DEVELOPMENT OF ANALYTICAL METHOD FOR THE DETERMINATION OF ALKYL HALIDES BY GAS CHROMATOGRAPHY IN ACTIVE PHARMACEUTICAL INGREDIENT

Glavna naloga farmacevtske industrije je pacientu zagotoviti varno in kvalitetno izdelano zdravilo. V zadnjem času se veliko pozornosti posveča kontroli potencialnih genotoksičnih nečistoč (PGI), saj le-te povzročajo genske mutacije, kromosomske prelome ter preureditve, ki lahko vodijo v rakava obolenja. Dovoljena meja za vsebnost PGI-jev v zdravilih je običajno zelo nizka (koncentracijski nivo µgg-1) in je odvisna od dnevnega odmerka. V skupino teh nečistoč uvrščamo tudi alkil halide, ki lahko nastajajo kot stranski produkti različnih sinteznih stopenj, postopkov čiščenja (tvorba API soli) ali pa so kot reagenti prisotni v sintezi. Cilj dela je bil razvoj občutljive in selektivne analizne metode, ki bo direktno omogočala določevanje alkil halidov (klorometana, kloroetana bromometana in bromoetana) v aktivni farmacevtski učinkovini (API). Zaradi izredne hlapnosti analitov smo se razvoja analizne metode lotili s tehniko plinske kromatografije. Za uspešno ločbo analitov smo z neposrednim injiciranjem standardnih raztopin v metanolu preizkusili polarne, srednje-polarne in tudi nepolarne »WCOT« kapilarne kolone v kombinaciji z različnimi detektorji. Največji izziv je bilo izbrati kolono, ki bo zagotovila utrezno ločitev med analiti in topilom (metanol). Ločitev smo dosegli na koloni DB-624 v kombinaciji z masno-selektivnim detektorjem (MSD). Po večkratnem neposrednem injiciranju raztopine vzorca smo ugotovilili slabo ponovljivost, nestabilnost bazne linije in slabo obliko kromatografskih vrhov. Neposredno injiciranje smo nato uspešno nadomestili z injiciranjem plinaste faze s tehniko nadprostora (HS - headspace). Analizno metodo smo optimirali in jo tudi validirali kot limitni test na nivoju 2 µgg-1 (µgml-1). Pri validaciji smo potrdili selektivnost analizne metode, ponovljivost injiciranja, ponovljivost metode (za vzorec in vzorec s standardnim dodatkom), točnost ter robustnost. Z metodo smo nato analizirali realne vzorce izdelanega API-ja in potrdili ustreznost le-tega (vsebnost vseh analiziranih alkil halidov je bila pod mejo poročanja). Dobljeni rezultati so uradni del kontrolne strategije in so vključeni v registracijsko dokumentacijo.The main focus of Pharmaceutical industry is to produce safe and quality manufactured drug substance, which will ensure patient\u27s safety. Recently, much attention is paid to the control of potential genotoxic impurities (PGI), since these cause gene mutations, chromosomal breaks and rearrangements, which may lead to cancer. Permissible limit for the content of PGI\u27s in medicines is usually very low (concentration level mgkg-1) and it depends on the daily dose. One group of these impurities are also alkyl halides, which may be formed as by-products of the various synthetic steps, cleaning processes (the formation of API salts) or they can be present as reagents in the synthesis. The aim of my work was to develop a sensitive and selective analytical method that will allow direct determination of alkyl halides (methyl chloride, ethyl chloride, methyl bromide and ethyl bromide) in active pharmaceutical ingredient (API). Because of the extremely volatile nature of analytes method was developed by using gas chromatography (GC) as an analytical technique. To confirm the appropriate separation of the analytes and solvent (methanol), a direct injection of standard solutions in methanol was performed. During this process we tested polar, medium polar and non-polar "WCOT" capillary columns in combination with a variety of GC detectors. The biggest challenge was to identify the column, which would provide suitable separation between the analytes and the solvent (methanol). Appropriate separation was obtained on GC column DB-624 in combination with mass-selective detector (MSD). After repeated direct injections of the sample solution poor repeatability, baseline instability and poor shape of peaks were identified. Direct injection was then successfully replaced by injecting a gas phase with headspace injection (HS). Analytical method was optimized and validated as a limit test at level 2 µgg-1 (µgml-1). During validation of the analytical method selectivity, reproducibility of injections, reproducibility (for pure and spiked sample), accuracy and robustness were confirmed. The method was then used for testing of real samples of produced API. It was shown that the assay of all analyzed alkyl halides was below reporting limit. The results are an official part of the control strategy and are included in the registration dossier (DMF)

A Highly Productive, Whole-Cell DERA Chemoenzymatic Process for Production of Key Lactonized Side-Chain Intermediates in Statin Synthesis

<div><p>Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with <i>Escherichia coli</i> BL21 (DE3) overexpressing the native <i>E. coli deoC</i> gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2<i>S</i>,4<i>R</i>)-4,6-dihydroxytetrahydro-2<i>H</i>-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L⁻¹ h⁻¹ (up to 50 g L⁻¹ h⁻¹) with >80% yield and >80% chromatographic purity with titers reaching 100 g L⁻¹. Stereochemical selectivity of DERA allows excellent enantiomeric purities (<i>ee</i> >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins.</p></div

DERA activity measurements of the whole-cell catalyst.

<p><b>A.</b> Fluorescence raw data for a DERA activity assay (dotted lines). Velocities for triplicate samples of the whole-cell catalyst were measured for 7 different loads (<i>b–h,</i> 3.16 µg–26.9 µg in 3.96 µg increments) of biomass. After normalization with the blank (<i>a</i>), maximum slopes were determined for each sample and averaged (solid lines) to yield velocity for a given biomass load. <b>B.</b> Velocity vs. biomass load plot. The first 5 points are taken for the specific activity calculation. Linear regression: y = 0.2366x+0.2073 R² = 0.9936 <b>C.</b> Comparison of velocities measured for cell-free lysate spiked with increasing loads of biomass. <b>D.</b> Validation of linearity of the activity assay within samples with constant biomass. The whole-cell catalyst <i>E. coli</i> BL21 (DE3) pET30/<i>deoC</i> was mixed with w.t. <i>E. coli</i> BL21 (DE3) biomass (•). Linear regression: y = 248.94x+1.3840, R² = 0.9995. In parallel, sonicated and cleared samples were measured (□). Linear regression: y = 235.00x+2.6433, R² = 0.9989.</p

Time course of whole-cell, DERA-catalyzed, fed-batch reactions yielding 3g.

<p>Reaction species data from three independent experiments using (in total) 550 mmol L⁻¹ of <b>2g</b> and 1200 mmol L⁻¹ of <b>1</b> are shown. Whole-cell catalyst (<i>E. coli</i> BL21 (DE3) pET30/<i>deoC</i> high-density culture) with 217 kRFU s⁻¹ g⁻¹ DERA specific activity and 182 g L⁻¹ WCW was used. Results are given as molar concentrations obtained from GC-FID analysis. The measured quantity of a particular compound, with the exception of the stable 6-ring hemiacetals (<b>3</b>), represents the sum of the corresponding equilibrium forms (hydrate, aldehyde and acetal/hemiacetal) which exist under the reaction conditions. <b>1</b> (<b>▪</b>, black), <b>3a</b> (▴, blue) <b>3g</b> (♦, green), <b>8g</b> (•, red), <b>10g</b> (Δ, orange), <b>2g</b> (◊, brown), cumulative molarity of reaction species originating from <b>2g</b> (□, grey; sum of <b>2g</b>, <b>8g</b>, <b>10g</b> and <b>3g</b> ). Secondary vertical axis shows in %: residual DERA activity (□, violet), cumulative feed of <b>2g</b> (dotted line), cumulative feed of <b>1</b> (dashed line).</p

The optimization of lactol (<b>3g</b>) oxidation yielding lactone <b>15</b>.

<p>The optimization of lactol (<b>3g</b>) oxidation yielding lactone <b>15</b>.</p

Inactivation of DERA whole-cell catalyst and DERA cell-free lysate with various aldehydes.

<p>Samples were treated with 75 mM, 150 mM and 225 mM substrate aldehydes for 15 minutes prior to the activity assay. The specific DERA activity was 226.8 kRFU s⁻¹ g⁻¹ and 226.6 kRFU s⁻¹ g⁻¹ for the whole-cell catalyst and for the cell-free lysate, respectively. Residual activities are given relative to non-treated whole-cell catalyst. Aldehydes used were acetaldehyde (A), <b>2b</b> (B) and <b>2g</b> (C).</p

Time course of exemplary whole-cell, DERA-catalyzed, fed-batch reactions with ∼50 g L⁻¹ h⁻¹ volumetric productivity.

<p>Whole-cell catalyst (E. coli BL21 (DE3) pET30/deoC high-density culture) with 247 kRFU s⁻¹ g⁻¹ DERA specific activity and 215 g L⁻¹ WCW was used. Results are given as mass concentrations obtained from GC-FID analysis. The measured quantity of a particular compound, with the exception of the stable 6-ring hemiacetals (<b>3</b>), represents the sum of the corresponding equilibrium forms (hydrate, aldehyde and acetal/hemiacetal) which exist under the reaction conditions. <b>A:</b> Reaction species data from reaction using (in total) 700 mmol L⁻¹ of <b>2g</b> and 1540 mmol L⁻¹ of <b>1</b> are shown. <b>1</b> (▪, black), <b>3a</b> (▴, blue) <b>3g</b> (♦, green), <b>8g</b> (•, red), <b>10g</b> (Δ, orange), <b>2g</b> (◊, brown), acetic acid (□, grey). Secondary vertical axis shows in %: cumulative feed of <b>2g</b> (dotted line), cumulative feed of <b>1</b> (dashed line). <b>B:</b> Reaction species data from reaction using (in total) 700 mmol L⁻¹ of <b>2b</b> and 1540 mmol L⁻¹ of <b>1</b> are shown. <b>1</b> (▪, black), <b>3a</b> (▴, blue) <b>3b</b> (♦, green), <b>8b</b> (•, red), <b>10b</b> (Δ, orange), <b>2b</b> (◊, brown), acetic acid (□, grey), 2,6-chloro-2,4-dideoxyhexose (□, purple). Secondary vertical axis shows in %: cumulative feed of <b>2b</b> (dotted line), cumulative feed of <b>1</b> (dashed line).</p

Time course of whole-cell, DERA-catalyzed batch reactions.

<p>Reactions were performed using <i>E. coli</i> BL21 (DE3) pET30/<i>deoC</i> fermentation cultures directly (DERA specific activity = 232 kRFU s⁻¹ g⁻¹, WCW = 207 g L⁻¹). Results are given as mass concentrations obtained from GC-FID analysis. The measured quantity of a particular compound, with the exception of the stable 6-ring hemiacetals (<b>3</b>), represents the sum of the corresponding equilibrium forms (hydrate, aldehyde and acetal/hemiacetal), which exist under the reaction conditions. <b>A:</b> Reaction species data from reactions using 400 mmol L⁻¹ of <b>2g</b> and 840 mmol L⁻¹ of <b>1</b> are shown. <b>1</b> (▪, black), <b>3a</b> (▴, blue) <b>3g</b> (♦, green), <b>8g</b> (•, red), <b>10g</b> (Δ, orange) and <b>2g</b> (◊, brown). <b>B:</b> Reaction species data from reactions using 400 mmol L⁻¹ of <b>2b</b> and 840 mmol L⁻¹ of <b>1</b> are shown. <b>1</b> (▪, black), <b>3a</b> (▴, blue) <b>3b</b> (♦, green), <b>8b</b> (•, red), <b>10b</b> (Δ, orange), <b>2b</b> (◊, brown), 2,6-chloro-2,4-dideoxyhexose (□, grey). Concentration of the latter (Information S8) is evaluated based on the assumption, that the GC-FID response factor is similar to that of <b>3b</b>.</p

Current art in the direct synthesis of optically pure super-statins from the lactol precursors.

<p>Current art in the direct synthesis of optically pure super-statins from the lactol precursors.</p