American preraaeeulleal review
`v. 13, no. & (Sept-Oct 2010)
`General Collection
`WiAMBSEM
`
`can Pharmaceutical
`
`VICW |
`
`The Review of American Pharmaceutical Business 6Technology
`
`MEDICINE Development Strategies
`
`PROPERTY OF THE
`NATIONAL
`LIBRARY OF
`
`AMERICAN PHARMACEUTICALREVIEW
`
`aneoa 13 ISSUE 6
`
`
` =|waaWIM) natyticalLaboratory
`
`rmaceutical Analysis
`
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`September/October 2010 | Volume 13, Issue 6
`
`R American Pharmaceutical
`
`COVER FEATURES
`kT 2 SRS ie
`
`12 Injectables
`Latest Developments in Injectable Drug Delivery
`Mathias Romacker, AmgenInc.
`:
`
`st a era
`
`28 Solubility
`Effective Formulation DevelopmentStrategies forPoorlySolubleActive
`Pharmaceutical Ingredients (APIs)
`David P. Elder, Ph.D., GlaxoSmithKline
`
`SLAS A ST
`
`RE SeLY
`
`XRDin PharmaceuticalAnalysis:A Versatile Toolfor Problem-Solving
`Cynthia S. Randall, William L. Rocco & Pierre Ricou, Sanofi-aventis US R&D
`
`2Sa A LESOR STE
`
`60 Chromatography-
`
`Informatics in theAnalytical Laboratory:Vision for a New Decade
`James M. Roberts, Ph.D., Mark F. Bean, Ph.D., Steve R. Cole, Ph.D.,
`William K. Young,Ph.D., & Helen E. Weston, GlaxoSmithKline
`
`
`
`September/October 2010 Review|7
`
`Page 2 of 13
`
`Page 2 of 13
`
`

`

`IN THIS ISSUE »
`ae
`111 SFC & LC/MS
`80 LYOPHILIZATION/
`UseofSFOMSiin thePurification of
`THERMAL ANALYSIs
`DevelopmentofFreeze-dried
`Achiral Pharmaceutical Compounds
`JenniferVan Anda, Ph.D.,
`Formulations Using ThermalAnalysis
`AstraZeneca Pharmaceuticals
`and Microscopy
`Vicky Ket Ph.D. , School of Pharmacy,
`Queen's University of Belfast
`
`116 RAMAN
`PracticalConsiderationsiinn Data
`Pre-treatment for NIR and Raman
`Spectroscopy
`Jun Huang, Ph.0., Saly Romero-Torres, Ph.D.
`& Mojgan Moshgbar, Process Analytical Sciences
`Group & Pfizer Global Manufacturing
`
`128 NIR
`PrimeronNIRSpectroscopy
`as a PATtoolin the Tablet
`Manufacture Process
`Maria Gerald Rajan, Ph.D.,
`Himanshu Bhattacharjee, Ph.D.,
`Sonia Bedi, Ph.D., & Joseph Reo, Ph.D.,
`Merck Consumer Care & University of Tennessee
`Health ScienceCenter, College of Pharmacy
`
`88 MICROBIOLOGY
`Introduction ofaRapid
`MicrobiologicalMethod as an
`Alternative to the Pharmacopoeial
`Methodforthe Sterility Test
`Jennifer C. Gray, Ph.0., Alexandra Staerk, Manfred
`Berchtold, Manuel Mercier, Gunther Neuhaus &
`Andreas Wirth
`
`95 PARTICLE SIZING
`The RoleofParticle SizeAnalysis.
`in the DevelopmentProcess of
`Nanosized Drug Products
`Jan Méschwitzer, Ph.D.,
`AbbotHealthcare Products B.V.
`
`102 UHPLis
`ApplicationofUltra--high
`PerformanceLiquid Chromatography
`for Chemical Characterization of
`Liposome-based Therapeutic Small-
`interfering RNA
`ZhongLi, Ph.D., Joe A. Schariter,
`Jingtao Zhang, Ph.D., Jared C Davis &
`Anthony M. Leone, Ph.D.,
`Merck Research Laboratories
`
`22 EXCIPIENTS
`Trendsiin| Pharmaceutical Excipients:
`An Update
`Chris Moreton, Ph.D., FinnBritt Consulting
`
`36 DISSOLUTION
`NovelAutomatedDissolution na
`Workflows Designed to Support
`PharmaceuticalScientists in
`Their Quest to Improve
`FormulationSelection
`Christopher John & Christina Bacci,
`Merck, Analytical Sciences, West Point, PA
`
`44 SINGLE-USE
`
`A Significant and Growing Market:
`Single Use Technologyin the
`BiopharmaceuticalIndustry
`Rick Stock, Ph.D. , BioProcess Technology Consultants
`
`68 HOT MELT EXTRUSION
`Detection ofPhaseSeparation in
`Hot Melt ExtrudedSolid Dispersion
`Formulations: Global vs. Localized
`Characterization
`LSheng Qi & Duncan QM. Craig,
`Schoolof Pharmacy, University of East Anglia
`
`75 TOC
`OnlineTotal OrganicCarbon Analysis
`for Cleaning Validation
`RiskManagement
`Keith Bader, Hyde Engineering + Consulting, Inc.
`
`SPECIAL FEATURES
`
`136 AAPSPreview & Company Profiles
`
`REGULAR FEATURES
`
`148 IndustryNews
`159 Classified Advertisements
`
`160 Advertiser's Index
`
`8 | RAVE | Seprembertoctober 2010
`Page 3 of 13
`
`Page 3 of 13
`
`

`

`
`
`
`
`Racial
`Sia
`
`intemational
`DrugDisccvery
`How to Accelercte Acceptance
`
`
`New
`Biomarkers?
`From Bids
`Utility of microRNAs
`ix ecet
`Imaging Based Assays
`
`set) eemncmm
`
`Pharmaceutical
`Outsourcing ‘temas cme:
`
`American Pharmaceutical
`
`Latest Developments in
`Injectable Drug Delivery
`Effective Formulation
`DevelopmentStrategies
`XRDin Pharmaceutical Analysis
`Informatics in the Analytical Laboratory
`
`
`
`American Pharmaceutical Review,
`Pharmaceutical Outsourcing, and
`International Drug Discovery bring the most up
`to date reviews, news, trends and comments
`to an ever changingindustry. With their
`Peri luculelsavecceceAe Rel
`MeecheeuAClmuMoelleccmelSoka
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`eyetinea inia iicea(ueMOLLIaLe
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`emily.johnson@russpub.com
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`

`

`UHPLC >
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`
`revolutionizing biomedical research and drug development. The use
`
`has become a major focus of industrial and academic laboratories.
`
`therapeutics is the efficient systemic delivery of siRNA to the target
`cells. Liposome-based formulations in lipid nanoparticles (LNPs)
`
`vivo delivery of siRNA. The complex nature of liposomal formulations
`
`Application of Ultra-high Te
`Performance Liquid
`eeeaaeteieae
`ChromMatog fd phy fol
`ofsmall interfering RNA (siRNA)asatargetvalidationtool andtherapy
`Che mM ica | Ch ad fa cte rizati 0 n of One of the primary challenges in realizing the full potential of siRNA
`Liposome-based Therapeutic have become the most promising and widely used strategy for in
`Sma | |-j nterferi n g RNA
`containing duplex siRNA oligonucleotides and multiple functional
`
`OicayiicovetMm Oua4CHT
`
`Zhong as Ph.D.*, aolWa NeACHR
`Jingtao Zhang, Ph.D., Jared C. Davis
`& Anthony M. Leone, Ph.D.
`RUBCMeeee eeauataue eCelEat
`
`a great challenge to the successful
`lipid excipients presents
`physicochemical evaluation of the siRNA LNPs. Ultra-high performance
`liquid chromatography (UHPLC) playsa critical role in characterizing
`the chemical properties of the LNP formulations. The high-throughput
`and superior separation efficiency offered by UHPLC enables the rapid
`and reliable determination of RNA,lipids, and related compoundsina
`complex formulation matrix. In this review article, an overview of the
`application of UHPLC for the chemical characterization of siRNA LNP
`formulations will be provided. Strategies for utilizing UHPLC potency
`andstability-indicating methodsfor siRNA oligonucleotides andlipids
`analyses will be described and future prospects will also be discussed.
`
`Introduction
`
`Discovered by Fire and Mello in 1998 [1], RNA interference (RNAi) is an
`endogenous processin which thetranslation of specific target messenger
`RNA (mRNA)in a cell is silenced in the presence of small complimentary
`RNA sequences which bind the mRNA and activate the RNA silencing
`complex (RISC). Targeting of exogenous RNA sequencesto cell cytosol
`also activates RISC and presents substantial opportunity as a therapeutic
`and target validation tool.
`Industrial and academic communities have
`quickly capitalized on the discovery and adopted RNAi as a powerful
`target validation tool and a promising new therapeutic modality (2, 3].
`As the primary focus of research into the utilization of RNAi as a therapy,
`siRNA (small interfering RNA) is a chemically synthesized RNA duplex
`and is generally delivered by nonviral delivery systems [4, 5]. Because
`of its poor pharmacological properties including serum instability, urine
`excretion, and inefficientcell delivery, effective and safe systemic delivery
`
`102 | Review | September/October 2010
`
`Page 5 of 13
`
`

`

`© KINETEX
`
`Revealing New
`
`Core-Shell Phases
`
`Find out whattheyare
`and get a specialoffer!
`www.phenomenex.com/reveal
`
`Phenomenex products are available worldwide. Email us at international@phenomenex.com.
`
`Page6 of 13
`
`PA82580910_L1
`
`Page 6 of 13
`
`

`

`UHPLC »
`
`of siRNA to the target tissues has become the major hurdle in realizing the
`full potential of RNAi as drug therapeutics. Among various approaches
`to overcomethe challenges, cationic liposome-based formulations in
`lipid nanoparticles (LNPs) represents the most promising and widely
`used strategy for in vivo delivery of siRNA [6,7]. A distinct advantage of
`the cationic LNPsis their ability to greatly improve the pharmacokinetic
`properties of synthetic oligonucleotides administered systemically.
`Efficient entrapment of the highly hydrophilic and negatively charged
`duplex siRNA oligonucleotides inside positively charged nano-particles
`protects siRNA from enzymatic degradation leading to significantly
`improved biodistribution and intracellular delivery [8, 9].
`
`the active
`as
`In addition to duplex siRNA oligonucleotides
`pharmaceutical
`ingredient, an siRNA LNP formulation generally
`consists of multiple lipids that play an importantrole in defining the
`LNP functions [Figure 1]: acationiclipid (CL) toenablethe encapsulation
`of oligonucleotides and the binding of LNP to anionic cell surface
`molecules;
`a “stealthy” pegylated-lipid conjugate (PEG-lipid)
`to
`control LNP particle size during formulation and prolong its systemic
`circulation time; and neutral “helper”lipids including a phospholipid
`to enhance the fusogenicity and cholesterol to minimize thermotropic
`phase transitions. A thorough physicochemical evaluation of LNP
`is essential for the siRNA liposomal formulation development and
`optimization. Furthermore, as RNAi
`therapies advance from pre-
`clinical to clinical development, demandfor rigor in CMC definition of
`the oligonucleotide and delivery componentsrises. To assure product
`quality and process capability, more advanced analytical methodology
`for the analysis of siRNA LNPs is evolving [10].
`
`In order to accomplish the full characterization of these complex
`formulations, multiple analytical
`techniques have typically been
`employed including chemical analysis, particle size analysis, and
`morphological analysis [8]. Rapid determination of RNA andlipid
`contents
`enables
`efficient
`formulation development, process
`optimization and manufacturing control. More important, reliable
`stability-indicating methods are required to ensure the purity and
`stability of the LNP products usedin the preclinical safety andclinical
`studies. High performance liquid chromatography (HPLC) is the primary
`tool for the chemical characterization of LNPs. The complex nature
`of liposomal formulations containing siRNA duplexes and multiple
`functional
`lipid excipeints presents a significant chromatographic
`challenge. Both siRNA oligonucleotides and lipids can degrade by
`various pathwaysincluding hydrolysis, oxidation, and desulfurization,
`potentially generating numerous impurities and degradates in the
`LNP formulations. The need to detect and quantitate RNA and lipids
`related degradation products has placed great demands on the
`chromatographic technologyfor the analysis of siRNA LNP. Ultra-high
`performanceliquid chromatography (UHPLC), which offers significant
`advantages over HPLC in terms of sample throughput and resolving
`power,is an excellent alternative for the analysis of siRNA LNPs.In this
`review article, an overview ofthe application of UHPLC for the chemical
`characterization of siRNA LNP formulationswill be provided. Strategies
`for developing UHPLC potencyassays and stability-indicating methods
`for RNA and lipids analyses will be described and future prospectswill
`also be discussed.
`
`
`
`UHPLCin Pharmaceutical Analysis
`Since the first commercial ultra-high pressure liquid chromatograph
`system was introduced in 2004, UHPLC has gained increasingly
`popularity and wide acceptance across many industries. Although it
`has yet to replace conventional HPLC systems as the work horse in the
`pharmaceutical laboratories, UHPLC has been implemented in many
`segments of drug discovery and development. UHPLC technology
`promises significantly faster separations in addition to increased
`chromatographic resolution and enhanced “mass”sensitivity. Specially
`plumbed UHPLC hardware offers low overall system dispersion for
`improved separation efficiency, small delay volume enabling rapid
`column re-equilibration for gradient analysis, fast detector response
`for narrowly eluting peaks. These benefits of UHPLC make it well
`suited for fast and ultrafast potency assays without significantloss of
`separation efficiency [11]. Normally, 4-10 folds of increase in speed can
`be obtained using UHPLC. Consequently, high laboratory productivity
`desired to speed up the drug discovery and pre-clinical development
`can be realized with UHPLC, ultimately lowering the ever-increasing
`drug developmentcostsin today’s pharmaceutical industry. Besidesits
`capability to achieve fast and ultrafast separation, another significant
`advantage of UHPLCis its capability of solving the most challenging
`separation tasks in pharmaceutical analysis. By offering much higher
`operating pressure limits (up to 1200-bar) compared to standard HPLC
`(400-bar), UHPLC allows the use of sub-2 um (STM) particle columns
`of lengths up to 150 mm to achieve very high separation efficiencies
`and increased sensitivity, resulting in considerable benefits in high-
`resolution LC analysis since the STM columns provides much higher
`theoretical plate counts than the conventional HPLC columns packed
`with 3-5 um particles. Moreover, when combined with high column
`temperature and column coupling, UHPLC can achieve ultra-high
`resolution separation for the analysis of very complex mixtures, where
`the high separation efficiency often far outweighs the selectivity
`optimization due to the presence of multiple critical pairs needed to
`be resolved chromatographically [11]. For example, more than 100,000
`plates were generated for heptanophenone in under 12 min with
`three linked C18, 2.1 x 150mm, 1.7m columns operated at 90 °C and
`14,100 psi [11]. The unique characteristics of UHPLC makeit especially
`suited for the analysis of siRNA LNP products as demonstrated in the
`following sections.
`
`
`
`Analysis of siRNA Oligoneucleotides
`BEeeI ne ccrae
`siRNA is a class of double-stranded RNA (dsRNA) molecules, 21-23
`nucleotides(nt) in length, with 2-nt 3’ overhangs on both passenger
`strand (PS) and guide strand (GS) [Figure 1]. Among manyanalytical
`techniques utilized for the analysis of oligonucleotides including
`radiolabel tracer methods, ELISA methods, gel electrophoresis-based
`methods, and HPLC, strong anion-exchange LC (SAX-LC) and ion-
`pairing reserved phase LC (IP-RP LC) with UV-visible detection at 260-
`nm have been the standard techniquesfor the analysis of therapeutic
`
`104 | Review Saptember/October 2010
`Page7 of 13
`
`Page 7 of 13
`
`

`

`Rl.ssi.shimadzu.
`Nexera UHPLC IsHere!
`At 19,000 psi, Shimadzu’s New Nexera
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`is the World’s Only No Compromise UHPLC
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`Shimadzu’s passion has always
`been to improve performance,
`ease of use and flexibility forall
`HPLC environments. That passion
`continues with Nexera, the next era
`in UHPLC, which offers the reliability,
`versatility, and superior basic
`performancefor all applications.
`
`Nexera features:
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`@ World’s fastest injection
`(10 second cycle time)
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`@ Near-zero carryover
`(0.0015%withoutrinsing)
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`H@ Maximum resolution with the
`widest pressure/flow range
`(130MPa up to 3mL/min)
`
`® Excellent injection reproducibility
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`i” Modular design enables numerous
`system configurations
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`Andthelist goes on!
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`ruggedness and reproducibility,
`oe
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`Page8 of 13
`
`Page 8 of 13
`
`

`

`UHPLC »>
`
`
`
`'
`
`siRNAsas a result of HPLC’s dominant presence in pharmaceutical labs.
`SAX-HPLCis a traditional technique for nucleic acid analysis and most
`commonly used for the identification and potency determination
`of siRNA duplex in LNPs against placebo siRNA sequences and/or
`undesirable non-hybridized single-stranded RNAs
`(ssRNA) which
`are often associated with a decrease in therapeutic potency. SAX
`analysis is normally performed using non-denaturing mobile phases
`and low separation temperature (< 40 °C) to maintain the integrity
`=
`I
`of non-covalent siRNA duplex throughoutthe analysis. Although the
`y Ohyk f
`ididii
`P aa
`:
`developmentofsmall particle SAX-LC columnshas laggedsignificantly
`
`
`behind the ones for RP-LC, the first commercial STM SAX columns : Resoaei I sits
`
`has been introduced very recently [12]. A preliminary evaluation of Bwindi BeIeS
`the column on the UHPLCin our lab has produced promising results,
`,
`!
`PLEAIAA.
`OF ORR
`indicating a potential significant improvementin resolution and speed
`1
`See
`PEG-DMA
`compared to the commonly used SAX HPLC columns as shownin
`Seerid iveetaieimegelMaaartis Racaliadged
`Figure 2. Three of four siRNAs with very similar structures were seen
`of example siRNA and functionallipid excipients. Cationiclipid:
`co-eluting on a 25-cm, 8-m SAX-HPLC column in a 20-min separation
`| DLinDMA(1,2-DiLinoleyloxy-N,N-dimethylaminopropane); Helper
`lipid: DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine); PEG-
`while a rapid separation with much improved resolution was achieved
`i
`lipid: PEG-DMA[Poly(ethylene glycol) dimethacrylate]. (Notes:
`Cholesterol structure not shown;Asterisks near the 3’ end of siRNA
`denoting phosphorothioatelinkages.)
`
`12,3
`
`|
`
`2
`
`14
`
`18
`
`18
`
`with a 5-cm STM column on UHPLC in less than 2 minutes.
`.SS T10
`
`UV-visAbsorbance
`I min. (B) SAX-UHPLC: 4.6x50mm,1.7 jim; 85-90%Bin 1.5-min; strand scission dueto oxidation,leading to truncated oligonucleotides (es eee
`
`Compared with SAX-LC for oligonucleotide analysis, IP-RP LC offers
`higher separation efficiency and compatibility with mass spectrometry
`(MS), and has increasingly become the methodofchoice for impurity
`profiling of siRNAs. Conventional
`IP-RP separation methods for
`oligonucleotides are based on the use of triethylammonium acetate
`(TEAA) containing mobile phases at pH 7.0. Mass transfer in the
`stationary phase was found to be a major factor contributing to peak
`broadening on porous C18 columnsfor analysis of oligonucleotides
`[13]. Small particle size, elevated temperature and a relatively slow
`flow-rate have been recommendedto maximize separationefficiency.
`Consequently, STM columns combined with UHPLC would provide
`significant advantages over IR-RP HPLC. Benefits of IP-RP UHPLC for
`Time (minutes)
`siRNA analysis including improved resolution and productivity have
`Figure 2. Comparative SAX-LC/UV chromatogramsof a mixture
`been demonstrated with Spproninistely four-fold increase of sample
`eeenteesheeaoe MPoe
`throughput compared totraditional IR-RP HPLC methods [14].
`ElelerAtmierieeetle ies)Ba amet
`*
`‘
`.
`ee
`Major sources of siRNA netsted impurities in LNPs are often the eenee eeeee
`result of multiple degradation pathways such as hydrolysis of the
`(A) SAX-HPLC: 4.0x250mm, 8 jim; 0-100%B in 20-min; 1.0-mL/
`phosphoester bond and the N-Glycosidic bond, cleavage of bases and
`
`i
`
`ass
`
`: tein
`
`[(N-x) impurities] [15]. Additionally, chemical modifications to siRNA
`backbone, sugars, and/or bases are usually made to enhance the
`performanceof siRNA, including increased serum stability, decreased
`off-target effects, and reduction of undesired immune stimulation.
`These modifications have made analysis and characterization of
`siRNAs more challenging than their native counterparts. For example,
`:
`integration of phosphorothioate (P-S) backbone linkages by the
`;
`Eecere
`i
`substitution a sulfur atom forone ofthe non-bridging oxygen atomsin
`
`each phosphodiester bond provides exonuclease resistance (Figure 3), ::es
`However, P-S backbone has been foundto be susceptible to oxidation
`i
`9
`and can undergo desulfurization to generate the phosphodiester (P-
`hd
`0) form which undermines siRNA drug in vivo stability. Therefore,it is
`lw
`critical to have a stability-indicating method which is specific against
`ps
`the P-O degradates as well as the (N-x)
`impurities. Furthermore,
`sulfurization of oligonucleotide backbone creates multiple new
`diastereometers, causing substantial peak broadening in IP-RP HPLC
`
`E
`
`cnARA
`i
`
`Base
`
`we
`é
`
`Gase
`
`i
`
`i
`:
`
`106 | Review | September/October2010
`
`Page9 of 13
`
`Figure 3. Schematic representation of a phosphorothioate
`(P-S) backbonelinkage and its desulfurization to form a P-O
`
`degradate.
`
`Page 9 of 13
`
`

`

`
`
`Gs
`
`PS
`
`«& UHPLC
`
`[
`
`analysis [16]. Chromatographic resolution of P-S diastereomers can
`be difficult or impossible to resolve by the conventional IP-RP HPLC
`method using TEAAion-pairing system [16].
`
`
`
`
`:
`|
`|
`hall
`ald 8,No .
`||
`
`:
`ad
`
`>2
`
`i
`2
`f
`&
`5
`
`2
`
`1
`
`I
`
`
`Figure 4. Comparative denaturing IP-RP UHPLC separationof an
`
`Tet hel ieteesulaurumesladle alte
`
`
`seeruCMN eeeeectimeaCELL
`Mee Bess eemCeeUeCULE ce
`PeGSR PAUP deuscelUnOM ok ee)
`
`
`min; ~ 6k psi. (B) C18, 2.1x400mm, 1.7um; gradient 10 to 20%Bin
`35 min; ~ 12k psi.
`a
`
`th i ee ct Sis beastie
`
`
`Direct impurity profiling of siRNA duplex is particularly challenging
`because of the small differences in size, charge and hydrophobicity
`between the full length duplex, ssRNAs, and impurities, which often
`co-elute. Therefore, IP-RP LC analysis of impurities in siRNAsis typically
`performed at elevated column temperature to fully denature the
`I
`|
`|
`|
`|
`|
`|
`duplex and generate two single-stranded counterparts. Figure 4
`
`rer l \ 8)ft
`illustrates the IP-RP analysis of a P-S siRNA on UHPLC utilizing in-
`He UM WAIL
`:
`ah
`-
`;
`situ denaturation of the siRNA duplex at 80 °C. Resolution of two GS
`“x er r
`|
`10
`|
`Time (minutes)
`diastereomers and four PS diastereomers were achieved on a 15-cm
`STM column within 20 mins. To further take the advantages of very
`high operatingpressure limit offered by UHPLC, column-coupling (40-
`cm) was employed to fully resolve the ssRNA peaks from their (N-x)
`impurities. The improved resolution achieved with a longer column
`provided for increased method robustness to ensure the detection
`andreliable quantitation of potential new impurities and degradates.
`The IP-RP UHPLC method with TEAA IP buffer is suitable for the
`impurity profiling of siRNA products as a routine process control
`and product
`release. However, monitoring degradation profiling
`in the stability testing can be challenging due to the presence of
`numerous diastereomer peaks for both full
`length RNA and (N-x)
`
`
`J\4 A4.
`impurities. IP aqueous buffers composedof triethlyamine (TEA) and
`hexafluoropropanol (HFIP), pH 7.9, and methanol have been found
`PS
`&
`Gs
`
`2
`|
`to tend to suppress the diastereomeric resolution, and therefore, are
`more suitable for stability testing of P-S oligonucleotides [16-17]. In
`P-O degradate|P-O degrad
`addition, the TEA/HFIP/methanol based mobile phases do not cause ion
`
`|
`\
`i
`
`\
`suppression and are more appropriate for LC/MSanalysis of unknown
`degradation products. Figure 5 compares the IP-RP separation
`achieved on HPLC and UHPLC with TEA/HFIP/methanol system under
`the fully-denaturing condition (80 °C). The figure illustrates that
`the UHPLC method provides significantly improved resolution and
`sensitivity for detection of potential degradation products including
`P-O and (N-x) degradates compared with the HPLC method. Separation
`performance of UHPLC makesit an ideal chromatographic technique
`for the analysis of therapeutic P-S oligoneucleotides.
`It should be
`pointed out
`that chromatography techniques alone, even with
`extremely high separation efficiency achieved with UHPLC, cannot
`solve all separation problems and unequivocally identify degradation
`products. LC coupled with electrospray ionization mass spectrometry
`(ESI-MS) has increasingly being used as an important characterization
`tool for oligonucleotides, enabling identification of process-related
`impurities and degradation products [16-18]. UHPLC coupled with MS
`to deliver superior LC performance for optimal LC/MS characterization
`of siRNAs,is especially suited for the quantitation of P-O degradates.
`The mass difference of 16 Da betweenall P-S oligonucleotides and
`their monodiester forms can be readily detected using deconvoluted
`ESI-MS signal [16].
`
`
`Figure 5. Comparative denaturing IP-RP LC separation of an
`TNEMesMa eeeele RSM Cetteee ea
`
`mM HFIP, pH 7.9. Mobile phase B: methanol; UV detectionat
`=|
`260nm; Column temperature: 80 °C. (A) HPLC: 4.6x150mm, 31m;
`
`| gradient 17 to 25%B in 30 min; 1.0-mL/min (B) C18, 2.1x300mm,
`;
`1.7m; gradient 17 to 25%B in 30 min; 0.6-mL/min.
`
`
`iat
`isles Slain
`se
`=
`
`|
`
`
`
`CADResponse
`
`Time (minutes)
`
`
`
`
`
`Figure 6. Comparative LC-CAD chromatograms of an siRNA
`eerout wsakerRUS RGM UtellHaharated ia
`
`in water; Mobile Phase B, 0.19%TFA in IPA; (A) HPLC: C-18,
`| 4.6x50mm,1.8 1m; 60-90%B in 20-min; 1,0-mL/min.(B) UHPLC:
`
`=
`| C8, 2.1x 50 mm,1.8 pm; 70-100%B in 3-min. Peaks: 1, PEG-lipid;
`
`2, cholesterol; 3, phospholipid; 4, cationic lipid.
`
`
`
`Page 10 of 13
`
`September/October 2010
`
`Review
`
`107
`
`
`
`
`
`SS
`
`PS
`
`
`
`=
`
`(Nex),
`
`\
`
`TI
`1000
`
`200
`Time (minutes)
`
`TF
`
`T
`
`-
`
`oO
`
`\ T
`
`>5
`
`4
`
`
`
`
`Page 10 of 13
`
`

`

`UHPLC »
`
`
`Analysis of Functional Lipid
`Excipientsby UHPLC
`
`Unlike the commonly used excipients in the conventional oral and
`parenteral formulations, the lipid composition as well as siRNA/total
`lipids ratio are the critical parameters to define the bio-performance
`of the siRNA LNPs. Therefore, development of reliable analytical
`methodsfor thoroughly unraveling the composition offunctional lipid
`excipients and related impurity profile is essential in the development
`of LNP formulations.
`
`The diversity in structural features along with the distinct hydrophobic
`and hydrophilic elements makeslipids one of most difficult group of
`substances to analyze. PEG-lipids can be particularly challenging due
`to their polydispersity as a result of molecular-weight distribution.
`Compared to RNA,one additional challengefor lipids analysis is that
`mostoflipids lack chromophores that mightfacilitate the use of the
`standard UV-vis detector for LC. The situation has madethe use of a
`universal HPLC detection technique necessary such as the ones based
`on aerosol particles produced via nebulization including evaporative
`light scattering detector (ELSD) and charged aerosol detector (CAD).
`Unlike ELSD which correlates the amountoflight scattered with analyte
`concentration, CAD utilizes a measurementof charge thatis carried by
`the analyte to an electrometerfor correlation [19]. Compared to the
`ELSD forthe lipid analysis, the CAD has been demonstrated to offer
`a more uniform responsecurve over a wider range of concentrations
`(over 4 orders of magnitude) with better accuracy, precision and lower
`limits of detections(typically an order of magnitude better than ELSD)
`[20-22], which are the critical attributes for HPLC methodsto quantitate
`low levels of impurities in pharmaceutical products. In addition, CAD
`has been found to be more user-friendly since it does not require any
`optimization or operating parameters to be set, except for the output
`range, while the ELSD requires some optimizationin nitrogen pressure,
`drift tube temperature, and nebulizer temperature in orderto attain
`maximum sensitivity [23]. Since the introduction ofthe first commercial
`CAD in 2004,
`it has been widely used throughout pharmaceutical
`and biopharmaceutical R&D. An HPLC-CAD method was recently
`developed and found suitable for the detection oflipids in liposomal
`formulations [24]. A comparison of HPLC and UHPLC separation for
`the analysis of lipid composition in an siRNA LNP product is shown
`in Figure 6. Compared to HPLC, a gain in analysis speed by factors of
`10 is realized withoutsacrificing the separation efficiency. In addition,
`the much sharper peak shape obtained in the UHPLC chromatogram,
`especially for the PEG-lipid, makes the peak integration more robust,
`leading to improved accuracy in quantitation.
`
`There are potentially several mechanisms by which the lipid
`components in an siRNA LNP formulation can undertake to generate
`degradates which will compromise the product stability and bio-
`performance. Free-radical-initiated lipid peroxidation is a potential
`route of degradation for the cationic lipid (CL) containing unsaturated
`linoeyl hydrocarbon chains such as DLinDMA (Figure 1) and can
`produce a variety of complex degradation products. CL with a tertiary
`amine head group may also undergo two-electron oxidation to form
`N-oxide. Hydrolysis is the primary degradation mechanism for ester-
`
`108 | Review | septemberOctober 2010
`
`Page 11 of 13
`
`containing lipids such as DSPC (Figure 1), which will produce lyso-
`phosphatidylcholine(lyso-PC) andstearic acid, resulting in a lower pH
`of the formulation.It is essential that a stability-indicating method for
`lipids is specific against all of these potential degradation products
`in addition to the synthetic impurities of the lipids such as the CL
`isomers. Figure 7 shows an impurity separation under conventional
`HPLC conditions with a 3-m particle, 4.6x150mm column, and
`the same separation performed via UHPLC using a 2.1x150 mm
`column with 1.7-um particles. Exceptional resolution ofall potential
`degradation products and synthetic impurities was obtained with the
`UHPLC method within 30 mins. Although no reduction in run time
`was achieved compared to the HPLC method, the high efficiency of
`the UHPLC separation allowed the increased resolution between the
`DLinDMAperoxidation degradates and stearic acid as well as closely
`eluting DLinDMA isomers and N-oxide peaks, which elute right after
`the parent peak.
`
`3
`
`‘
`mH Ny
`ae/ .
`ews
`3
`
`4
`A
`ff
`
`DLINOMA
`perondative
`Georadates
`
`7
`
`5
`
`(8 A
`
`5
`
`CL isomers
`
`'
`
`7
`
`=
`
`A
`Hogi
`
`\
`
`AKA
`
`|
`|
`|
`|
`|
`
`2
`e
`|
`&
`°
`<
`oO
`
`0
`
`
`5, CL; 6, CL N-oxide; 7, DSPC.
`
`4
`
`‘4
`B
`i
`\
`«lhe
`/
`oe
`\
`4 i (haTe
`|
`\
`WL ee
`\
`
`cee
`:
`hi rac
`6
`20
`25
`30
`5
`0
`Time (minutes)
`
`|
`|
`E
`
`I
`EE
`
`|
`
`ateWelleReeeuelscurtiesii) eee)
`Tred eeROeG teleaeeeERCee lelle
`Phase B, 0.1%TFA in THF; 50-75%B in 30-min. Column temperature
`@ 60°C. (A) HPLC: C-18, 4.6x150mm,311m; (B) UHPLC: 2.1x150mm,
`1.7m; Peaks:1, lyso-PC; 2, Stearic acid; 3, Cholesterol; 4, PEG-lipid;
`
`
`
`Conclusion
`
`Gene silencing by RNA interference has shown great promise in
`biomedical research and therapeutic applications. The development
`of safe and efficient in vivo systemic delivery systems remains as a
`primary hurdle for realizing the full potential of siRNA therapeutics
`and necessitates a thorough physicochemical characterization of
`siRNA LNPs. Ultra-high performance liquid chromatography plays
`a critical role in characterizing the chemical properties of the LNP
`formulations. In comparison to traditional HPLC, the high-throughput
`capability offered by UHPLC along with UV-vis and charged aerosol
`detectors enable the accurate and rapid potency determination
`of siRNA oligonucleotides and functional
`lipid excipients in the
`chromatographically challenging sample matrices,
`leading to
`greater overall laboratory throughput and efficiency. The superior
`resolving power of UHPLC providesfor the reliable impurity profiling
`
`Page 11 of 13
`
`

`

`
`
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`MRM? webinar:
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`
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`For Research Use Only. Notfor use in diagnostic procedures.
`©2010 AB SCIEX. The trademarks mentioned hereinare the property of AB Sciex Pte. Ltd. or their respective owners, AB SCIEX™js be used underlicense.
`
`Page 12 of 13
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`Page 12 of 13
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`

`

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`10.
`
`11.
`
`12.
`13.
`
`Dr.