2-diastereomeric mixtures of enantiomers using coupled chiral and achiral stationary phases under SFC conditions. The achiral selectivity of DEAP stationary phase was used to modify the resolution of compounds on a Chiralcel AS-H chiral stationary phase by combining the chiral and achiral columns in series

Multiphase Column Technique

Column: AS-H+ Ethyl Pyridine

Column: AS-H+ + Pyridyl Amide

Mobile Phase: CO2:Ethanol (90:10), flow rate 4mL/min


RegisPack + DEAP in series

AS-H+ Ethyl Pyridine in series

Co-solvent: CO2:EtOH (90:10), flow rate 4ml/min

RegisPack + Cyano in series


DEAP + AS-H in series

Co-solvent: CO2:Ethanol (90:10), flow rate 4mL/min


AS-H + DEAP in series

Loading Estimation to be completed next

Overlay Chromatogram of Achiral Column

Cyano, Diol and AmideDEAP Ethyl Pyridine

Mobile Phase: CO2:Ethanol (90:10)Flow Rate: 4mL/min

SFC Chromatogram of Collected Individual Diastereomers 1

Column: AS-H

Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min

SFC Chromatogram of Collected Diastereomers 2

Column: AS-H


Overlay Chromatogram of Collected Individual Diastereomers

Column: AS-H



Column: AS+DEAP in series



AS-H+DEAP in series


Overlay Chromatogram of Collected Individual Diastereomers

AS-H+DEAP in series



DEAP + AS-H in series


Conclusion

– SFC systems at Regis Technologies were successfully implemented as a scale up tool for purification of chiral and achiral molecules from mg to multi-kg scale

– Regis has found no correlation between RPLC and SFC

– Method development at Regis is generally done by trial and error procedures, or quite often by inspired guess work using the columns available in our lab

THANKS

• ES Industries for Tech support and achiral columns

• THAR/Waters for Tech support• Regis’ SFC Lab Team (Francis

Mannerino, Jeffrey Wheeler and Ted Szczerba)

regis technologies, inc. - waters corporationmilligram scale purification one sfc 350 unit for...

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