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

Outline Slide 2

• Drug Development Overview and introduction

• RPLC screening tool

• SFC as a fast normal phase screening tool

• LC/MS applications

• Acknowledgements

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

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

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

Confidential – do not distribute

GDC-0068

RP HPLC Method Development

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

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

GDC-0068 Potential Impurities in SM Slide 9 Slide 9

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

Purity method for Enamide Acid (G-02859109) HPLC Purity Method Slide 11

LOQ: 0.1%

Slide 11

Confidential – do not distribute

GDC-0068

Chiral SFC Method Development

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

Agilent 1260 SFC-MS System Slide 14

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

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

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

Chirality Control Slide 18

Compound A

Compound B

Cl and Br

analogues

of Compound C

Compound D

Chiral Method Slide 19

Chirobiotic V2 Column

Enantiomer 1 Enantiomer 2

Reversed Phase Chiral HPLC

Chiral LC/MS Application

Alanine Coupling Reaction Slide 21

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

Alanine Chiral Method Development Slide 23

(25%)

Goal: A method can detect Alanine in sample matrix

GDC-0077 Alanine Coupling Slide 24

Alanine Coupling Slide 25

L-alanine

D-alanine

Step 1 Intermediate

Step 2 Intermediate

GDC-0077 Alanine Coupling Slide 26

L-Alanine D-Alanine

• Need to resolve this matrix interference.

• LC- SIM-MS ??

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

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

Chiral LC/MS method development Slide 29

Linearity Sensitivity Recovery Precision

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.

Acknowledgments Slide 31

Genentech

• Jin Wang

• Matthew Janson

• Francis Gosselin

• Chong Han

• Jack Pellett

• Pete Yehl

• Kelly Zhang

Agilent Technologies

• Lisa Wang

• Michael Yap