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1 RP HPLC Purification of Small Molecules Lou Cheng Astrazeneca R&D Boston.

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Presentation on theme: "1 RP HPLC Purification of Small Molecules Lou Cheng Astrazeneca R&D Boston."— Presentation transcript:

1 1 RP HPLC Purification of Small Molecules Lou Cheng Astrazeneca R&D Boston

2 2 HPLC Basics: Classification, k, α, N, R s Reversed-Phase HPLC – Isocratic and Gradient Analytical Method Development – Screening & Optimization Analytical Scale-up of Optimized HPLC Method From Analytical Scale-up to PrepLC Purification PrepLC Purification – Fraction Collection & Recovery Summary Outline

3 3 HPLC Basics - Classification Nonionic polar Nonpolar Ionic Water-insolubleWater-soluble Increasing polarity Adsorption Partition Ion Exchange [ NP Partition] [ RP Partition] Size Exclusion [Gel Permeation] [Gel Filtration] Molecular Weight

4 4 HPLC Basics - Classification Nonionic polar Nonpolar Ionic Water-insolubleWater-soluble Increasing polarity Adsorption Partition Ion Exchange [ NP Partition] [ RP Partition] Size Exclusion [Gel Permeation] [Gel Filtration] Molecular Weight

5 5 Retention factor t Ri - t 0 k = t0t0 k2k2 α =α = k1k1 Selectivity N = 16 2 tWitWi Efficiency R s = 0.25 ( -1)N 0.5 k k + 1 Resolution tR1tR1 tR2tR2 t0t0 w1w1 w2w2 Minutes 010 mVolts HPLC Basics – k, α, N, R s

6 6 k: retention factor k w : retention factor by 100% water as mobile phase (φ = 0) S: a constant for a given sample compound φ : organic fraction (volume) in binary mobile phase log k = log k w – Sφ Eq. 1* K w: Hydrophobicity Reversed-Phase HPLC – Isocratic S: Sensitivity to change of mobile phase strength * Ref: Practical HPLC Method Development, Lloyd Snyder, etc. Wiley, New York, 1997.

7 7 ID: (BCL2) (MW = 426.5) Log k = log k w – Sφ (MW = 454.3) Code: (CoaD) Conditions: HPChem 10, XBridge C8, 4.6 × 50 mm, 10 mM NH 4 Form/AcN, 1.0 mL /min Rule of Adjusting Isocratic Retention by φ: 5% Ξ 100 % k Isocratic RP HPLC - AZ Example 1

8 8 K w = 28 S = 3.35 K w = 62, S = 3.67 K w = 72, S = , 10 g (HE-TMK) M w = 163 M w = log k = log k w – Sφ Conditions: XBridge C18, 4.6 × 50 mm, 0.1% NH 4 OH/MeOH Flow Rate: 1.0 ml /min, room temperature. The K w and S of the two isomers are too close to be separable by RP gradient runs. Isocratic RP HPLC - AZ Example 2

9 9 Reversed-Phase HPLC – Gradient *By assuming S = 4 for all small molecules, one gradient run is sufficient to resolve k w Two gradient runs can solve k w and S, by assuming log k/φ linear relationship Automatic method development Retention Prediction (Drylabs, Chromsword) b = SV m /t G F log k 0 = log k w - S 0 t R = (t 0 /b) log [2.3k 0 b(t s /t 0 ) + 1] + t s + t D Eq. 2* T 0 : column dead volumn; b: gradient steepness; k 0 : k at the beginning of the gradient; t s : value of t R for a nonretained solute; t D : dwell time for gradient elution; : change of organic percentage in the mobile phase; S: system constant; V m : column dead volumn; t G : gradient time; F: flow rate. For Linear Solvent Strength (LSS) Gradient: * Ref: Practical HPLC Method Development, Lloyd Snyder, etc. Wiley, New York, 1997.

10 10 Retention Prediction of Gradient RP HPLC - AZ Examples Sample b tGtG t R, predicted t R, Exp Error (%) (%/min)(%)(min.) (BCL2) (K w = 933, S = 4.53) (CoaD) (K w = 2290, S = 7.85) Conditions: HPChem 10, XBridge C8, 4.6 × 50 mm, 10 mM NH 4 Form/AcN, 1.0 mL /min, room temperature. t 0 = t s = 0.6 min, t D = 1.2 min. t R = (t 0 /b) log [2.3k 0 b(t s /t 0 ) + 1] + t s + t D

11 11 Why Gradient? Flexibility (b,, 0 ) to optimize separation with minimal effects on efficiency (N) Samples with a wide k range, sometimes containing late-eluting interferences that can either kill the column or carryover to subsequent runs More precise, robust, and automatable Dilute solutions of sample dissolved in a weak solvent Gradient RP run is the best starting point for method development Reversed-Phase HPLC – Gradient Over Isocratic

12 12 B: ACN C: MeOH D: THF/H 2 O (9/1) Solvents Column Examples 1: XBridge C 18 2: Gemini C 6 -Phenyl 3: Atlantic dC 18 4: YMC ODS AQ 5: Synergi Hydro-RP 6: YMC Carotenoid (C 30 ) 162 conditions can be screened for one sample if necessary RP HPLC Analytical Method Development – Screening E: 0.1% TFA F: 0.1% Formic Acid G: 10 mM NH 4 Ac pH8 H: 0.1% TEA J: 0.1% NH 4 Form K: 0.1% NH 4 OH L: 10 mM NH 4 Ac/HOAc pH5 M: 20 mM NH 4 Ac N: 10 mM (NH 4 ) 2 CO 3 Valves: G1159A 6-ColSelector, G1160A, 12/13 Selvalves Detectors: Agilent MSD and Sedex 75 ESDL Detector, Finnigan AQA mass spectrometer by closed contact Waters MicroMass Massspectrometer NP, chiral, SFC have similar settings

13 13 Criteria for Evaluating and Optimizing HPLC Methods General: Low k, low tailing factor, high N High α, high R s Client-specific: Clear communication with clients is a prerequisite to successful method development MPS/library (universal applicability) Fraction collection for one component, multiple components, or all components? Purity/Recovery? pH stability of the desired components? Amount of the sample (high loading)

14 14 AZ Example of Screening Sequence ( )

15 15 From Screening To PrepLC Anal. Scale-up 100 mg/ml, 4.6 × 100 mm XBridge C % MeOH, 10 min, 1 ml/min Screening (QuinFF) 0.8 mg/ml, XBridge C18 (4.6 × 50 mm) 5-95% MeOH/10 mM HCOONH 4, 5 minutes, 1.0 ml/min, 240 nm. Tracked by MSD * * * R s 1.8 R s 2.2 Optimization 30-80% MeOH R s 3.6 R s 4.8 PrepLC 19 × 100 mm XBridge C % MeOH, 10 min 20 ml/min, 100 mg/ml Baseline Resolution

16 16 From Screening To PrepLC Scale-up/loading 100 mg/ml, 4.6 × 100 mm XBridge C % MeOH, 10 min, 1 ml/min Screening (QuinFF) 0.8 mg/ml, XBridge C18 (4.6 × 50 mm) 5-95% MeOH/10 mM HCOONH 4, 5 minutes, 1.0 ml/min, 240 nm. Tracked by MSD * * * R s 1.8 R s 2.2 Optimization 30-80% MeOH R s 3.6 R s 4.8 PrepLC 19 × 100 mm XBridge C % MeOH, 10 min 20 ml/min, 100 mg/ml Baseline Resolution

17 17 Analytical Scale-up of Optimized HPLC Method Goal: 1) Is the optimized analytical HPLC method good for preparative one? 2) If it is, what is the maximum loading for touching-band separation? Optimization EN Synergi Hydro-RP (4.6 × 50 mm) 30-60% MeOH/TFA, 5 min, 1.0 ml/min, 240 nm, 0.6 mg/ml. Product (MW =471.5) Anal. Scale-up Synergi Hydro-RP (4.6 × 100 mm) 30-60% MeOH/TFA, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml. Product This method is not practical for separation of 5 gram samples! (125 prep injections for 19 × 100mm column!)

18 18 Scale-up of Optimized HPLC Method – Search for the Best Optimization Anal. Scale-up Gemini C6-Phenyl (4.6 × 50 mm) 50-70% MeOH/NH4OH, 5 min, 1.0 ml/min, 240 nm, 0.6 mg/ml. Gemini C6-Phenyl (4.6 × 100 mm) 50-70% MeOH/NH4OH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml. XBridge C18 (4.6 × 100 mm) 30-60% CH3OH/NH4OH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml. XBridge C18 (4.6 × 50 mm) 30-60% CH3CN/NH4OH, 5 min, 1.0 ml/min, 240 nm, 5ul inj, 0.6 mg/ml

19 19 Gilson PrepLC via UV-triggered Fraction Collection EN XBridge C18 (50 × 250 mm) 30-60% CH3CN/NH4OH, 25 min, 100 ml/min, 240 nm, 6.0 ml, 160 mg/ml. Only 5 injections!

20 20 Scale-up of Optimized HPLC Method – Theoretical Aspects W TRTR W 2 = W W th 2 Eq. 3 = 16 N -1 t 0 2 ( k) t 0 2 k 2 w w s -1 (column effect)(sample-weight effect) W s : column saturation capacity ( 0.4 surface area) Overloading in reality may include mass overloading, and others. volume overloading,

21 21 Optimized Analytical (BCL2) Xbridge C8 (4.6 × 50mm) 20-50% CH3CN/NH 4 Ac (pH 8) 5min, 1 ml/min, 254 nm, ~1 mg/ml 5 μl injection Loading of Analytical Scale-up - Theoretical vs. Practical Scale Up (BCL2) Xbridge C8, 4.6 × 100mm 20-50% CH3CN/NH 4 Ac (pH 8) 10 min, 1 ml/min, 254 nm, ~70 mg/ml Inj Vol: 20 μl (V th = 90 μl) Rs 3.5 Product What is the maximum loading of touching-band separation? * *

22 (BCL2) Xbridge C8, 4.6 × 100mm 20-50% CH3CN/NH 4 Ac (pH 8) 10 min, 1 ml/min, 254 nm, ~70 mg/ml Inj Vol: 5 μl, 10 μl, 15 μl, 20 μl 5 μl 10 μl 15 μl 20 μl 10 μl: Baseline separation Analytical Scale-up of Optimized HPLC Method - Mass Loading Studies 15 μl: Touching-band separation 20 μl: No separation * * * RtRt

23 23 From Analytical Scale-up to PrepLC F = (d prep /d anal ) 2 x L prep /L anal Constant: Column chemistryColumn chemistry Particle sizeParticle size Sample concentration To scale: Flow rate Injection volume 4.6 × 100 mm 19 × 100 mm 50 × 100 mm V TB, 1 ml/min 17 V TB, 17 ml/min 118 V TB, 118 ml/min × F semi × F large (BCL2) XBridge C8, 20-50% CH3CN/NH 4 Ac (pH 8), 10 min, 20 ml/min, ~70 mg/ml, Gilson TM LC Baseline Separation 200 (255) μl, 20 (17) ml/min, 19 × 100 mm 15 μl, 1 ml/min, 4.6 × 100 mm (BCL2) XBridge C % CH3CN/NH 4 Ac (pH 8) 10 min, 1.0 ml/min ~70 mg/ml, Agilent 1100

24 24 PrepLC Purification - UV-triggered Fraction Collection XBridge C18 (50 × 250 mm) 30-60% CH3CN/NH4OH, 25 min, 100 ml/min, 240 nm, 6.0 ml, 160 mg/ml.

25 25 97 % Recovery (91 % without first fraction) PrepLC Purification - Fraction Analysis & Recovery

26 26 PrepLC Purification - MS-triggered Fraction Collection

27 27 PrepLC Purification - MS-triggered Fraction Collection Recovery 80 %

28 28 Summary Anal. Scale Up Screening Optimization Anal. Scale-up Prep LC Gradient RP analytical run is the best starting point for developing PrepLC method Screening, optimization, and scale-up are effective steps toward PrepLC method development The best analytical methods are not always the best PrepLC methods, and scale-up experiments are imperative to validate the performance and loading of the analytical method under PrepLC conditions UV-triggered fraction collection has high recovery and lower purity than MS- triggered fraction collection.

29 29 Acknowledgment Members of Analytical Group: Tatyana, Camil, Nancy, Mark, Sharon, Milena, Ziling. Randstad USA: Yushen Chang, Vincent Cianciaruso.

30 30

31 31 Analytical Method Screening - Results XBridge C18 (4.6 × 50 mm) 5-95% NH4OH/AcN, 5 min, 1.0 ml/min, 240 nm. Gemini C6-Phenyl (4.6 × 50 mm) 5-95% NH4OH/MeOH, 5 min, 1.0 ml/min, 240 nm. Atlantis dC18 (4.6 × 50 mm) 5-95% NH4Fm/MeOH, 5 min, 1.0 ml/min, 240 nm. Synergi Hydro-RP (4.6 × 50 mm) 5-95% TFA/MeOH, 5 min, 1.0 ml/min, 240 nm. Luna C6-Phenyl (4.6 × 50 mm) 5-95% AcONH4/MeOH, 5 min, 1.0 ml/min, 240 nm. Curosil PFP (4.6 × 50 mm) 5-95% HCOOH/CH3CN, 5 min, 1.0 ml/min, 240 nm.

32 32 Analytical Method Optimization XBridge C18 (4.6 × 50 mm) 30-60% NH4OH/AcN, 5 min, 1.0 ml/min, 240 nm, 5ul inj, 0.5 mg/ml Gemini C6-Phenyl (4.6 × 50 mm) 50-70% NH4OH/MeOH, 5 min, 1.0 ml/min, 240 nm. Atlantis dC18 (4.6 × 50 mm) 50-95% NH4Fm/MeOH, 5 min, 1.0 ml/min, 240 nm. Synergi Hydro-RP (4.6 × 50 mm) 30-60% TFA/MeOH, 5 min, 1.0 ml/min, 240 nm.

33 33 Analytical Scale-up of Optimized Analytical Methods XBridge C18 (4.6 × 100 mm) 30-60% NH4OH/AcN, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml. Gemini C6-Phenyl (4.6 × 100 mm) 50-70% NH4OH/MeOH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml. Synergi Hydro-RP (4.6 × 100 mm) 30-60% TFA/MeOH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

34 34 EN c Gemini C6-Phenyl (4.6X100mm) 40-50% CH3CN/0.1% HCOOH 14 min, 1 ml/min, 254 nm, ~30 mg/ml Inj Vol: 3 μl, 6 μl, 12.5 μl, 25 μl Analytical Scale-up of Optimized HPLC Method - Volume Loading RtRt

35 35 Effects of Buffer Concentration on Preparative Loadability AG-166 Gemini C6-Phenyl (4.6X100mm), 0-20% CH3CN, 20 min, 1 ml/min, 254 nm, 12 ul, ~100 mg/ml 0% HCOOH 0.05% HCOOH 0.1% HCOOH 0.2% HCOOH

36 36 Screening (CoaD) 1JB JB JB50955 Optimization 1JB JB JB40405 Scale-up/loading 4.6 × 100 mm XBridge C18 40% AcN, 60% NH 4 Form, 30 min, 1 ml/min, 200 mg/ml Purification 19 × 250 mm XBridge C18 40 % AcN, 100 ml/min 100 min, 1.0 ml (200mg/ml) Difficult Purifications: Example × 50 mm XBridge C18 10mM NH4Form, 5min, 1 ml/min, 200 mg/mL 4.6 × 50 mm XBridge C18 10mM NH4Form, 5min, 1 ml/min, 200 mg/mL

37 37 Valves: G1159A 6-ColSelector, G1160A, 12/13 Selvalves Detectors: Agilent MSD and Sedex 75 ESDL Detector Finnigan AQA mass spectrometer by closed contact NP Chiral HPLC Solvent: HX, MeOH/EtOH(1/1), IPA, 0.1% Diethylamine Detectors: Advanced Laser Polarimeter, PDR_Chiral Inc Columns: Chiralpak AD Chiralpak OD, Chiralpak AS, Chiralpak IA, Chiralpak IB, Chiralcel OD, Chiralcel OJ, Regis Pirkle covalent (S,S) whelk O2 10/100 FEC, Regis Pirkle covalent (S,S) whelk O1 5/100, Regis (S,S) ULMO 5/100, Regis (S,S) DACH DNB 5/100, Phenomenex Chirex ®-PGLY and DNB, Large 5cm X 50cm Prep Columns: Chiralpak AD Chiralpak OD, Chiralpak AS, Chiralcel OJ NP Columns: Luna silica 10/100, YMC-PVA-sil 5/120, YMC-Pak Diol 5/60, YMC-Pak CN 5/120, Luna NH2 5/100, PrincetonSFC Pyridine 5/60 SFC Columns: Berger silica, Diol, CN, Pyridine, Chiralpak AD-H

38 38 Method Optimization Summary for

39 Gradient Runs Gradient and Isocratic Runs for % MeOH, R s = % MeOH, R s = 1.8 XBridge C18, 4.6 × 50 mm, 0.1% NH 4 OH, 5 min, 1.0 ml /min Isocratic Runs 40 % MeOH, R s = % MeOH, R s = % MeOH, R s = 2.30 XBridge C18, 4.6 × 50 mm, 0.1% NH 4 OH, 1.0 ml /min. 20 % MeOH 10 % 40 %

40 40 From Scale-up to Gilson Separation for (HE-TMK) XBridge C18, 4.6 × 100mm 20 % MeOH/0.1% NH 4 OH 20 min, 1 ml/min, 254 nm, 100 mg/ml Inj Vol: 8 μl, 12.5 μl, 25 μl Agilent HP (HE-TMK) XBridge C18, 50 × 250mm 20 % MeOH/0.1% NH 4 OH 50 min, 100 ml/min, 254 nm, Inj Vol: 1.5 (1.6) ml,100 mg/ml) Gilson TM LS System Touching-band loading still low Retention still too large NP HPLC in progress!


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