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
Achiral SFC Screening Result
Column: Cyano (250X4.6) (ES Industries)Mobile phase: CO2:MeOH (85:15)Flow rate 4mL/min
Achiral SFC Screening Result
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
Chiral SFC Screening Result
(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
Chiral SFC Screening Result
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
Multiphase Column Technique
RegisPack + DEAP in series
AS-H+ Ethyl Pyridine in series
Co-solvent: CO2:EtOH (90:10), flow rate 4ml/min
RegisPack + Cyano in series
Multiphase Column Technique
DEAP + AS-H in series
Co-solvent: CO2:Ethanol (90:10), flow rate 4mL/min
Multiphase Column Technique
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
Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min
Overlay Chromatogram of Collected Individual Diastereomers
Column: AS-H
Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min
SFC Chromatogram of Collected Individual Diastereomers 1
Column: AS+DEAP in series
Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min
SFC Chromatogram of Collected Individual Diastereomers 2
AS-H+DEAP in series
Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min
Overlay Chromatogram of Collected Individual Diastereomers
AS-H+DEAP in series
Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min
Multiphase Column Technique
DEAP + AS-H in series
Mobile Phase: CO2:Ethanol (90:10) Flow Rate: 4mL/min
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)