Antisense Oligonucleotides at Biogen Biogens entry into ASOs with a - - PDF document

2/24/2017 1

Antisense Oligonucleotide Purification Platform: How does it measure up?

March 2, 2017

Kris Ruanjaikaen, Hien Nguyen, Ratnesh Joshi, Yannick Fillon, Robert Gronke

2 Biogen | Confidential and Proprietary

• Biogen’s entry into ASO’s with a strategic alliance with Ionis

Pharmaceuticals (Carlsbad, CA)

• Pipeline: 8+ drug candidates with a neurology focus including SMA,

ALS, Parkinson’s, and Alzheimer's diseases

• Our lead ASO drug Spinraza: FDA approval granted in December

2016 for Spinal Muscular Atrophy (SMA)

Antisense Oligonucleotides at Biogen

2/24/2017 2

3 Biogen | Confidential and Proprietary

• Introduction: Antisense oligonucleotides (ASOs)
• What are they?
• How are they made?
• Current process vs. Biogen’s platform
• Structure and key impurities
• Purification platform development
• Purification Strategies
• Evaluation of Biogen’s pipeline against platform
• Process development
• Conclusions

Outline

4 Biogen | Confidential and Proprietary

What are ASOs?

Rigo F, et al. The Journal of Cell Biology. 2012. 199(1):21-25.

• ASO are short, synthetic

nucleic acid chain 8-50 units in length

• Designed to bind to

complementary mRNA

• Has a broad range of effects
• n protein expression

Intrathecal Delivery

• Biogen has a strong interest in

diseases of the brain

• Most of our ASO’s require

intrathecal (IT) delivery (lumbar puncture) into cerebrospinal fluid

• Low concentration DP (< 10 g/L)

2/24/2017 3

5 Biogen | Confidential and Proprietary

ASOs : Structure

1 2 3 5

Chain length varies: 16 – 20 unit 5’ DMT blocking group

• Used for synthesis

reaction sequences

• Hydrophobic handle
• Will be cleaved off

from product Phosphorothioate linkage

• S provides resistance to

nuclease degradation

• Negative charge

Bases

• Can be modified for stability

2’ modifications

• Added stability and potency

6 Biogen | Confidential and Proprietary

ASO Manufacturing Overview: Existing Process

Automated Solid-Phase Synthesis grow chemically (3’ to 5’)

• n a resin bead

G G U C

…

…one unit at a time…

Purification

1) Reverse- Phase Chromatography 2) Detritylation 3) Precipitation 4) Lyophilization

Biogen’s assessment

• High yielding process
• Good purity
• Downstream = bottleneck
• Solvent intensive
• Not fit into our MFG facility

A good process with improvement opportunities

Image courtesy of Ionis Pharmaceuticals

2/24/2017 4

7 Biogen | Confidential and Proprietary

ASOs : Key Impurities (Process-Related)

Chain-length Impurities

• Failure sequences (N-x)
• N-1
• N+1

1 2 3 5

Modification Impurities

• DMT-on
• P=O
• Abasic

DMT group not cleaved S replaced by O in phosphorothiate bond A base is cleaved off

8 Biogen | Confidential and Proprietary

ASO Impurities: LC-MS Analysis

LC Chromatogram (Reverse Phase) Mass Spectrum

Failure sequences DMT-on

2/24/2017 5

9 Biogen | Confidential and Proprietary

Biogen’s Purification Process

Synthesis RP-HPLC Detritylation Rxn Multiple Precipitations Lyophilized API

Existing Process

• Aqueous downstream
• Leverage

hydrophobicity similar to RP-HPLC

• Cleave DMT group

after HIC

• Polishing

chromatography

• Robust purity
• Streamline formulation
• Ready-to-fill DS
• Reduce process time

Hydrophobic Interaction Chromatography (B/E) UF/DF Detritylation Rxn Anion Exchange Chromatography (B/E) Liquid DS Synthesis (improved rxns)

New Process (ASO-A) Key Rationales

10 Biogen | Confidential and Proprietary

Development of ASO Purification Platform Process

2/24/2017 6

11 Biogen | Confidential and Proprietary

Variables explored (yield vs. purity, process fit)

• Resin screening
• Kosmotropic salt screening  ammonium sulfate
• Leave DMT group on or off?
• Location of HIC before detritylation
• Optimization
• Binding capacity
• Wash conditions
• Elution conditions

HIC Overview : Development for first ASO

HIC as a capture step to replace RP-HPLC

• Facility fit: aqueous process, reduce waste issues
• Bind/Elute mode: High resolution of product-related impurities

12 Biogen | Confidential and Proprietary

HIC Chromatography

Typical chromatogram: ASO-A

Load / chase Load / chase Wash Wash Elution Elution Strip / CIP Strip / CIP EQ EQ

• 0.002

HPLC Purity: ASO-A

Load Elution U.S. Patent filled: 62/349,970

• Powerful step for removal of

failure sequences (≥ N ± 2) and small organics

• Step yield ≥85%
• Partially remove impurities

similar to product: P=O and N-1

• - UV
• - Conductivity

Does HIC fit across our ASO pipeline? How can we develop a process efficiently?

AS added to load to increase hydrophobicity (binding)

2/24/2017 7

13 Biogen | Confidential and Proprietary

HIC Chromatography:

Platform Feasibility-- Exploratory Mapping

• A range of solubility: hydrophobicity

depends on ASO sequences

• Guideline for loading condition

ASO solubility in (NH4)2SO4

• HIC in B/E mode will work for ASOs without major changes

UV Absorbance or Conductivity

• All ASOs bind to HIC
• Elution at similar AS concentration

Linear (NH4)2SO4 gradient

Process Volume

ASO-A ASO-B ASO-C ASO-D ASO-E ASO-F

Conductivity

14 Biogen | Confidential and Proprietary

HIC Chromatography: Platform Development Approach

• Dynamic binding capacity
• Wash conditions – yield vs. impurity removal
• Elution conditions – yield vs. impurity removal

Key Developments HIC Purification: ASO- B

• Rapid development w/ platform approach
• Similar HIC process and yield/ purity performance

Step Gradient Chrom

• - UV
• - Conductivity

yield vs. impurity

Elution Impurity Wash

LC Data

2/24/2017 8

15 Biogen | Confidential and Proprietary

Detritylation Reaction: Platform Evaluation

• Simple kinetic model for conversion vs time.
• Reaction rates are dependent on 5’ nucleotide (G>U>C)

Rxn: platform acidic condition

Removal of DMT:

X W Y X Z W Y Z Z W X Y Z W W Y Z Z

3’ 5’

• Platform rxn condition works for all ASO w/ reasonable process time

l

• 16

Biogen | Confidential and Proprietary

Variables explored (yield vs. purity, process fit)

• Resin screening
• Yield vs. Impurity resolution
• P=O and DMT-on impurities
• Buffer types
• Optimization
• Binding capacity, Wash and Elution conditions

AEX Overview: Development for first ASO

AEX evaluated as a polishing step

• Bind/Elute mode: High resolution of product-related impurities
• Removal of impurities very similar to product (P=O, N-1), DMT-on

2/24/2017 9

17 Biogen | Confidential and Proprietary

AEX Process & Platform Evaluation

• The ASOs had similar elution salt
• Biogen AEX process will work for the

ASO pipeline

• Removal of P=O and N-1 in wash
• Removal of DMT-on in strip
• ≥ 90% yield

18 Biogen | Confidential and Proprietary

AEX Chromatography: Platform Development Approach

• Binding capacity
• Wash conditions – yield vs. impurity removal
• Elution conditions – yield vs. impurity removal

Key Developments

 Step Gradient Approach

AEX Purification: ASO- B

• Impurities N+1, N-1, P=O can be partially removed.
• Similar process and performance for 2 ASOs

2/24/2017 10

19 Biogen | Confidential and Proprietary

UF/DF Overview

UF/DF as final formulation

• Replacing lengthy precipitation and lyophilization (6 hr vs. several days)
• Removal of residual small impurities

10 PSI 20 PSI 40 PSI 40 PSI

So = Cfiltrate / Cfeed

Membrane Screening

Membrane 1, PES Membrane 2, RC

20 Biogen | Confidential and Proprietary

UF/DF: Platform Evaluation

Design equations

• Loss during concentration

Loss 1

• Loss during diafiltration

Loss 1 expDV ∗

Process Yield Estimation

ASO-A ASO-B ASO-E So 0.006 0.004 0.007

• 3 kDa UF/DF deliver ≥ 90%

yield

MWCO = 3 kDa Filtrate Feed

2/24/2017 11

21 Biogen | Confidential and Proprietary

UF/DF

Diafiltration Concentration

• Good model agreement
• High overall yield, 95%
• Product concentration, pH, osmolality met target

22 Biogen | Confidential and Proprietary

Overall Process Performance: Pilot Data

• Overall downstream: high purity and acceptable yield (60-70%)
• Consistent platform performance across molecules

2/24/2017 12

23 Biogen | Confidential and Proprietary

Conclusions

• Assessment of platform feasibility using exploratory mapping approach
• ASOs effectively bind to HIC below solubility limit in (NH4)2SO4 solution
• Linear gradients for HIC and the AEX show similar elution profiles
• Simple, predictive detritylation kinetics.
• Sieving experiment as a quick tool for UF/DF performance
• Future ASO pipeline fits the purification platform
• Requires minor tweaks for each molecule
• Fast process development with reduced time and resources
• Future improvements to come.
• A robust aqueous purification process was successfully developed
• Orthogonal chromatography + UF/DF
• High yield and purity

How does it measure up?

24 Biogen | Confidential and Proprietary

Acknowledgements

Special Acknowledgements

• Ionis Pharmaceutical colleagues

ASO Synthesis Development

• Austen Ng
• Jim Yang
• Xianglin Shi

ASO Leadership

• Firoz Antia
• William Kiesman

ASO Analytical Development

• Jessica Stolee
• Ruiting Liang
• Hong Jiang
• Xiaoye Su
• Bing Guan
• Amy Moran
• Richard Smart