Oligonucleotide Process Considerations and Controls

Agilent Confidential

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Joe Guiles, PhD

Agilent Technologies

PQRI-FDA , Rockville, MD

March 21-23, 2017

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Talk Outline

1) Oligonucleotide Overview

a. Research and Clinical Program Trends

b. Functional & Molecular Types of Oligo’s

c. Process Types

d. Process Models (Origin and Control of Impurities)

2) QbD Approach

a. Risk-based Criticality

b. Group or Individual Unit Op approaches

3) Discussion

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Oligo Therapeutic Research & Preclinical Programs Vs.Clinical Trials Programs

4232 38

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144 145 150 152165

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7971 68 65 70 69

86 83 84 91100 98

112 109

143133

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Number of Research & Preclinical Programs Number of Clinical Programs

From 2016 to 2017 oligo therapeutics had largest ever increase

in Research and Preclinical programs

Types of Oligonucleotide Therapeutics

Other Classes of

Oligo Therapeutics:

Antisense, Aptamers,

Decoys, Exon-Skipping

siRNA

miRNA

shRNA & mRNA

Synthetic Oligo Therapeutics RNAi Therapeutics

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TranscriptionTranslation

DNA

mRNA

mRNA

message

blockedPROTEIN

X

Double Strand siRNA molecule

enters RISC complex

RISC

siRNA’s integrated into RISC

complex bind to mRNA to block

translation

Non-synthetic

Molecular Types of Oligonucleotide Therapeutics

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miRNA (n= 8-16)

siRNA (n= 18-27)

Double Strand siRNA, Modified Single, Modified Double Single Strand,

miRNA

3’

5’

3’ or 5’, Saccharide,

Cholesterol, PEG, other

A C G T A C G T A C G T A C G T A C G T 21 FLP = Full Length Product

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Types of Oligo MFG Processes

Synthesis

Cleavage &

Deprotection

Ultrafiltration

Purification

All Solid Phase to FLP(natural or modified nucleotides)

(Single Strand)

Lyophilization

Synthesis

Cleavage &

Deprotection

Lyophilization

UF/Conc

Modified FLP (After SP)

Ultrafiltration

Purification

Conjugate

Synthesis

Cleavage &

Deprotection

Ultrafiltration

Purification

Duplex Anneal

Lyophilization

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Oligo MFG Processes

Solid Phase

Synthesis

Cleavage &

Deprotection

A C G T A C G T A C G 12

PG1Solid phase

polymer or glass bead

Purification

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Oligo MFG Processes

Cleavage &

Deprotection

Purification

Ultrafiltration

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Oligo MFG Processes

Ultrafiltration

Lyophilization

Purification

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Impurities: Origin & Removal

(3) Iso

(2) RI

(1) OoI

Synthesis w/

non-Nucleic

Acids

Cleavage &

Deprotection

Ultrafiltration

Lyophilization

Purification

Raw Material Impurities (synthesis impact)

Synthetic Impurities (side products)

Deprotection Impurities (side products)

Species with reasonable

Charge difference

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Impurity Types

Synthetic Impurities

(n-x), Shortmer, missing “n”(s)

(n+x), Longmer, extra “n”(s)

Deprotection Impurities

FLP(PG1), FLP(PG2), FLP(PG3)PG (protecting group)

FLP(Deg)Deg (degraded structure)

1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2014

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PG1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20FLP (n=20)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

DEG

1 2 3 4 5 6 7 8 9 10 11 12 13 Cap(n-x), Shortmer, capped

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Coupling Yield Perspective

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120 140

99.9% CY

99.5% CY

99.0% CY

98.5% CY

98.0% CY

97.5% CY

Relative Amounts of FLP vs. Oligonucleotide Length

Oligonucleotide Length (X)

Pe

rce

nto

f F

LP

(Y

)

Theoretical

Coupling Yield

(Y)

Example: 99% CY ~ 1% Impurities / (n)x

66.8%

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Removal of Impurities

Difficulty of Removal

(n-x), Shortmer, missing Base(s)

(n+x), Longmer, extra Base(s) AX

FLP(PG1)x, FLP(PG2)x, FLP(PG3)x

FLP(Deg)*

High Low

Imp R

em

oval

Similarity to FLP

X = 1

X = 2

X = 3 or >

AXX = 1

X = 2

X = 3 or >

* = unique

Process Property Summary

• Total Impurity Level is greatly impacted by chain length,

coupling yield and Deprotection yield

• Major impurities which remain in the FLP are similar

molecular structures, (n-1,n+1, PG1, Deg, etc)

• Removal of impurities to low levels (<10% total) is restricted

by similarity of structure and charge to that of the Full Length

Product

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Control Points & IP Controls

Temperature & Time Controllers

(2) HPLC

(Purity)

(1) HPLC

(Purity)

IPT

Raw Material Specs

Parameter control via Computer

Time/Flow/Conductivity/UV/pH/Temp

Parameter control via Computer

Time / Flow / UV

IPAC(acceptance

criteria

Yes

Yes

Synthesis w/

non-Nucleic

Acids

Cleavage &

Deprotection

Ultrafiltration

Lyophilization

Purification

Key Considerations of In Process Assays

• Use a Representative Sample

• Prepare & Handle Sample for Accurate Analysis

❖ Understand impact on analysis (e.g. degradation)

❖ Some Oligo matrixes require careful handling

• Assay type fits the need

• Review the data for soundness (integrity)

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❖ Consider and Implement in Development

Categorization of Process Parameters –Range Impact to Criticality

Operating Range

LowerFailureLimit

UpperFailureLimit

Acceptable Range

Critical

Parameter

Scenario: Parameters that impact CQA

Non-critical

Parameter

???

Operating Range

Lower

Failure

Limit

Upper

Failure

Limit

Acceptable Range

Scenario: Parameters w/minor impact to CQA

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Design Space Options

Synthesis

Cleavage &

Deprotection

Ultrafiltration

Lyophilization

Purification

Individual Unit Ops

Synthesis w/

non-Nucleic

Acids

Cleavage &

Deprotection

Ultrafiltration

Lyophilization

Purification

Groups of Unit Ops

Discussion Items

❖ Basis for Setting Impurity Specifications• Total Impurity Level is greatly impacted by coupling

efficiency, chain length and charge similarity to FLP Major impurities with high structure similarity to FLP are difficult to remove

• Removal of impurities to low levels (<10% total) is restricted

❖ Basis for defining criticality• Does the range D between PAR and NOR and PAR and

failure play a role in defining criticality of a parameter

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AcknowledgementsLori TroupTodd KreutzianBob GronebergJeff BeckvermitGary Carter

NASD Development Department

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Extra Slides

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Key Considerations of In Process Controls

• Use simple physical parameters (visual, computer triggered)

• Use IPC’s for Physicochemical parameters wherever

possible (pH, UV, Conductivity, FTIR..)

• Use in process tools (test strips, etc)

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