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Mohammad Al-Sayah, Meenakshi Goel, and Tina Nguyen
Small Molecule Analytical Chemistry
Genentech Inc. USA
June, 23, 2016
Phase-appropriate Method Development to
Expedite Entry to Clinic
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Outline Slide 2
• Drug Development Overview and introduction
• RPLC screening tool
• SFC as a fast normal phase screening tool
• LC/MS applications
• Acknowledgements
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Phase-appropriate Drug Development Slide 3
Pre-Clinical Late Stage
Research Phase I
Non-GLP
GLP
GMP
Phase II
GMP
Phase III
GMP
• Process related impurities (Organic and inorganic impurities)
• Impurities present in starting materials
• Degradation products
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Confidential – do not distribute
GDC-0068 Phase-appropriate Drug Development Slide 4
Target: Total impurities ≤ 3.0%,
No individual impurity > 1.5%)
Total impurities ≤ 3.0%
Individual Impurity ≤ 0.25% Total impurities ≤ 2.0%
Individual Impurity ≤ 0.15%
ID Threshold: 0.10%
Pre-Clinical Late Stage
Research Phase I
Non-GLP
GLP
GMP
Phase II
GMP
Phase III
GMP
Slide 4
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Confidential – do not distribute
GDC-0068 Genentech Development Compound 1 Slide 5
• API is a complex molecule with 3 chiral centers
• 2N sterioisomers, 4 pairs of diasteriomers
Slide 5
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Confidential – do not distribute
GDC-0068
RP HPLC Method Development
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RP-HPLC Screening Conditions Slide 7
Mobile Phase A
Low pH, pH 2.0
Mid pH, pH 4-5
Neutral pH, pH 6.8
Stationary Phases with small column
dimensions
CN
C8
Polar Embedded Phase
Fused Core
PFP
Phenyl Mobile Phase B
1. Acetonitrile
2. Methanol
Agilent 1260 HPLC/MS
Slide 7
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8
Phase 3 API Method
SRS
SSR
SRR
RRR
Genentech Development Compound 1 Slide 8
Process Related
Imp 1
Process Related
Imp 2
Process Related
Imp 3
SM
Slide 8
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GDC-0068 Potential Impurities in SM Slide 9 Slide 9
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10
Potential Impurities in G-0044355 and G-02859109 Potential Impurities in Starting Material Slide 10 Slide 10
Isopropyl amine
impurities
Cloro positional isomers
Di-Cloro positional isomers
Des-Cloro impurity
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Purity method for Enamide Acid (G-02859109) HPLC Purity Method Slide 11
LOQ: 0.1%
Slide 11
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Confidential – do not distribute
GDC-0068
Chiral SFC Method Development
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Properties of Supercritical CO2 (scCO2)
• Critical Temp: ~ 31 ˚C
• Critical Pressure: ~ 73 bar
• Highly compressible, low viscosity, high diffusivity
• Solvating properties similar to liquids
• Inexpensive, non-toxic, and Green! Typical phase diagram of a pure substance
Properties of supercritical CO2 (scCO2) Slide 13
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Agilent 1260 SFC-MS System Slide 14
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Column: Choice of 8 different stationary phases per screen Mobile phase A: scCO2
Modifier: MeOH, EtOH, or IPA Additive: 0.1% NH4OH or DMEA Flow rate: 4-5ml/min BPR: 130 bar Column Temp: 40oC Detection: UV at220, 254, & 280nm MS scan: 150 -1000 m/z
SFC Screen: 24 conditions ~ 1 hour
NP-LC Screen: 72 hours!!!
SFC Screening Conditions Slide 15
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Position Chiral Primary Chiral Secondary Achiral
1 Whelk-o1 AY-H Epic Diol
2 IA-3 OD-3 DEAP
3 IC-3 Cell-3 Basic
4 AD-3 Regis Pack Ethyl-pyridine
5 Cell-4 CLA-1 PFP
6 AS-3 CC5 Nitro
7 OJ-3 CC4 Pyridyl amide
8 OZ-3 Cell-2 Zorbax Rx-Sil
Available SFC Screens Slide 16
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Parameter SFC NP-HPLC
Column Chiral or Achiral No change
MPA CO2 Heptane or Hexanes
MPB 0.1% additive in Alcohol No change
Flow Rate 4 ml/min 1-1.5 ml/min
MP Program Isocratic Isocratic
BPR 130 bar N/A
Column Temp 40oC No change
Wavelength 220nm No change
SFC Method Transfer to NPLC Slide 17
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Chirality Control Slide 18
Compound A
Compound B
Cl and Br
analogues
of Compound C
Compound D
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Chiral Method Slide 19
Chirobiotic V2 Column
Enantiomer 1 Enantiomer 2
Reversed Phase Chiral HPLC
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Chiral LC/MS Application
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Alanine Coupling Reaction Slide 21
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Alanine Chiral Method Development Slide 22
Parameter Condition
Column Astec Chirobiotic T, 4.6x250mm, 5um
Column temp 30⁰C
Injection volume 15µL
Mobile phases A = 5mM Ammonium Acetate pH4.1 B = Acetonitrile
Detection Wavelength UV at 210 nm DAD: Bandwidth: 4nm
Isocratic 35/65 MPA/MPB (V/V)
Flow rate 1.0mL/min
Run time 10 minutes
Alanine 20mg/mL
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Alanine Chiral Method Development Slide 23
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(25%)
Goal: A method can detect Alanine in sample matrix
GDC-0077 Alanine Coupling Slide 24
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Alanine Coupling Slide 25
L-alanine
D-alanine
Step 1 Intermediate
Step 2 Intermediate
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GDC-0077 Alanine Coupling Slide 26
L-Alanine D-Alanine
• Need to resolve this matrix interference.
• LC- SIM-MS ??
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Chiral LC/MS method development Slide 27
Parameter Condition
Column Astec Chirobiotic T, 4.6x250mm, 5um (Cat # 12024AST)
Column temp 30⁰C
Injection volume 10µL
Mobile phases (premixed, optional)
A = 5mM Ammonium Acetate pH4.1 B = Acetonitrile
MS-SIM Detection Positive mode (M+H = 90) AND Negative mode (M-H = 88)
Isocratic 30/70 MPA/MPB (V/V)
Flow rate 1.0mL/min (back pressure 67 bars)
Run time 25 minutes
Alanine concentration 0.001 to 0.02mg/mL
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Chiral LC/MS method development Slide 28
0.015mg/mL
0.02mg/mL
0.01mg/mL (target conc.)
0.002mg/mL
0.001mg/mL (LOQ)
Stream diverted to MS from 6-12min, otherwise to waste
SIM-MS negative mode
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Chiral LC/MS method development Slide 29
Linearity Sensitivity Recovery Precision
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Conclusions Slide 30
• RPLC screening system can facilitate method development
• SFC is a very efficient technique and can expedite chiral method development
• The LC/MS method helped guide process development to understand the epimerization of L-alanine in solution ( prior to coupling).
• The reaction turned out to be very oxygen sensitive.
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Acknowledgments Slide 31
Genentech
• Jin Wang
• Matthew Janson
• Francis Gosselin
• Chong Han
• Jack Pellett
• Pete Yehl
• Kelly Zhang
Agilent Technologies
• Lisa Wang
• Michael Yap