Characterization of therapeutic oligonucleotides by liquid chromatography

Journal Pre-proof Characterization of therapeutic oligonucleotides by liquid chromatography Alexandre Goyon, Peter Yehl, Kelly Zhang

PII:

S0731-7085(19)32877-8

DOI:

https://doi.org/10.1016/j.jpba.2020.113105

Reference:

PBA 113105

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

26 November 2019

Revised Date:

21 December 2019

Accepted Date:

8 January 2020

Please cite this article as: Goyon A, Yehl P, Zhang K, Characterization of therapeutic oligonucleotides by liquid chromatography, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113105

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Characterization

of

therapeutic

oligonucleotides

by

liquid

chromatography Alexandre Goyon, Peter Yehl, Kelly Zhang*

Small Molecules Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

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*CORRESPONDENCE: Kelly Zhang Phone: +1 650 467 8470 E-mail: [email protected]

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Characterization of therapeutic oligonucleotides by liquid chromatography

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Alexandre Goyon, Peter Yehl, Kelly Zhang*

Francisco, CA 94080, USA

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ur

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Graphical abstract

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Small Molecules Pharmaceutical Sciences, Genentech Inc., 1 DNA Way, South San

1

Highlights

  

Reviewed current status of therapeutic oligonucleotides, including different types, the FDA approvals, typical chemical modifications, delivery systems and common impurities. Reviewed the characterization of oligonucleotides by ion-pair reversed phase, ion exchange, mixed-mode chromatography, HILIC, SEC and 2DLC. Discussed the analytical challenges and future perspectives.

ABSTRACT

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Marketed therapies in the pharmaceutical landscape are rapidly evolving and getting more diverse. Small molecule medicines have dominated in the past while antibodies have grown dramatically in recent years. However, the failure of traditional small and large molecules in accessing certain targets has led to increased R&D efforts to develop alternative modalities. Therapeutic oligonucleotides (ONs) can accurately be directed against their ribonucleic acid

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(RNA) target and represent a promising approach in previously untreated diseases. Established automated synthesis of ONs coupled with chemical improvements and the

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advance of new drug delivery technologies has recently brought ONs to a heightened level of interest. The first part of the present review describes the different classes of oligonucleotides, namely antisense oligonucleotide (ASO), small interfering RNA (siRNA),

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microRNA (miRNA), aptamer and immunostimulatory ON, with a focus on their delivery systems relevant for future analytical characterization. The second part reviews the typical impurities in therapeutic ON products. The third part discusses the use of historical methods

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anion exchange chromatography (AEX), ion-pair reversed phase liquid chromatography (IPRP), mixed-mode chromatography (MMC) and recent analytical methodologies of hydrophilic interaction liquid chromatography (HILIC), two-dimensional liquid chromatography (2D-LC)

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mass spectrometry for the characterization of ASO and siRNA modalities. The effects of physiochemical properties of RPLC columns and ion-pair agents on ON separation are

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specifically addressed with possible future directions for method development provided. Finally, some innovative analytical developments for the analysis of siRNAs and their delivery materials to pave the way toward the use of multi-attribute methods in the near future are discussed.

Keywords: 2D-LC; AEX; HILIC; ion-pair RPLC; MMC; oligonucleotides

Abbreviations

2

Two-dimensional liquid chromatography

AEX

Anion exchange chromatography

Ago2

Argonaute 2

AMD

Age-related macular degeneration

Anti-miRs

Antagomirs

API

Active pharmaceutical ingredient

ASO

Antisense oligonucleotide

BNA

Bridged nucleic acid

CMV

Cytomegalovirus

CNET

N3‐(2-cyanoethyl)thymine

CpG

Cytosine-phosphate-guanosine

CPP

Cell Penetrating Peptides

CRISPR

Clustered regularly interspaced short palindromic repeats

DBAA

Dibutylamine acetate

DEAE

Diethylaminoethyl

DMCHA

N,N-dimethylcyclohexylamine

DMD

Duchenne muscular dystrophy

DMT

Dimethoxytrityl

DNA

Deoxyribonucleic acid

dsON

Double-stranded oligonucleotide

EMA

European Medicines Agency

FCS

Familial chylomicronemia syndrome

FDA

Food and Drug Administration

GalNAc

N-acetylgalactosamine

HAA

Hexylamine acetate

HFIP

1,1,1,3,3,3-hexafluoro-2-propanol

HILIC

Hydrophilic interaction liquid chromatography

HoFH

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1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol Homozygous Familial Hypercholesterolemia International Council for Harmonisation

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ICH

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HFMIP

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2D-LC

IP

Ion-pairing

IP-RP

Ion-pairing reversed phase liquid chromatography

miRNA

microRNA

LDL

Low-density lipoprotein

LNA

Locked nucleic acid

LNP

Lipid nanoparticle

LOQ

Limit of quantification

3

MS

Mass spectrometry

MMC

Mixed-mode chromatography

NMR

Nuclear Magnetic Resonance

ODN

Oligodeoxynucleotide

ON

Oligonucleotide

OPC

3-(2-oxopropyl)imidazopyrimidinone

PA

Propylamine

PEG

Polyethylene glycol

PLGA

Poly lactic-co-glycolic acid

PEI

Polyethylenimine

PDase-II

Phosphodiesterase-II

PMO

Phosphorodiamidate morpholino oligomer

PO

Phosphodiester

PS

Phosphorothioate

RNase

Ribonuclease

RNA

Ribonucleic acid

RPLC

Reversed phase liquid chromatography

SAX

Strong anion exchange

SEC

Size exclusion chromatography

siRNA

Small interfering RNA

SMA

Spinal muscular atrophy

SPOS

Solid phase oligonucleotide synthesis

SPP

Superficially porous particles

SPS

Solid phase synthesis

ssON

Single-stranded oligonucleotide

sVOD

Severe hepatic veno-occlusive disease

TEA

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Tributylamine Triethylamine

Triethylammonium acetate

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TEAA

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TBA

TLR9 WAX

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methoxyethyl

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MOE

Toll-like receptor 9 Weak anion exchange

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Introduction Traditional small molecule drugs are primarily composed of hydrophobic organic molecules and typically aim to deactivate or inhibit target proteins through competitive binding [1]. However, proteins that possess accessible binding pockets are estimated to represent only 2-5% of the protein–coding human genome [2]. Conversely, antibody-based drugs can bind with a high specificity to different types of targets or replace mutated or missing proteins but their size and stability can limit their use toward many potential disease targets [1]. The central dogma of biology stipulates that DNA makes RNA and RNA makes proteins, therefore the RNA or DNA represent promising upstream targets for previously undruggable diseases. Single-stranded ASOs and aptamers first entered clinical trials 30 years ago while

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double-stranded siRNAs were initiated 15 years ago [3]. Although only nine ON-based products have been approved by the Food and Drug Administration (FDA) to date, acceleration has been observed recently with 6 of these therapeutic ONs approved between 2016 and 2019. The recent clinical successes can be attributed to a better knowledge of the human genome acquired since 2000 [4] and the improvement of ON technology with i) the

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improvement of ON pharmacokinetic properties [5]; and ii) the development of innovative delivery materials and methods [6]. Synthetic therapeutic ONs are short and linear polymers

(DNA) sugars and phosphate groups.

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of nucleotides comprised of purine or pyrimidine nucleobases, ribose (RNA) or deoxyribose

Critically, plethora of impurities can be formed at various steps of the ON synthesis [7]. The

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main impurities reported in the literature are shortmers (n-1, n-2) [8–10], longmers (n+1) [11], residual amounts of phosphodiester (PO) ONs in phosphorothioate (PS) products [12] and abasic ONs [13]. AEX and ion-pairing (IP)-RP have been the gold standard liquid

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chromatography methods for the analysis of ONs [14]. The need to identify impurities and elucidate their structures via mass spectrometry has brought IP-RP to a next level over the last 20 years. Fundamental and practical aspects of AEX and RP are well-established.

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However, the increasing diversity of delivery materials and the need to identify a large set of impurities have challenged the predominant AEX and IP-RP modes. Mixed-mode

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chromatography (MMC), HILIC modes and 2D-LC methods have been developed in the last 10 years to address these challenges and unambiguously identify ON impurities. The present review aims to provide a comprehensive perspective of the fundamental and practical aspects of the analysis of ONs by the conventional AEX, IP-RP, and MMC methods, as well as innovative multi-dimensional LC/MS and HILIC methods. The first part discusses the different classes of therapeutic ONs, key chemical modifications to improve their pharmacokinetic properties, and the associated delivery materials. In the second part, the different chromatographic modes used for ON analysis are critically reviewed and possible future directions provided.

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1. Introduction to therapeutic oligonucleotides 1.1 Types of therapeutic oligonucleotides

Figure 1 shows the different types of therapeutic ONs and in particular those which have already reached the clinical trials. ASOs are single-stranded ONs generally containing 16 to 20 nucleotides (7-8 kDa) and designed to bind complementary RNA targets [16]. ASOs have their hydrophobic bases exposed to the solvent making them amphipathic molecules [17]. Following their binding to the RNA through Watson-Crick base pairing, ASO can regulate

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RNA modulation via two main mechanisms: RNA degradation or occupancy-only mechanisms [16]. Most ASOs in clinical development act by RNA degradation which is mediated via the ribonuclease (RNase) H1 or argonaute 2 (Ago2) endonuclease [17]. ASO ‘gapmers’ typically contain a central eight to ten base DNA gap which serve as a substrate for the RNase H [5]. Occupancy-only mechanisms include an alteration in RNA processing

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such as exon skipping which is used to restore the reading frame, the inhibition or enhancement of protein translation and interference of the RNA/proteins interactions [16,18].

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Small interfering RNA (siRNA) are double-stranded ONs usually containing around 21 base pairs and comprising an antisense strand which is the pharmacology active strand and a complementary sense strand which helps transport the antisense strand to the intracellular

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RNA endonuclease Ago2 [19]. Therefore, siRNAs predominantly act via RNA degradation. The two strands of a siRNA are not covalently attached and can separate or melt at high temperature, depending on the ON composition. siRNAs have their hydrophobic bases

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shielded from water in duplex structures making them more hydrophilic molecules [17]. Due to their relatively large size of around 13 kDa and hydrophilicity, it is difficult to achieve cellular uptake of siRNAs without any carrier or conjugation with a ligand [17]. microRNA

(miRNA)

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Endogenous

contains

approximately

18–25

nucleotides.

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development of miRNA therapeutics is a promising approach particularly in oncology where

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miRNA dysregulation is known to cause several types of cancers [20]. The dysregulation of miRNA can be addressed by miRNA therapy replacement either using mimic miRNA or inhibition of miRNA function by using anti-miRs [20]. Similar to siRNAs, mimic miRNAs are duplex ONs that aim to replace the loss of naturally-occurring miRNA due to disease whereas anti-miRs are single-stranded ASO-based molecules binding the complementary miRNA [20]. Next to the development of RNA therapeutics, ONs binding to other than RNA targets have also been investigated [21]. Nucleic acid aptamers are short (6-30 kDa), single-stranded (20 to 100 nucleotides) DNA or RNA molecules with a high affinity specific to a target [22]. Their

6

ability to form 2D and 3D structures makes them capable of recognizing various targets including metal ions, peptides, proteins and cells [22]. Aptamers can achieve superior specificity to antibodies in discriminating closely related molecules such as conformational isomers [23] or proteins containing a single amino acid modification [24]. Unmethylated cytosine-phosphate-guanosine (CpG) dinucleotides are common in bacterial DNA and they have shown immune-stimulatory effects by activating the toll-like receptor 9 (TLR9) [25]. Conversely, CpG motifs are methylated in mammalian DNA and their frequency is low [25]. To trigger an immune response, single-stranded CpG oligodeoxynucleotides (ODNs) have been developed with a PO backbone usually containing a least some PS modifications to resist nuclease degradation [26]. The immunostimulatory oligonucleotides have been

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investigated in clinical trials for cancer and allergic diseases [26]. Finally, it is worth mentioning the critical role of the RNA component which guides the Cas 9 endonuclease to the DNA in the well-known CRISPR/Cas9 genome editing technology [27].

1.2 Chemical modifications and delivery systems for ASOs and siRNAs

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ASOs and siRNAs are the most clinically advanced ONs, which is why this part of the review focuses on these two classes. To improve the stability of ASOs against nuclease

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degradation, their base, sugar, and inter-nucleoside linkages are usually modified, while the conjugation of small or large molecules to enhance delivery has recently attracted attention

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(Figure 2) [16,17].

Among the thousands of nucleic acid analogs of RNA and DNA that have been tested [17],

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Shen and Corey highlighted the chemical modifications that mostly impacted drug development [5]. The modification of PO linkages, which consist of the replacement of a nonbridging oxygen atom by a sulphur atom to form a PS linkage, is widely used to increase

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stability against nuclease digestion (to increase half-lives from minutes to days) [28]. The substitution of an oxygen atom by a sulfur atom introduces a chiral center and therefore the

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presence of two stereoisomers per PS linkage. Interestingly, it has been suggested that PS stereochemistry can affect the pharmacological properties and efficacy of ASOs. For example, the chirality of PS linkages has been shown to critically affect the metabolic stability in vitro and durability of in vivo response of an FDA-approved ASO [29]. Though standard solid phase oligonucleotide synthesis (SPOS) do not address the stereochemistry issue, the work of Iwamoto et al. published in 2017 provided a way for the synthesis of stereopure ASOs [29]. In 2018, a fruitful collaboration between the Scripps Research Institute and Bristol-Myers Squibb subsequently led to the development of a new class of P(V)-based reagents which also enabled the stereo-control of SPOS [30]. To further improve the stability

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of ONs against nuclease digestion, the nucleophilic hydroxyl moiety at the 2’-ribose position can be replaced by O-methyl (2’-OMe) or O-methoxyethyl (2’-MOE) groups, for example [31,32]. Finally, in order to increase the strength of Watson-Crick hybridization of ON analogs to RNA, the 2’ oxygen can be linked to the 4’ carbon of the ribose to form a bridged nucleic acid (BNA) [5]. Examples of BNAs used for ASO drug candidates investigated in clinical trials include the locked nucleic acid (LNA) sugars consisting in a 2’, 4’ methylene linkage and the 2′,4′-constrained ethyl nucleic acid ((S)-cEt) [17]. Of the seven ON products approved by the FDA between 2013 and 2019 (see section 1.3), only two siRNA products, namely Onpattro® and Givlaari™, involve the use of a carrier or a ligand. Conversely, ASO targeting organs other than the liver do not use either carrier or

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ligand. A major difference between the chemical conjugation vs carrier approaches relies in their delivery scale, from molecular to macromolecular scale with ligand-ON conjugates to nanoscale with lipid or polymeric carriers [6].

The use of lipid nanoparticles (LNP) as carrier usually involves the complexation of anionic ONs with cationic lipids and the addition of a neutral lipid such as cholesterol [6,33].

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Challenges associated with the use of LNPs include i) production of anti-PEG antibodies by the immune system, especially following multiple administrations, which compromise the

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long-term circulation [34–36]; ii) reduced uptake by the targeted cell due to the polyethylene glycol (PEG) coating used to limit non-specific interactions with plasma proteins in order to increase circulation time; iii) lack of specificity of the carrier to the targeted cell; and iv)

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accumulation of high amounts of potentially toxic cationic nanoparticles [37]. To increase the effectiveness of lipid carriers, PEG-stabilized LNPs [38] and lipid-like molecules [39] have been developed. To improve recognition by target cell populations, LNPs have been coated

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with antibodies [40], small molecules ligands [41] and, more recently, with aptamers [42]. Neutral liposomes have been also investigated since cationic lipids have been reported to be toxic when interacting with the cellular membrane [43]. Polymeric nanocarriers are widely

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used but they are less advanced in clinical phases than LNPs [6]. The use of poly lactic-coglycolic acid (PLGA) has been early reported [44] as well as other vehicles such as

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dendrimers or polyethylenimine (PEI) [6]. Ligand-ON conjugates can be divided into two categories: non-targeted conjugates vs targeted ones [6]. The ligand can be attached to the ON via a solid-phase approach or an insolution method [45]. Non-targeted lipid conjugates were earlier developed through conjugation of the oligonucleotide with cholesterol in order to enhance cell uptake but they lacked efficacy [6]. The conjugation of fatty acids to ASO has been shown to improve the pharmacokinetic properties of ASOs through non-covalent binding to the human serum albumin (HSA) [46] and can also enhance their potency in muscles [47]. The advantages of fatty acid conjugation can be extended to siRNAs to direct them against alternative targets

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than the hepatics cells (N-Acetylgalactosamine (GalNAc) conjugates) such as the lung, muscle or heart [48]. Cell-penetrating peptide (CPP) conjugates have also been tested with anionic ONs and uncharged ONs such as phosphorodiamidate morpholino oligomers (PMOs) [49], whereas their cellular targeting mostly relies on non-specific, charge-mediated interactions with cell membranes. However, R&D efforts in ligand-ON conjugates have primarily focused on the development of targeted conjugates. Most siRNAs currently evaluated in clinical trials use a ligand for delivery [17]. GalNAc is a moiety with high recognition specificity for the asialoglycoprotein receptor expressed by hepatocytes cells (6). Numerous ASOs chemically modified with the GalNAc moiety have been recently evaluated in

clinical

phases.

The

development

of

GalNAc-siRNA

conjugates

by

Alnylam

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Pharmaceuticals is considered to be a major breakthrough with several candidates now evaluated in patients [17]. In particular, GalNAc-siRNAs enable the use of siRNAs without the need of pro-inflammatory liposome formulations [50]. However, the termination of the Revusiran clinical phase 3 trial has evidenced some potential hepatic toxicity issues [17]. An alternative delivery approach involves the use of dynamic polyconjugates [51], but one

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pharmaceutical company stopped all clinical programs using the technology in 2016 [52]. Alternatively, nucleic acid aptamers form three-dimensional (3D) structures via intra-

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molecular base pairing and can theoretically bind to any receptors [6,53]. The aptamermediated delivery of ONs has been investigated for a plethora of therapies including direct killing of cancer cells [54,55], cancer immunotherapy [56,57], and HIV [58]. However, the lack

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of stability of the aptamer-ON conjugate against nucleases and/or low efficient internalization into cells remains to be tackled in order to convert the promise of aptamers into clinical success [6]. Alternative forms of macromolecular ON-ligand conjugates involve the use of

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serum proteins where the molecules are covalently linked [59]. Antibody-mediated delivery of ONs was first described by Song et al. in 2005 [60] but initially suffered from the formation of heterogeneous aggregates [61] and later from insufficient tissue penetration due to the large

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size of the THIOMABTM used [6]. Conjugation of ONs to peptides or small organic molecules has also been tested but suffers from several limitations, including i) change of the small

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molecule affinity to a target, once conjugated to the ON; and ii) rapid clearance of the relatively small conjugates by the kidney [6]. Finally, it is worth mentioning that the combination of RNA drugs with small molecule therapeutic enhancers has recently been shown to improve the pharmacological properties of ASOs and siRNAs [62] and could potentially pave the way for combination therapy.

1.3 FDA-approved oligonucleotides

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As of November 2019, the FDA approved a total of 9 oligonucleotide drugs as shown in Figure 3. The first two ON-based therapies approved in 1998 and 2004 aimed to treat ophthalmic diseases. Vitravene® was the first ON drug approved by the Food and Drug Administration in 1998 to treat cytomegalovirus (CMV) retinitis. Macugen® was then approved in 2004 for the treatment of age-related macular degeneration (AMD) of the retina. More recently, 7 ON drugs were approved by the FDA between 2013 and 2019 to treat a variety of rare and/or orphan diseases. Kynamro® was approved by the FDA in 2013 and is indicated for patients suffering from homozygous familial hypercholesterolemia (HoFH) which is characterized by very high plasma concentration of low-density lipoprotein (LDL) [3]. Exondys 51® was then approved by the FDA in 2016 for the treatment of Duchenne muscular

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dystrophy (DMD) after a controversial debate due to the limited number of twelve patients involved in the clinical trials [63] and lack of efficacy evidence presented at the FDA drug review meeting [64]. A polydisperse mixture of single-stranded oligonucleotides (ssONs) (90%) and double-stranded oligonucleotides (dsONs) (10%) (Defitelio®) indicated for the severe hepatic veno-occlusive disease (sVOD) was approved in the same year. Finally,

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Spinraza® was also approved in 2016 for the treatment of spinal muscular atrophy (SMA) in children. With worldwide sales of $1.7 billion in 2018, Spinraza® became the first blockbuster

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ON therapy [65].

Two additional ONs were approved for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis in 2018, namely Onpattro® and Tegsedi® [66]. Onpattro®

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is the first siRNA encapsulated in a lipid nanoparticles approved by the FDA. Tegsedi® is of the more common ASO genre. In addition, a 20-mer 2’-MOE ASO (Volanesorsen/Waylivra®) was approved by the EMA in 2019 for the treatment of Familial Chylomicronemia Syndrome

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(FCS) [67]. Finally, Givlaari™ was the first GalNac-siRNA product approved by the FDA in 2019.

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2. Impurities in therapeutic oligonucleotides ICH guidelines Q3A(R2) and Q3B(R2) classify nongenotoxic impurities as organic, inorganic,

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or residual solvents. Starting raw materials, inorganic impurities, and residual solvent are addressed by current ICH guidelines for small molecules whereas structural analogs of the API also known as organic product-related impurities are not specifically addressed. Productrelated impurities due to side reactions or storage can be classified into different categories based on structural similarities, e.g., modified inter-nucleotide linkage, sugar or base residue, shortmers, longmers and high molecular weight species [7]. This classification proposed by Capaldi et al. has the advantage to reflect the mechanism of impurity formation and it can therefore provide meaningful information on the efficacy of a particular synthesis step or control strategy [7]. Common impurities found in ssONs include the n – 1 shortmers [8–10], n

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+ 1 longmers [11] and presence of the PO impurities in PS ONs (12). Shortmers can be formed due to reaction failures during oligonucleotide synthesis with an incomplete base coupling followed by a failed capping reaction. Krotz et al. indicated the n +1 longmers were formed during a single coupling step by successive addition of two molecules of phosphoramidite [11]. PO impurities may form due to incomplete sulfurization or/and possible side reactions related to the reagent used [68]. Among the other classes of impurities, several different types can occur at the base residues, such as i) presence of a N3‐(2cyanoethyl)thymine (CNET) impurity, which can be formed by the addition of acrylonitrile to a thymine residue during the ammonium hydroxide deprotection step [69]; ii) deamination or depurination of adenine, cytosine, or guanine residues, which can be induced by thermal,

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basic, and acidic conditions [70,71]; and iii) oxidation, primarily occurring at guanine residues [70,72]. Conversion of a isobutyryl-protected guanosine base into an acetyl-modified diaminopurine moiety or the formation of N2-acetyl-2,6-diaminopurine impurity during the capping reaction have been also reported [73,74]. With double stranded siRNAs, analytical scientists should assess: i) the purity at the single strand and double strand levels; and ii) the

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residual amounts of single strands [75]. For conjugated oligonucleotides, such as the GalNAc constructs, the purity of the unconjugated oligonucleotide, conjugated oligonucleotide and

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free conjugating moiety should be assessed [76]. The main impurities in GalNAc-conjugated ON usually related to the GalNAc cluster itself may include the loss of one of the GalNAc sugar with its aminohexyl linker due to hydrolysis or ammonolysis, loss of one of the three

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cluster branches and loss of acetyl. Figure 4 shows the structures representative of common impurities found in oligonucleotide products. Detailed explanations on the formation of product-related impurities and their characterization can be found in comprehensive reviews

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[77–79]. In particular, Pourshahian published a thorough review in 2019 on the characterization of ON impurities by mass spectrometry with multiple insights on their formation mechanism [79]. In addition, a table was provided and summarized the average

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and monoisotopic masses differences produced by different impurities and degradants in

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comparison to the full-length product.

Pioneering works of Stec et al. using oxathiaphospholanes [80,81] and Iyer et al. using oxazaphospholidine derivatives [82] have enabled SPOS with defined PS stereoselectivity. In 2017, Iwamoto et al. further improved the oxazaphospholidine approach and developed a generic “stereo-controlled synthesis platform” which was successfully applied to produce Kynamro® with a defined PS stereochemistry [29]. The next challenge in ON characterization could well relate to the distinction of diastereoisomers known to potentially affect the pharmacological properties and efficacy of ASOs [29] but cannot be removed during the

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purification step. To the best of our knowledge, there is no reported method for the separation, or at least partial separation, of the 2n-1 diastereosiomers present in n-mer ONs with full PS backbone. In 2017, a consortium of pharmaceutical companies listed the main impurities usually found in therapeutic ONs and proposed identification and qualification thresholds for the product-related impurities [7]. Authors proposed to set a reporting threshold at 0.2% for ON impurities based on the limits of quantification (LOQs) found in the literature of 0.1% to 0.3% [83,84]. Impurities that are not specific to therapeutic ONs, such as residual solvents, elemental impurities, extractables and leachables are not included in this review; the same is true of

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genotoxic impurities such as acrylonitrile and dichloroacetic acid.

3. Analysis of oligonucleotide impurities by liquid chromatography

Table 1 lists the relevant analytical methods published over the last 15 years for the analysis

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of the impurities in single-stranded and double-stranded oligonucleotides.

3.1 Ion-Pair Reversed Phase Liquid Chromatography (IP-RP)

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3.1.1 Fundamental aspects

The effect of chromatographic parameters on the achievable peak capacity for PO ONs has been deeply studied by Gilar and Neue with model 15- to 60-mer ONs [101]. As predicted by

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theoretical calculations, higher peak capacities were experimentally obtained for shorter PO ONs (15-20 mer). A limited gain in resolution was evidenced when using longer columns, which was not proportional to column length, whereas the gradient slope has a major effect

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on resolution. For these reasons, the use of a short column should be preferred over sharp gradients for fast separation of PO ONs as shown in Figure 5. The effect of particle size has been shown to significantly impact the peak broadening of PO ONs due to their slow mass

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transfer (C term of the Van Deemter equation) [102]. The authors concluded that the particle size should be minimized and the mobile phase flow rate optimized in order to achieve the

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highest peak capacities [102] in agreement with another study aiming at separating impurities from PO ONs [71].

In 2016, Close et al. compared numerous RPLC silica-based columns packed with superficially porous particles (SPP) with different pore sizes and chemistries for the analysis of PO, PO/PS and PS ONs [86]. The pore size of SPP particles was shown to significantly affect the resolution of nucleic acids and a nominal value of 150 Å was found to be the most suitable for the analysis of >19-mer ONs. A smaller pore size of 80 Å provided the best results for shorter ONs such as those generated during RNase mapping experiments. An

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Accucore™ C18 column packed with 2.6 µm SPP particles (150 Å pore size) successfully separated the two diastereoisomers of an PO/PS ON containing a single PS linkage [86]. In 2014, Biba et al. evaluated a new core-shell particle SunShell C18 column, which was shown to provide similar chromatographic separation for PO ONs compared to other columns packed with SPP particles previously evaluated [103]. Better long-term column stability was reported with the SunShell C18 column with > 300 injections compared to < 200 injections with a Kinetex® C18 column when operating the columns at neutral pH and elevated temperatures (> 60°C) [103].

3.1.2 Stationary phase chemical properties

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C18 stationary phases have been predominantly used, in particular the XTerra MS C18 [87,88,102,104–106], Xbridge BEH C18 [71,88,97,107,108], and more recently the Acquity UPLC BEH C18 [84,94,97,98,100,101,109]. The choice of C8 and C18 sorbents has been shown in multiple literature reports to have limited effect on the separation of PO ONs according to their chain lengths [102,104,110–112]. Roussis et al. evaluated C18, C4, C30,

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phenyl, fluoro, and cyano stationary phases in 2019 and concluded on the absence of significant advantages from the use of C4 over C18 columns for the separation of common

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CNET, 3-(2-oxopropyl) imidazopyrimidinone (OPC), deaminated and n-1 impurities from PO or PO/PS ASOs [71]. PO ONs were less retained and resolved on polar stationary phases, e.g. fluoro and cyano, while they were more retained on columns modified with amide groups

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[71]. Another study indicated a better resolution of PO/PS siRNA duplex diastereosiomers on a cyano column with a mobile phase containing 0.2M TEAA while single-stranded denatured siRNA stereoisomers were better resolved on C18 and phenyl stationary phases [100].

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Acquity BEH, CSH and HSS UPLC® columns with different chemistries (phenyl, C8, C18, CSH fluoro phenyl and HSS cyano) were also investigated for the separation of PO/PS siRNAs from lipids [99]. Longer siRNA retention times were observed on phenyl or fluoro

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phenyl stationary phases likely due to additional π-π interactions according to the authors. Therefore, phenyl-based stationary phases successfully reduced the retention gap between

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the siRNAs and the lipids investigated. A similar increase of PO ON retention was also reported for a phenyl-hexyl column packed with SPP particles while better resolution was obtained on a column modified with an amide with a mobile phase containing 0.1M TEAA [103].

In these studies, it is important to keep in mind that differences were observed for PO ONs containing no or a limited number of PS linkages with mobile phases containing TEA which is a weak IP agent only partially covering the stationary phase, thus allowing hydrophobic interactions (see next section 3.1.3 for explanations).

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3.1.3 Ion-pair agents

Due to the very polar and charged nature of ONs, IP agents have to be added in the mobile phase to retain them on RPLC columns. However, their use limits MS sensitivity and robustness, therefore dedicated MS instruments are often needed. Common IP agents used for ON analysis are listed in Table 2. TEAA has been often used in the past but at a concentration of 100 mM which is not suitable for ESI-MS detection. Counterintuitively, the acetic acid component of the TEAA IP agent was identified as the principal component responsible for ion-suppression [112]. This effect was attributed to the preferential

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evaporation of TEA, leaving acetic acid in the droplets and reducing the ionization efficiency of the dissolved ONs [113]. Yet, a study showed that acetate still provided higher MS sensitivity in comparison to the bicarbonate and formate counter-anions [112]. This observation, which was not correlated with the volatility of the counter-anion but its conductivity instead, was described by a competition between the anions and the ON during

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the ESI process, at a roughly constant ion current generated by the ESI source. Only pH values higher than 9.0 provided high MS intensities, but the basic pH would not be

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compatible with the use of pure silica column particles [112]. To overcome the low MScompatibility of TEAA, Apffel proposed the use of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) in 1997 [113]. Of particular interest, Fountain et al. indicated a significant increase of MS

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signal intensity (by a 20- to 100-fold factor) when combining TEA with HFIP [105]. In addition, the combination of TEA/HFIP provided a better desalting of PS ON samples with fewer sodium adduct ions formed in comparison with a mobile phase containing only pure water

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[88,113]. Similarly, sufficient PS ON desalting and reasonable ionization efficiency were achieved with a mobile phase containing 15 mM hexylamine acetate (HAA) using an Orbitrap instrument [84]. It was found that the use of a low in-source energy helped to reduce the

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multiple HA+ adducts observed in the absence of in-source energy [84]. However, care should be taken when optimizing ESI source parameters to limit artificial in-source

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depurination or desulfurization [105,114]. In order to limit common sodium and potassium adducts which have a high affinity with PS ONs, it is possible to add a combination of imidazole and TEA in the dissolution solvent as shown in the past [115]. It is worth mentioning that HFIP is insoluble in acetonitrile, thus limiting the choice of the organic mobile phase to methanol during method development.

Several studies demonstrated that the use of TEA with HFIP was also more efficient for the chromatographic separation of ONs in comparison to the ammonium salt of TEA [87,101,102,106]. For example, the separation of native and chemically modified PS ASOs

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from their metabolites or failure products was significantly improved with the combination of TEA and HFIP [87]. Gilar et al. explained this as that HFIP enhances the ion-pairing efficiency of TEA due to the limited solubility of TEA in HFIP resulting in higher adsorbed TEA concentration on the RP stationary phase [87]. Since phosphorothioate diastereomers possess different hydrophobicity, the combination of TEA/HFIP can therefore reduce the peak band broadening resulting from the different hydrophobicity of the numerous diastereomers [87,88,105]. An increase in TEA or/and HFIP concentrations was shown to improve the peak capacity but the improvement did not strictly correlate with the concentrations of TEA [106]. Theoretically, the concentration of the TEA adsorbed on the stationary phase rather than in the mobile phase should affect the IP efficiency. Therefore,

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the limited solubility of TEA in HFIP was considered to affect the partitioning of TEA between the stationary phase and the mobile phase by enhancing the adsorption of TEA on the C18 sorbent [106]. Furthermore, the combination of hexylamine (HA) with HFIP has been shown to improve the LC and MS performance for a PO/PS siRNA sample in comparison to TEA/HFIP or HAA [94]. However, HAA still provided a robust separation of the n-1 shortmer

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from the single-stranded species. In another study, the use of 25 mM of HAA allowed the separation of partially truncated siRNA species with suitable MS sensitivity [97]. More

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recently, an alternative fluorinated alcohol 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) combined with DMCHA provided higher LC and MS performance for a 33-mer PS ON than HFIP [109].

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Short alkyl chain IP agents such as TEAA only partially cover the stationary phase and allow hydrophobic interactions between the stationary phase and the ONs [86,102]. As a result, the separation is based on both the size and the hydrophobicity of the ON when using TEAA.

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The use of low amounts (≤ 20 mM) of propyl-, isopropyl- and diethylamine provided suitable chromatographic separation of n-1, CNET, deaminated and OPC impurities from PO ONs [71]. A combination of 10 mM TEA and PA has been shown to allow the separation of n-1,

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n+1 and methoxy to hydroxyl swap at the 2’ ribose position for several denatured PO siRNA samples [98]. The modification extended the applicability of their conventional methods

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involving the use of a single IP agent but was not applicable for PS ONs [98]. However, Roussis et al. later reported the absence of significant improvement when combining IP agents due to the predominant effect of the most hydrophobic component [71]. Weak IP agents such as TEAA can resolve a limited number of diastereomers as demonstrated by Close et al. for a PO/PS ON containing a single PS linkage [86]. Strong IP agents were added to suppress the diastereomeric separation to prevent the co-elution of diastereomers when analyze other impurities of interest. To prevent a potential co-elution of the two diastereomers with single/double PO impurities and the n-1 shortmer, a strong IP agent (tetrabutylammonium bromide) and a combination of TEA and HFIP were used to elute the

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diastereomers as a single peak. Tributylamine is another mobile phase modifier widely used to suppress the unwanted resolution of diastereomers in PS ONs [83,86,116]. Finally, the six diastereoisomeric species of a denatured PO/PS siRNA could be distinguished on a BEH C18 stationary phase with a mobile phase containing 0.2 M TEAA at high column temperature of 80°C (separation of the sense and antisense strands) [100]. At the end, Roussis et al. proposed a desulfurization procedure adapted from the literature for PS ONs, which allowed using only weak IP agents in the mobile phase [71]. The procedure was shown to achieve a complete desulfurization without inter-nucleotide cleavage and limited overall degradation [71].

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3.1.4 Applications The degradation products from four different generations of PS ASOs subjected to pH and oxidative stresses were studied in 2018 by IP-RP/MS using an XBridge C18 column and a mobile phase containing 10 mM DMCHA/25 mM HFIP [108]. Shortmers, depurinated base residues, PO impurities were induced by acidic and basic stresses while desulfurization of

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PS linkages was observed after oxidative stress [108].

In 2010, an IP-RP method was developed for the separation and quantification of two PO/PS

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siRNA duplexes formulated in a liposome without sample pretreatment [107]. An XBridge C18 column heated at 60-65°C was used with a mobile phase containing 385 mM HFIP and 14.5 mM TEA. The denaturing conditions were required to disrupt the lipid layer and release

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the two encapsulated siRNAs denatured into four sense and antisense strands [107]. The authors indicated that the stability-indicating method met the requirement for QC analysis and was suitable for the characterization of impurities and determination of degradation

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pathways when coupled to MS [107]. Multicomponent LNP is an alternative carrier to deliver siRNA-based therapeutic to the targeted RNA. In 2019, Li et al. developed an IP-RP method including a sample preparation for the separation of a PO/PS siRNA and functional lipids in

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LNPs by IP-RP under non-denaturing conditions [99]. A BEH phenyl column was selected to reduce the retention gap between the ONs and lipids possessing significant differences in

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hydrophobicity. Column temperature was maintained below the on-column melting temperature of the siRNA duplex (70°C for most samples) to maintain a sharp peak shape. As expected, the nature of the alkyl ammonium agent had a significant effect only on the ON retention. DBAA (0.1M) was selected due to the better chemical purity in comparison to other agents. An application of the developed method was presented with the successful separation of the double-stranded siRNA, two different types of phospholipids, cholesterol, and a PEGylated short-chain lipid. Finally, LC-MS and NMR techniques were compared for the quantitation of a low trace amount of co-eluting single PO linkage impurity, which could not be separated from the full

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PS ON by IP-RP with a mobile phase containing 15 mM HAA [84]. A sensitivity of 0.3% (w/w) for the monophosphate substituted impurity was achieved by LC-MS and excellent agreement was found between the LC/MS and NMR methods on the amount of impurity quantified.

3.2 Anion Exchange Chromatography (AEX) AEX is an historical technique used to separate molecules based on their differences in local charge distribution. In 1979, Alpert and Regnier described the preparation of porous silicabased anion exchangers coated with polyethylenimine (PEI) for the separation of proteins and nucleotides [117]. Polymeric-based materials such as polystyrene [118] and

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diethylaminoethyl (DEAE) derivatives of Sephadex and cellulose [119–122] were first used for the separation of PO ONs but they suffered from low mechanical stability and poor sample resolution/recovery [123]. In 1983, a silica-based SAX column was shown to allow the analysis of larger PO ODNs (20 vs 15 and 10 nucleotides, respectively) within 30-min in comparison to the IP-RP and RPLC modes [110]. Drager et al. further improved the

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chromatographic performance of PEI coated silica-based material by synthesizing quaternized PEI supports. An optimal quaternization of amines by 40 – 60% improved the

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separation of PO ONs according to their chain lengths from 30 bases with unquaternized PEI coating [124] to 50 bases with the quaternized material [123]. In 1986, two porous silicabased columns bonded with DEAE were compared to polymeric columns bonded with DEAE

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and quaternary amines by Muller for the separation of DNA restriction fragments (7 to 650 pair bases) [124]. The polymeric Mono-Q® column bonded with quaternary amines was suitable for the analysis of large DNA fragments (more than 200 pair bases) and an increase

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of column temperature from 20°C to 60°C further improved the reproducibility of the method [124]. Following the successful application of non-porous ion exchangers for protein analysis, the first commercial non-porous AEX column was described in 1988 for the analysis of PO

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ONs [125]. Authors reported a significant reduction of analysis time needed for the high resolution analysis of a hydrolysate of polyadenylic acid up to a 70-mer [125]. In contradiction

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with early reports published in the 80s, in 1993 Cohen et al. indicated a better reproducibility and efficiency using commercial strong anion exchange (SAX) polymeric-based columns compared to silica-based materials [126] due to their ability to work at higher temperature (60-70°C) and the absence of secondary interactions with residual silanols [127]. Superior chromatographic separation was obtained with AEX in comparison to RPLC or IP-RP using tetrabutylammonium phosphate for the separation of the failure sequences from the full length 25-mer PS ODN; similar resolutions were obtained when using WAX or SAX columns [127]. In 1992, the performance of new non-porous AEX columns, including the DEAE-NPR and Pak Fax WAX columns functionalized with DEAE groups, and the NucleoPak PA100

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SCX column functionalized with quaternary amino groups was reported to improve the chromatographic performance in comparison to the former generation of AEX columns [128].

Novel polymeric-based AEX columns were subsequently commercialized in the 2000s. A polymeric-based Mono-Q® 10/10 column functionalized with quaternary amine groups was used by Yang et al. for the analysis of PO and PO/PS ON dithioates using NaCl or NaSCN salt gradients at pH 8.0 [129]. In 2005, Thayer et al. presented a new class of methacrylate polymer developed to improve the stability of the DNAPac™ PA100 (formerly called NucleoPak PA100) column made of methacrylate polymer, which were known to hydrolyze under alkaline conditions [130]. The novel DNAPac™ PA200 column improved the

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chromatographic performance for ssONs. However, a combination of acetonitrile and sodium perchlorate at pH 6.5 was still suggested by the authors to limit possible secondary hydrophobic interactions, which hamper the separation of impurities [130]. The choice of mobile phase pH in the salt-gradient elution mode was shown to impact the chromatographic performance by i) significantly improving the recovery of PS ONs at pH 12.4 compared to pH

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8.0, probably thanks to the inhibition of hydrogen bonding [128]; ii) alternative selectivity between pH 8.0 and 12.4; and iii) higher retention times for G- and T-containing ssONs at

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basic pH 12.4. It is worth noting that the use of non-linear curved gradients could further improve sample throughput according to the authors [128]. The kinetic performance of the DNAPac™ PA200 column was further improved with the DNAPac™ PA200 RS column

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packed with 4 µm particles. Finally, the separation of diastereoisomers was described in 2011 by Thayer et al. for a large PO/PS aptamer containing 37 bases and two PS linkages [93]. As shown on Figure 6, the four different possible diastereoisomers were partially

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aptamer [93].

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separated on a DNA Pac™ PA200 column and they were all eluted later than the full PO

3.3 Mixed mode chromatography (MMC)

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In 1982, mixed-mode HPLC phases with two or more dominant retention mechanisms occurring simultaneously were introduced and synthetized by Crowther et al. for the analysis of PO ON fragments [131,132]. They were followed by McLaughlin and Bischoff who modified a commercial aminopropylsilyl bonded-phase with different types of organic acids, each containing a hydrophobic moiety and an amine group [133]. The phenylalanine moiety bound to the commercial APS-Hypersil™ support via an amide bond (APS-PHE) was found the most suitable for the separation of two PO ONs with the same chain length but different hydrophobicity [133]. The authors further described two different approaches for the synthesis of mixed mode AEX/RPLC columns by binding hydrophobic moieties or ionic

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amines to commercial ASP-Hypersil™ and ODS-Hypersil™, respectively [134]. An alternative

AEX/RPLC

silica-based

stationary

phase

bonded

with

C8

and

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chlorobutyldimethylsilane was described in 1986 by Floyd et al. and applied for the characterization of single ssON and double-stranded DNA restriction fragments [135]. In 1991, the performance of a commercial Neosorb-LC-N-7R mixed-mode column (RPC-5 type) was compared to TSKgel® OligoDNA RP, TSKgel® DEAE-NRP and Shim-pack WAX-1 columns for the separation of polynucleotides [136]. The mixed-mode Neosorb-LC-N-7R column was found to achieve complete separation within 60 min of larger PO ONs than AEX (40-mer vs 30-mer) and PO ONs of similar sizes as RPLC [136]. More recently in 2013, Biba et al. presented the separation of PO ONs (including n-X

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deletion from the 5’ end) using commercial Sherzo columns with three different stationary phase chemistries providing low to high ion-exchange capacities (Figure 7) [90]. Chromatographic peak shapes were more suitable when using the SM-C18 column functionalized with C18, WCX and WAX groups. The use of a NaCl salt gradient significantly improved the chromatographic peak shape compared to an ammonium acetate salt gradient

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recommended by the column provider or a TEAA salt gradient. In 2014, Zimmermann et al. synthesized a RPLC/WAX stationary phase packed with 5 µm thiol-silica particles and

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covalently bonded with an N’-(10-undecenoyl)-3-aminoquinuclidine functional group [91].The mixed mode column was compared to the commercial Zorbax RRHD Eclipse Plus C18, Gemini C18 RPLC columns and to a synthesized silica-based WAX material for the

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separation of an PO ON from two potential impurities. The RPLC/WAX column was the only column suitable for the simultaneous separation of the three PO ONs by applying a TEAA salt-mediated pH gradient (pH comprised between 7 and 8) and adding 20% (v/v) acetonitrile

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in the mobile phase. Two pore sizes were compared (200Å vs 100Å) and due to the smaller surface area of the 200 Å material, the phosphate buffer concentration could be reduced by

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two (lower ligand density) [91].

3.4 Hydrophilic Interaction Liquid Chromatography (HILIC)

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HILIC is a technique of choice for the analysis of polar molecules and it has the advantage to be MS-compatible. In addition, HILIC can provide higher MS intensity compared to RPLC due to the higher amount of volatile organic solvent in the mobile phase. The first application of HILIC for the separation of ONs was presented by Alpert in 1990 for the separation of oligothymidylic acids with different chain lengths (from 12 to 30) on a polyhydroxyethyl A column using a TEA/phosphate buffer [137]. Later, Holdšvendová et al. synthesized hydroxymethyl methacrylate-based monolithic columns to separate PO ONs by capillary HILIC using TEAA [138]. The first HILIC separation of PO ONs using commercial columns, i.e., a diol-bonded stationary phase Luna® HILIC and a TSKgel® Amide-80 column

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functionalized with an amide group, were reported in 2010 [139]. Due to robustness issues observed with the Luna® HILIC column, the authors decided to select the TSKGel® Amide-80 column with a volatile mobile phase containing 5 mM of ammonium acetate (pH 5.8) for the analysis of polythymidylic acids of various lengths (10, 15, 20, and 30 nucleotides) [139]. Five commercial HILIC columns with different stationary phase chemistries (zwitterion, diol bonding, or non-bonded silica chemistry), namely the ZIC HILIC, YMC Pack silica, Luna® HILIC, Kinetex HILIC and ZORBAX HILIC Plus, were evaluated in 2011 for the separation of 15- to 30-mer PO ONs [140]. Authors mentioned the ZIC HILIC column was the only column to provide sufficient signal sensitivity for the analysis of a specific PO ON with a mobile phase containing 100 mM ammonium acetate at pH 5.8 [140]. Overall, the developed HILIC-

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MS method provided excellent MS sensitivity with an effective chromatographic desalting of alkali cations and detection limits in the picomole range [140]. Alternatively, the same authors reported another HILIC-MS analysis of PS ONs with detection limit of 50 nM [141]. In 2019, a recently-commercialized diol-bonded HILICpak VN-50 column was evaluated for the separation of DNA, RNA and PS ONs with a volatile mobile phase containing 15 mM

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ammonium acetate at pH 5.5 [89]. Ammonium acetate was preferred to ammonium formate due to better MS sensitivity and lower HILIC retention time. Similar LOQs were found for the

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HILIC and IP-RP modes despite the higher MS sensitivity using HILIC for all ONs studied [89]. Suitable column lifetime was reported (> 300 injections) but further development of the HILIC method was needed according to the authors to improve the peak shape in

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comparison to the IP-RP method [89]. Another HILIC column was successfully used in 2019 for the MS/MS quantification of a PO ON in plasma with a mobile phase containing 10 mM ammonium formate [142]. It is also worth noting the electrostatic repulsion hydrophilic

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interaction chromatography (ERLIC) provides an alternative selectivity to HILIC and may also be of interest for the analysis of ON as suggested by Alpert [143].

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3.5 Size Exclusion Chromatography (SEC) In theory, SEC separates molecules solely based on their difference in hydrodynamic radius.

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SEC and IP-RP have been compared by Noll et al. for the quantification of single-stranded species contained in a PO/PS siRNA sample [94]. Authors reported a good agreement between the two LC modes with a similar relative amount of the single strand measured. The same authors published another study with the Superdex 75 column to characterize the formation of impurities during the annealing of siRNA [95]. Slightly higher relative percentages of single-strand species were measured by IP-RP compared to SEC thanks to the better resolving power of IP-RP according to the authors. However, it is worth noting IPRP involves the use of denaturing conditions, which may result in artificially higher amounts of single-stranded species. SEC has been investigated by Shimoyama et al. for the

20

separation of higher order structures of PS ONs [96]. Secondary hydrophobic interactions between PS ON and the SEC stationary phase were evidenced. The addition of acetonitrile in the mobile phase limited the non-specific hydrophobic interactions but only a separation of the single from the double stranded ONs was achieved whereas high order structures (such as G-quadruplexes) were not separated. 3.6 Two dimensional LC/MS 2D-LC approaches can increase the peak capacity without sacrificing run time as they allow the fast on-line characterization of impurities separated by non-MS compatible techniques such as IEX. Off-line 2D-LC requires time-consuming fraction collection step while on-line 2D-LC is particularly appealing due to the automation of the different steps. On-line 2D-LC

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can be performed in the heart cutting, multiple heart cutting or comprehensive modes. With the comprehensive mode, fractions are collected during the entire 1D separation while only one fraction or a limited number of fractions are collected in the heart and multiple heart cutting modes, respectively. Therefore, the comprehensive mode provides the most detailed

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information but often requires tedious data treatment and the time constraint in the 2nd dimension can have deleterious effects on the chromatographic performance. An off-line comprehensive analysis of polyadenosine, thymidine, cytosine and uracil

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homodeoxyoligonucleotides of different sizes was performed by IP-RP x capillary gel electrophoresis (CGE) and AEX x CGE [144]. High effective peak capacities of 852 and 1474

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were obtained with the IP-RP x CGE and AEX x CGE modes, respectively. However, the use of the off-line comprehensive IP-RP x CGE and AEX x CGE modes is a time consuming approach and the use of CGE in the second dimension prevents the on-line MS identification

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of the species.

The location of the linkage in a RNA isomer was identified by Thayer et al. using a biocompatible LC system, consisting of a fraction-collecting autosampler and a column selection

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valve [145]. The authors reported the use of the phosphodiesterase-II (PDase-II) enzyme capable of cleaving 2′–5′ linkages to identify the location of the linkage in the RNA isomer [145]. The LC setup allowed the purification and the desalting of the AEX fraction for the on-

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line characterization of the PDase-II digests by MS [145]. A first comprehensive on-line 2DLC/MS method (HILIC x IP-RP) was reported in 2012 by Li et al. for the characterization of di- to deca- PO ONs [146]. The two dimensions were connected via a ten-port valve and ONs fractionated from 1D HILIC were trapped on the C18 cartridge by using an aqueous make-up flow to retain and focus the ONs on the 2D RPLC column head. An overall chromatographic peak capacity of 500 was obtained with the 2D-LC/MS setup. However, the use of 0.1M TEAA in the 2D IP-RP mobile phase at high flow-rate of 3.5 mL/min was likely to significantly hamper the MS sensitivity. The versatility of a commercial 2D-LC/MS system

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operated in the heart-cutting and comprehensive modes was presented by Roussis et al. in 2018 for the characterization of PO and PS ON impurities using 1D AEX, SEC, IP-RP or RPLC and 2D IP-RP [92]. Trapping columns were used instead of loops due to the fact that only 40 µL loop was available when the study was performed. The SAX x IP-RP mode allowed the separation of the “n+16” dithioate impurity resulting from the substitution of an oxygen atom by a sulphur atom in a dimethoxytrityl PS ON (Figure 8). However, the SEC x IP-RP mode did not provide significant additional benefits according to the authors.

4. Conclusions and future perspectives Breakthrough innovations in ON chemistry and delivery over the last 10 years have led to the

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rebirth of this class of therapeutics. In particular, the advance in ON technology has led to 6 FDA approvals between 2016 and 2019 out of a total of 9 ONs approved all time. The characterization of ONs by AEX, IP-RP and mixed-mode chromatography has been thoroughly reviewed. While in the past, most method development focused on the

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chromatographic separation, increasing efforts have been made to identify the impurities by MS. Since 2010, numerous IP-RP/MS methods have been published by pharmaceutical companies in order to identify ON impurities. Of particular importance, the choice of IP

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agents has been shown to be one of the most critical factors in IP-RP method development, and a combination of HFIP/TEA generally provided the most suitable LC/MS performance.

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A few online 2D-LC/MS methodologies have recently been published in order to identify the impurities separated by non-MS compatible methods such as AEX. Surprisingly, only a few publications reported the use of HILIC for the analysis of ONs despite some obvious

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advantages over IP-RP including the absence of IP agents and a higher amount of organic solvent beneficial for the ESI process. In addition, acetonitrile is an aprotic solvent which may also contribute to a better ON ionization in the negative ion mode compared to methanol

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which is required when using TEA/HFIP (due to the insolubility of HFIP in acetonitrile). For these reasons, 2D-LC methodologies involving the use of HILIC mode in the second

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dimension are also expected to rise in the near future despite some technical challenges related to solvent compatibility. In conclusion, multi-dimensional LC/MS methods and innovative strategies are anticipated to grow in the short term to better characterize and understand complex ON products.

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements The authors thank Dr. Stefan Koenig and Dr. Yuchen Fan of Genentech for reviewing the

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manuscript and their helpful comments and discussions.

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Figure 1. Classification of the different therapeutic oligonucleotide types according to their

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target. The names of the oligonucleotide types which have not reached the clinical trials yet

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ur

na

lP

re

are written in gray. Reprinted with permission from [15].

35

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Figure 2. Chemical modifications of antisense oligonucleotides and their effect on

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pharmacological properties and receptor affinity. Adapted with permission from [16].

36

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Figure 3. Therapeutic oligonucleotides approved to date by FDA. The type, main chemical modifications used and administration routes are shown below the name of the therapeutic

Jo

ur

na

lP

re

-p

oligonucleotide.

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Figure 4. Structures of the typical impurities found in ASO and siRNA samples.

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Figure 5. Fast UHPLC separation of PO ONs according to their size with a short BEH C18 column (50 mm × 2.1 mm, 1.7 μm) using a mobile phase A containing 15 mM TEA and 400 mM HFIP and mobile phase B combining mobile phase A and methanol (50:50, v/v) (A) and

na

(B) 15–35mer oligodeoxythymidine ladder. (C) 30–60mer oligodeoxythymidine ladder.

Jo

ur

Reprinted with permission from [101].

38

ro of -p

Figure 6. Separation of the four different diastereoisomers present in an aptamer sample containing 37 bases with two PS linkages. The full PO was also separated from the different

Jo

ur

na

lP

re

diastereoisomers on the DNAPac™ PA200 column. Reprinted with permission from ref [93].

39

Figure 7. Chromatographic separation of RNA oligonucleotide samples by mixed mode chromatography using a Scherzo SM-C18 column with a NaCl gradient. (a) Separation of nX shortmers and (b) separation of ‘base-flip’ isomer standards. Reprinted with permission

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ro of

from [90].

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Figure 8. 2D-LC (AEX x IP-RP) separation of a “n+16” dithioate impurity. Top panel: 1D AEX and 2D IP-RP separations for two AEX fraction collected between 10.0–11.0 min and 11.0–

lP

12.3 min. Bottom panel: MS spectra of components 1 and 2. The dithioate “[+S-O]” impurity was identified in the second fraction collected from AEX component 2 fraction. Reprinted with

Jo

ur

na

permission from [92].

40

Table 1. Relevant analytical methods published for the analysis of impurities in ssONs and dsONs

Modified inter-nucleotide linkage Diastereosiomers

Double-stranded oligonucleotide

Separation of single strands from duplex siRNA Shortmers Longmers Modified base

Denaturing IP-RP (two PS on the sense strand; one PS on the antisense strand)

[100]

re

Modified inter-nucleotide linkage Separation of lipids and siRNA in LNP

Reference [85] [71,86–88] [89] [90,91] [71] [90] [86] [85] [92] [93] [86] [94–96] [94,95,97] [95,98] [94,97] [98] [94] [95,98] [94] [94] [99]

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Modified base

Analytical method AEX IP-RP HILIC Mixed-mode IP-RP Mixed mode IP-RP AEX AEX x IP-RP AEX (two PS) IP-RP (one PS) SEC Non denaturing IP-RP Denaturing IP-RP Non-denaturing IP-RP Denaturing IP-RP Non-denaturing IP-RP Denaturing IP-RP Non-denaturing IP-RP Non-denaturing IP-RP IP-RP

-p

Single-stranded oligonucleotide

Type of impurity Shortmers

lP

Diastereosiomers

Table 2. Structure and chemical properties of mobile phase modifiers commonly used for IP-

1

logDpH 8.0 pKa

1

Boiling point (°C) 

-3.59

4.27

101

Acetic acid

-3.41

4.54

118

Triethylamine (TEA)

-0.93

10.21

89

Hexylamine (HA)

-0.56

10.21

131

ur

Formic acid

Structure

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Modifiers

na

RP analysis of oligonucleotides.

41

N,N-dimethylcyclohexylamine (DMCHA)

-0.21

10.22

160

Dibutylamine (DBA)

-0.17

10.75

160

1,1,1,3,3,3-Hexafluoro-2propanol (HFIP)

1.14

7.97

59

Tributylamine (TBA)

1.42

10.44

216

-p

1.47

8.08

61

Theoretical values of the pKa and logDpH8.0 were calculated using an online tool at https://chemicalize.com/.

Jo

ur

na

lP

re

1

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1,1,1,3,3,3-hexafluoro-2methyl-2-propanol (HFMIP)

42