Impurity profiling and in-process testing of drugs for injection by fast liquid chromatography

Journal of Pharmaceutical Analysis 2012;2(S):372- -377Contents lists available at ScienceDirectPharmacouticalJournal of Pharmaceutical AnalysisPA3011www .elsevier.com/locate/jpawww.sciencedirect.comORIGINAL ARTICLEImpurity profiling and in-process testing of drugsfor injection by fast liquid chromatographyMarie Josee Rocheleau*,1, Elaine Larouche', Cristina Salamu, Mihaela CurcaOmega Laboratories Lld., 1177 Hamon, Montreal, QC H3M 3E4, CanadaReceived 4 January 2012; accepted 11 July 2012Available online 24 July 2012KEYWORDSAbstract Liquid chromatography (LC) is considered by many as a mature technique. Nonetheless.LC technology continues to evolve driven by the need for high-throughput and high- resolutionLiquid chromatography;analyses. Over the past several years, small particle size packing materials have been introduced byUPLC;Pharmaceutical;several column manufacturers to enable fast and efficient LC separations. Several examples ofTaxanpharmaceutical analyses, including impurity profiling of taxanes and atracurium besylate,Atracurium besylate;in-process testing of peptides in injectable dosage form, using sub-2 μm column technology alPeptidespresented in this paper, demonstrating some of the capabilities and limitations of the technology.。2012 Xian Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved.1. Introductionind speed of analysis have made the use of modern liquidchromatography (LC) very popular in the pharmaceutical indus-Ease of use, adaptability to a wide range of detectors, availabilitytry. Liquid chromatography is typically employed throughout theof a wide selection of stationary phases, its high resolving powerentire pharmaceutical development process, from drug discoveryto raw material analysis, impurity profiling, stability studies andfinal product testing [-3]. The development of LC methods for*Corresponding author. Tel: +1 4186511716x213;analysis of drugs and their related substances is typically al time-fax: +1 4186514847.ming process in an analytical laboratory, Therefore, separa-E-mail address: marie josee.rocheleau@ endoceutics.comtion scientists are continually driven towards the use of more(M.-J. Rocheleau).'Now at Endoceutics Inc, 1405 Boulevard du Parc Technologiquc,efficient LC technology to speed up the method development andvalidation process, as well as productivity in quality controlQuebec City, QC GIP 4P5, Canada.2Now at Groupe Parima, 4450 Cousens, Montreal, QC H4S 1X6,laboratories. Eficiency and speed of analysis become particularlyCanada.important while performing in-process pharmaceutical esting. aPeer review under responsibility of Xi' an Jiaotong University.necessary中国煤化工，achive high qulitstandards_nufacturing [4.5]Over tMHC N M H Gals have evolved fromirregular shaped 10 μum silica particles to small spherical particles,LUSEVIER | Production and hosting by Elsevierwhich are now less than 2 pum in diameter [6]. Spherical small2095-1779 。2012 Xi'an Jiaotong University. Production and hosting by EIsevier B.V. All rights reserved.oiorg/10.1016/jpha 20207.004Testing drugs for injection by fast liquid chromatography373particles can be packed more uniformly and homogeneously than3. Results and discussiontheir irregular shaped counterparts, thus providing better effi-ciency and resolution for chromatographic separations.3.I. Impurity profiling of drug substances and products withHowever column permeability tends to decrease significantly,sub-2 um LC columnswhich is accompanied with an increase in column back pressure[1,7]. High back pressure requires use of high-pressure LImpurity and degradation profiling is an integral part of thesystems allowing operation that exceeds 400 bars. The use ofpharmaceutical quality control of drug substances and pro-much shorter LC columns ( < 50 cm in length) at low flow rates,ducts. Impurity profiling often requires the use of longer LCor the use of high separation temperatures to reduce mobilecolumns, along with a mobile phase gradient [9,10]. A typicalphase viscosity may alternatively be used to reduce backexample involves the impurity profiling of an analog ofpressure, often at the cost of adequate separation efficiencyTaxol⑧requiring the use of a 250-mm YMCTM ODS-AQ[6,8]. Although short columns with small particles provide rapidcolumn with a water/acetonitrile gradient, to achieve theLC separations, the column plate number is not always suficientchromatographie separation of the taxane drug substanceto allow complex multi-components analysis. In this paper,from its many impurities [1]. Such a challenging separationseveral examples of pharmaceutical analyses, including impuritymay take upto 90 min per sample injection, including mobileprofling of taxanes and atracurium besylate, in-process testing ofphase gradient and column re-cquilibration.peptides in injectable dosage form, using sub-2 um columnUse of conventional LC is excessively time-consuming andtechnology will be presented, demonstrating some of the cap-could not practically be used for process testing duringabilities and limitations of the technology.pharmaceutical manufacturing, which typically requires rapidtesting methodologies. A Waters Acquity UPLC8 BEH C18column with 1.7 um silica hybrid particles was used advanta-2. Materials and methodgeously to achieve a 7-fold reduction in analysis time (Fig. 1).An equivalent gradient separation was achieved in 10 min withThe experimental results reported in this paper were obtained ona sub-2 um column [12], as compared to the 75 min run timeWaters Acquity UPLC& (Milford, MA, USA) systems withwith the conventional 3 μm ODS-AQ column. Such a shorteither a Photodiode Array Detector or tunable UV (TUV)analysis run time with UPLC is much more compatible withdetector. The first example of application of a sub-2 μm columnimpurity profiling while performing in process testing.with a high pressure LC system to drug compound impurityFor a typical analysis sequence requiring replicate injectionsprofiling involved the use of an Acquity UPLC BEH C18,of blank, standard (for system suitability), and sample solu-100 mmx 2.1 mm column with 1.7 μum hybrid silica particlestions, this represents a significant reduction in analysis time(Waters, Milford, MA, USA). The second example involved aand total volume of mobile phases consurmed: 2.8 h of analysisZorbax Eclipse XDB-C18, 50 mm x 4.6 mm column with 1.8 μmtime for 12 injections and a total volume of mobile phases ofparticle size (Agilent Technologies, Santa Clara, CA, USA).100 mL using the Acquity UPLCmethodology, as comparedAnalyses of peptides were performed on an Acquity UPLC8to 18h of analysis time and more than 1 L of mobile phaseBEH C18, 50 mm x 2.1 mm column with 1.7 um hybrid silicausing the conventional LC method. This represents not only a .particles, an Acquity UPLC8 BEH 300 C18, 50 mmx 2.1 mmsignificant gain in efficiency, but also a significant reduction incolumn with 1.7 um hybrid silica particles with 300 A pores, andoperating costs for solvents used to prepare mobile phases anda Kinetex@ CI8, 100 mm x 2.1 mm column with 1.7 um core-a reduction in waste disposal.shell particles from Phenomenex (Torrance, CA, USA). High-Another example of a challenging LC separation using aquality HPLC grade reagents and solvents were used throughoutsub-2 μm column involves the impurity and degradationthis study. Mobile phases and samples were filtered on 0.2 umprofiling of atracurium besylate injection. Atracurium besylateGHP membrane and syringe filters (Pall Corporation, Portis a muscle relaxant used in anesthesia [13]. This moleculeWashington, NY, USA).has four chiral stereocenters and may occur in 10 different0.100.08 10.06之0.04翳0.02 .器中国煤化工0.00MYHCNMH G46101214TIme (min)Figure 1 Example of chromatographic separation of a taxane from known impurties and degradants on a Waters Acquity UPLCRBEH C坍門数据(100 mm x 2.1 mm), using a water/aceonitrile gradient at a flow rate of 0.6 mL/min and UV detection at 228 nm.374M.-J. Rocheleau el al.stercoisomeric forms, which makes impurity and degradationsomatostatin [19]. Separation of octreotide acetate from theprofiling particularly challenging. Atracurium besylate injec-phenol preservative in a multi-dose formulation was achievedtion is typically composed of 55.0 60.0% of the cis- -cis isomer,using a Waters Acquity UPLC BEH C18 column, in34.5- -38.5% of the cis -trans isomer, and 5.0 -6.5% of the :combination with a rapid mobile phase gradient allowing intrans- -trans isomer [14]. The most biologically active isomer isprocess analysis to be performed in less than 3 min (Fig. 2).the cis- cis isomer, also known as cisatracurium besylate [15].The unidose formulation contains mannitol and sodiumThe LC methodology in the United States Pharmacopeiaacetate buffered with acetic acid at pH 4.2; the multi-for atracurium besylate describes use of a 250 mm x 4.6 mmdose formulation additionally contains 0.5% of phenoloctadecylsilane base-deactivated column with a 30 min mobilepreservative.phase gradient at 1.0 mL/min [14]. However, we have demon-Results from the dosage of the cyclic octapeptide instrated that separation of the main isomers of atracuriumrepresentative batches of several formulations and strengthsbesylate can be readily achieved on a much shorter column,are detailed in Table I. Results from the Acquity UPLC50 mm x 4.6 mm with a 1.8 um C18 stationary phase, in onemethodology and conventional LC methodology involving athird of the time with excellent resolution [16]; the resolution250 mm x 4.6 mm YMCM ODS-AQ column and a phospho-between the trans- -trans, cis trans, and cis cis isomers inric acid- acetonitrile gradient are compared. Although suffi-Fig. 2 is superior to 2.4. In addition to being more efficient,ciently sensitive, inaccurate dosing of the octapeptide at lowthe dimensions of the Eclipse XDB column allow the fast LCdosage strength was systematically observed with the Acquitymethodology for atracurium besylate to be operated on aUPLC methodology. While providing assay values consistentconventional system since the maximum column back pressurewith conventional LC at 200 and 500 μg/mL, inaccuracydoes not exceed 300 bars. However, care must be taken togrcater than 15% was observed in the peptide assay atminimize the internal volume of the system to reduce band50μg/L with a result lower than the specification limit [16].spreading and optimize peak resolution. It is important toDespite evident gain in efficiency, inconsistencies in assayminimize connecting tubing as part of instrument optimiza-values did not allow use of the UPLC methodology for intion, selecting the narrowest diameter and shortest lengthprocess testing of octreotide acetate formulation.tubing to prevent peak dispersion and loss in resolution.Non-linear response is not uncommon in the analysis ofAtracurium besylate injectable solution is refrigerated topeptides and proteins. On-column system adsorption is parti-extend its shelf-life, as it degrades when exposed to roomcularly common in the analysis of proteins by capillarytemperature for just few hours. The solution is also particu-clectrophoresis [20]. The use of low pH, high ionic strengthlarly sensitive to solution alkalinity [17,18]. Major degradantsbuffers, and a higher sample concentration or injection volumeof atracurium besylate include carly-eluting laudanosine and anay help reduce the effect of adsorption on the analysis ofmonoquaternary acrylate of laudanosine besylate [18], whichpeptides and proteins by LC. While higher dosage strengths atelutes near the end of the mobile phase gradient. In Fig. 2, all200 and 500 ug/mL are diluted by a factor of 4 and 10degradants eluted within 8 min allowing rapid assay andrespectively, with 0.1% aqueous phosphoric acid. the lowdegradation profling. While laudanosine elutes at a relativedosage strength at 50 ug/mL must be injected without furtherretention time (RRT) of 0.27 with respect to cisatracuriumdilution at pH 4.2 to maintain adequate sensitivity. Adsorp-besylate, monoquaternary laudanosine besylate elutes at RRTtion of octreotide acetate from the low-strength unidose1.26 after the cis cis isomer of atracurium besylate. Therefore,formulation，injected at a higher pH， may explain thethis methodology may not only be sucessfully applied to ininaccurate dosage results obtained by UPLC. Use of a largeprocess assay of atracurium besylatc, but also to the drugpore Acquity UPLC BEH 300 C18 column did not helpproduct degradation profling.improving the accuracy of the measurement at low-dose ofoctreotide acetate, with measured inaccuracy of 12% ascompared to the theoretical concentration [16]. The low3.2. Use of sub-2 pum column technology for in process testinginternal volume of small particle size columns does not allowof drugs for injectioninjection volumes above a few microliters without riskingoverloading the UPLC column. Use of conventional LC withIn process testing after compounding and formulating dosagea 50 uL injection volume proved to be more reliable for theforms is becoming a common practice in manufacturing ofdetermination of the low-dose peptide formulation.drugs for injection. Testing prior fitration and vial fllingInaccurate measurement of the main impurity of the anti-allows in process determination indicating whether or not theplatclet peptide drug eptiflbatide was also observed using afinal products will meet specifications, allowing process opti-Phenomenex Kinetex CI8 sub-2 μm column [16]. In Fig. 3. themization and concentration adjustment to maintain high-impurity eluting immediately after the main peak cannot bequality drug manufacturing. Speed of analysis is particularlydetected when injected alone at a concentration of 0.5 ug/mL.important while performing in process testing， since therepresenting a concentration equivalent to 0.1% of the samplemanufacturing process must be interrupted until results fromconcentration of eptifibatide. Injected along with eptifbatide.assay of the drug formulation are obtained. In processthe same i中国煤化工ame concentration canpharmaceutical testing may take full advantage of the smallbe casily dYHsignal-to-noise ratio ofparticle size column technology and high-pressure LC systemsat least 10c N M H Gimes higher, rprenrto gain separation speed, while maintaining adequate effi-ing an equivalent concentration of 1% or 5 ug/mL, significantciency and resolution.loss in sensitivity is observed for eptifbatide main impurity.Unfortunately, application of sub-2 um column technologyWhen peptide analysis is involved, low concentrationis not always successful as exemplified in the analysis ofmaterial appears to be difficult to recover from the high月咒鹦辖° acetate, a eyelic octapeptide mimicking naturalpressure LC system used along with sub-2 um columns,中国煤化工MYHCNMH G .376M.-J. Rocheleau et al.Table 1 Dosage of an octapeptide in a multi- strength drug product formulation.Ooctapeptide formulationSpecificationsPsiseleselesisg(ug/mLiing by UPLCDifference(%)(μg/mL)Unidose low-strength batch #145-5554217.6Multiidose batch #2180- 220209061.Unidose high-strength batch #3450 -55052170.8limiting detectability and method sensitivity. On-system adsorptionA 119(1.2) (2006) 140- 146.[2] M.W. Dong, Ulrahigh-pressure LC in pharmaceutical analysis:sensitivity at low concentration. Results from the analysis ofperformance and practical issues, LCGC North America 25 (7)octreotide acetate and Eptifibatide have indicated that response(2007) 1-19.signal is lost when injecting only a few nanograms of peptide.[3] B.L. Ackermann, M.J. Berna, J.A. 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