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Antisense Oligonucleotide Purification Platform: How does it measure up?

March 2, 2017

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

2Biogen | 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

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Introduction: Antisense oligonucleotides (ASOs)

o What are they?

o How are they made?

o Current process vs. Biogen’s platform

Structure and key impurities

Purification platform development

o Purification Strategies

o Evaluation of Biogen’s pipeline against platform

o Process development

Conclusions

Outline

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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 on 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)

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ASOs : Structure

1 235

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

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ASO Manufacturing Overview: Existing Process

Automated Solid-Phase Synthesis

grow chemically (3’ to 5’) on a resin bead

G G U C …

…one unit at a time…

Purification

1) Reverse- Phase Chromatography

2) Detritylation3) Precipitation4) 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

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ASOs : Key Impurities (Process-Related)

Chain-length Impurities• Failure sequences (N-x)• N-1• N+1

1 235

Modification Impurities• DMT-on

• P=O

• Abasic

DMT group not cleaved

S replaced by O in phosphorothiate bond

A base is cleaved off

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ASO Impurities: LC-MS Analysis

LC Chromatogram (Reverse Phase)

Mass Spectrum

Failure sequences

DMT-on

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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

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Development of ASO Purification Platform Process

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Variables explored (yield vs. purity, process fit)

• Resin screening

• Kosmotropic salt screening ammonium sulfate

• Leave DMT group on or off?

o Location of HIC before detritylation

• Optimization

o Binding capacity

o Wash conditions

o 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

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HIC ChromatographyTypical chromatogram: ASO-A

Load / chaseLoad / chase WashWash ElutionElution Strip / CIPStrip / CIPEQEQ

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00

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)

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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 Absorban

ce or Conductivity 

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

Linear (NH4)2SO4 gradient

Process Volume

ASO-AASO-BASO-CASO-DASO-EASO-F

Conductivity

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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 WashLC Data

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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: XW YX Z W YZ Z W X Y Z W W Y Z Z

3’ 5’

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

l

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Variables explored (yield vs. purity, process fit)

•Resin screening

• Yield vs. Impurity resolution

• P=O and DMT-on impurities

• Buffer types

• Optimization

o 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

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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

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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

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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, PESMembrane 2, RC

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UF/DF: Platform Evaluation

Design equationso Loss during concentration

Loss 1o Loss during diafiltration

Loss 1 exp DV ∗

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

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UF/DF

DiafiltrationConcentration

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

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Overall Process Performance: Pilot Data

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

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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?

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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