characterization of a novel cross-linked lipase: impact of cross-linking on solubility and release...

Characterization of a Novel Cross-Linked Lipase: Impact of Cross-Linking on Solubility and Release from Drug ProductEvan M. Hetrick, David C. Sperry, Hung K. Nguyen, and Mark A. Strege*

Small Molecule Design and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, UnitedStates

ABSTRACT: Liprotamase is a novel non-porcine pancreatic enzyme replacement therapy containing purified biotechnology-derived lipase, protease, and amylase together with excipients in a capsule formulation. To preserve the structural integrity andbiological activity of lipase (the primary drug substance) through exposure of the drug product to the low-pH gastricenvironment, the enzyme was processed through the use of cross-linked enzyme crystal (CLEC) technology, making the lipase-CLEC drug substance insoluble under acidic conditions but fully soluble at neutral pH and in alkaline environments. In thisreport we characterize the degree of cross-linking for lipase-CLEC and demonstrate its impact on lipase-CLEC solubility andrelease from the drug product under relevant physiological pH conditions. Cross-linked lipase-CLEC was characterized via sizeexclusion chromatography (SEC) and capillary electrophoresis sodium dodecyl sulfate polyacrylamide gel electrophoresis (CE-SDS−PAGE). A combination of methodologies was developed to understand the impact of cross-linking on drug productrelease. Dissolution evaluation using USP Apparatus 2 at pH 5.0 with an enzyme activity-based end point demonstrated solubilitydiscrimination based on degree of cross-linking, while full release was demonstrated at pH 6.5. The dissolution of the drugproduct was also evaluated using a dual-stage test employing a USP Apparatus 4 flow-through system to mimic the changing pHenvironments experienced in the stomach and intestine to understand the impact of cross-linking on drug product performance.Use of USP Apparatus 4 to characterize the pH-dependent release of lipase-CLEC represents a novel approach compared to theApparatus 1 test employing an acid-challenge stage outlined in the USP for delayed-release pancrelipase, and the advantages ofthis approach may prove useful for understanding the pH-dependence of release for other drug products. Collectively, thesestudies confirmed that degree of cross-linking is a critical parameter that may impact in vivo release of lipase-CLEC, and alsoprovided a risk assessment tool for understanding the potential impact of under- and over-cross-linked drug substance.

KEYWORDS: lipase, cross-linking, enzyme, dissolution, liprotamase, solubility, Apparatus 4

■ INTRODUCTION

Liprotamase is a drug product under development for thetreatment of exocrine pancreatic insufficiency, a disease statecommon to patients with cystic fibrosis.1,2 Exocrine pancreaticinsufficiency results from the lack or reduction of exocrinesecretions of the pancreas, requiring patients to use pancreaticenzyme replacement therapy (PERT) to aid in digestion andnutrient absorption.1 Liprotamase is a replacement therapydesigned to provide the missing enzymatic activity and, unlikeexisting porcine-derived therapies, consists of purified micro-bially derived enzymes: a lipase, protease, and amylase. Each ofthe enzymes has a separate function in the digestive process.Lipase, protease, and amylase break down lipids, proteins, andcarbohydrates, respectively. These three enzymes are individ-ually produced through microbial fermentation and subse-quently purified using conventional biotechnological processesto generate the three drug substances as dry powders. The drug

substance powders are formulated and filled into a gelatincapsule.Following ingestion of the drug product by the patient, the

enzymes in liprotamase are stable in the acidic proteolyticenvironment of the stomach and are active following passageinto the duodenum, where they act on food substrates. Lipase isthe most important enzyme in the drug product for energyintake since more than twice the energy is derived per gram offat versus energy per gram of protein or carbohydrate.3 Stabilityin the gastric environment in the presence of the other enzymes(specifically protease, which rapidly digests other proteins insolution) is conferred to lipase through cross-linking of the

Received: November 1, 2013Revised: January 27, 2014Accepted: March 7, 2014Published: March 7, 2014

Article

pubs.acs.org/molecularpharmaceutics

© 2014 American Chemical Society 1189 dx.doi.org/10.1021/mp4006529 | Mol. Pharmaceutics 2014, 11, 1189−1200

pubs.acs.org/molecularpharmaceutics

crystallized enzyme to form a cross-linked enzyme crystal(CLEC).4 Cross-linking is an example of the broader field ofchemical modification of enzymes, which has received a greatdeal of interest and has direct applications to chemicalsynthesis, biosensors, pharmaceuticals, food processing, anddetergents.5 It has been demonstrated that chemicalmodification of enzymes can impact key properties includingactivity, stability, and solubility.5 Indeed, it is these propertiesthat cross-linking of lipase-CLEC seeks to modulate to improveperformance of liprotamase. Enzyme cross-linking has beendescribed in the literature6,7 and was first performed withglutaraldehyde by Quiocho and Richards.8 While glutaralde-hyde remains the most commonly used enzyme cross-linkingagent,6 other bi- and polyfunctional cross-linking agents havebeen described, including dextran dialdehydes, sucrosealdehydes, bis(sulfosuccinimidyl)suberate (BS3), and others.5,9

Lipase-CLEC is synthesized by cross-linking lipase, anonglycosylated single chain serine hydrolase produced bythe cell line Burkholderia cepacia, with BS3, which contains anamine-reactive N-hydroxysulfosuccinimide (NHS) ester at eachend of an 8-carbon spacer arm. N-Hydroxysulfosuccinimideesters react with primary amines at pH 7−9 to form stablecovalent amide bonds to cross-link proteins through thereaction of primary amines in the side chain of lysine residuesand the N-terminus of each polypeptide.10 Figure 1 is a

schematic of B. cepacia annotated with the location of the lysineresidues (K) and N-terminus (N), the potential sites for cross-linking with BS3. In the preparation of lipase-CLEC, the cross-linking reaction generates a distribution of cross-linked lipaseunits ranging in size from intra-cross-linked monomer to inter-cross-linked components of n > 15. Because multiple sites inthe protein are integrated into the cross-linking (the lipase

monomer contains seven lysines plus the N-terminus,11 for atotal of eight potential reactive sites as depicted in theschematic in Figure 1), the lipase-CLEC material consists of acomplex distribution of numerous inter- and intra-cross-linkedconfigurations. Due to the BS3 cross-linking of primary amineswithin the enzyme structure, solubility of lipase-CLEC isreduced at conditions of pH less than 4.5. The removal of theamine functionalities reduces the solubility of the enzyme byincreasing its hydrophobicity, and therefore the enzyme activityis similarly modulated as a function of pH. Therefore, thedegree of lipase-CLEC insolubility at gastric pH correlates tothe stability of the enzyme in this environment, and dissolution(i.e., release from drug product) is expected to be directlydependent upon the degree of cross-linking. Since dissolution istypically considered a Critical Quality Attribute for drugproducts as defined by the International Conference onHarmonization (ICH),12 a thorough study to understandimpact of cross-linking on dissolution is warranted.In this report we characterize the lipase-CLEC drug

substance and demonstrate the impact of cross-linking ondrug substance solubility and release from the drug productunder relevant physiological pH conditions. Lipase-CLEC withdiffering degrees of cross-linking was generated and evaluatedby size exclusion chromatography (SEC) and capillaryelectrophoresis sodium dodecyl sulfate polyacrylamide gelelectrophoresis (CE-SDS−PAGE). A combination of method-ologies was developed to understand the impact of cross-linkingon drug product release. Dissolution evaluation using USPApparatus 2 at pH 5.0 with an enzyme activity-based end pointdemonstrated solubility discrimination based on degree ofcross-linking, while full release was demonstrated at pH 6.5.The dissolution of the drug product was also evaluated using anovel dual-stage test with a USP Apparatus 4 flow-throughsystem to mimic the changing pH environments experienced inthe stomach and intestine to understand the impact of cross-linking on drug product performance.

■ EXPERIMENTAL SECTIONSynthesis of Cross-Linked Lipase. Samples of lipase-

CLEC varying in the degree of cross-linking were prepared foruse in this study in the following manner: Lipase was providedby Eli Lilly and Company (Indianapolis, IN), and BS3 wasobtained from ChemWerth (Woodbridge, CT). Aqueousslurries of 13.2 g of crystallized lipase in 200 mL reactionvessels under agitation were mixed with solutions of BS3 in 10mM sodium acetate buffer pH 4.5 so that the finalconcentration of lipase in the reaction mixture was 54 g/Land the concentration of BS3 was at four levels (20.8, 31.1,41.5, and 70.0 g/L) resulting in BS3:lipase molar ratios of 2.0,3.0, 4.0, and 6.6. These samples were labeled “A”, “B”, “C”, and“D”, respectively. For the purposes of this study, the “B”conditions are considered representative of the nominal lipase-CLEC drug substance. Following completion of the cross-linking reaction at 21 h, the lipase-CLEC samples were washedin a succession of rinses in 40% ethanol, 50 mM glycine, 10mM calcium acetate to quench the reaction and centrifuged toisolate the solids and eliminate excess BS3 and N-hydroxysuccinimide, a degradation product of BS3 generatedthrough hydrolysis. The final lipase-CLEC cakes were thenlyophilized with a VirTis AdVantage 2.0 BenchTop Freeze-Dryer (SP Scientific, Warminster, PA). Initial freezingconditions were −45 °C for 3 h with a ramp time of 1.8 h.For primary drying, the pressure was set at 100 mTorr and the

Figure 1. A stereoview schematic of B. cepacia lipase. The lysines (K)and N-terminus (N) amino acid residues represent the potential sitesfor cross-linking with BS3. The catalytic sites are also labeled, andevery twentieth residue is marked by a dot. Reprinted with permissionfrom ref 11. Copyright 2002 Elsevier Science Ltd.

Molecular Pharmaceutics Article

dx.doi.org/10.1021/mp4006529 | Mol. Pharmaceutics 2014, 11, 1189−12001190

shelf set point was −15 °C for 3 h with a ramp rate of 0.5 °Cper min, followed by a shelf set point of 0 °C for 19 h with aramp rate of 0.5 °C per min. For secondary drying, the shelf setpoint was 33 °C for 20 h with a ramp rate of 0.5 °C per min.For end-of-freeze-drying, the set point was 4 °C.Drug Product Capsule Manufacture. Lipase-CLEC,

crystallized protease, and amorphous amylase were individuallyproduced and formulated into drug product. A preblendcontaining amylase, protease, microcrystalline cellulose, mal-trodextrin, crospovidone, colloidal silicon dioxide, and talc wasprepared by blending in a turbula mixer for 10 min. The lipase-CLEC lots with varying degrees of cross-linking were thenmixed with appropriate aliquots of preblend for 10 min. Finally,magnesium stearate was mixed with each blend to produce 40 gbatches of powder. Size 2 gelatin capsules were filled to a targetweight of 200 mg using a capsule filler (Bonapace andCompany, Limbiate, Italy). The resulting capsules hadtheoretical potencies of approximately 16,000−44,000 USPunits of lipase-CLEC (the range of activity is a result of thediffering degrees of cross-linking of individual lots of lipase-CLEC), 25,000 USP units of protease, and 3,750 USP units ofamylase based on the activity of each enzyme batch per unitweight.Capillary Electrophoresis Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis (CE-SDS−PAGE).The samples of lipase-CLEC were evaluated by CE-SDS−PAGE to determine the degree of cross-linking for each batch.Lipase-CLEC reference standard was provided by Eli Lilly. Allmeasurements were performed with a Beckman CoulterProteomeLab PA 800 capillary electrophoresis system (Beck-man Coulter, Fullerton, CA). Prior to each analytical run a baresilica capillary (30.2 cm length, 20.2 cm effective length × 50μm i.d.) was rinsed with 1 M sodium hydroxide for 5 min, 1 Mhydrochloric acid for 2 min, and purified water for 2 min (all at50 psi) followed by a filling with the SDS-MW Gel Buffersolution for 10 min at 70 psi (all solutions including SDS-MWSample Buffer are components of the Beckman Coulter SDS-MW Analysis Kit). The samples were prepared by combining200 μL of 1 mg/mL solutions in SDS-MW Sample Buffer with2 μL of Internal Standard (a 10 kDa protein used as a mobilitymarker, provided in the SDS-MW Analysis Kit) and wereinjected for 60 s at 5 psi. Before analysis all buffers and sampleswere degassed and filtered through 0.22 μm filters. Thecapillary cartridge temperature was maintained at 25 °C. Theelectrophoretic separations were conducted at 10 kV for 50 minunder reverse polarity (i.e., outlet = anode).Following the assignment of peak identity by relative

migration time in comparison to the MW Marker profile andinternal standard, the average degree of cross-linking wascalculated for each sample based on time-corrected peak areacorresponding to each component (TCAk) and the totalsummed TCA of all components in the profile (TCAtotal). A“degree of cross-linking contribution” corresponding to eachcomponent observed in the separation profile was calculated bymultiplying the number of lipase units (k) represented from 1to 11 by the corresponding fraction of total TCA, by the % oftotal TCA, and a total average degree of cross-linking (DoC)representing the average number of inter-cross-linked lipasesubunits in a sample was determined by summing the degree ofcross-linking contributions of each component, as per eq 1:

∑==

kDoC (TCA /TCA )k

k1

11

total(1)

Size Exclusion Chromatography. Size exclusion chroma-tography (SEC) was performed on an Agilent model 1100HPLC system monitoring elution using ultraviolet absorbancedetection at 280 nm. The column was a YMC Diol 300 mm × 6mm, 5 μm, 300 Å pore size packing held at ambienttemperature. The mobile phase consisted of 35 mM Trisbuffer pH 7.4, the flow rate was 0.5 mL/min, and the sampleinjection volume was 15 μL. Samples were prepared at aconcentration of 5.0 mg/mL in 6 M urea, 35 mM glycine, pH9.0, and a lipase monomer standard was prepared at the sameconcentration in 20 mM Tris, 200 mM NaCl, pH 8.0 buffer.Following the introduction of the solids to the sample buffers,the samples were agitated by orbital mixing at 10 rpm for 20min and then centrifuged at 12,000 rpm to remove anyremaining insoluble material prior to injection. Within the SECprofiles, all peaks corresponding to components of size greaterthan or equal to the monomer (eluting at approximately 11.4min) were integrated and the monomer % area and lipase-CLEC “high MW peak % area” eluting between 6.0 and 8.0 min(arbitrarily chosen to represent the higher states of cross-linking) were determined to facilitate an estimation of theextent of cross-linking.

Solubility Determination. Lipase-CLEC solubility deter-minations were performed at room temperature by preparingsamples of approximately 20 mg in 15 mL conical tubes at atarget concentration of approximately 2 mg/mL in two buffers,(1) 20 mM glycine, 20 mM L-glutamic acid, 20 mM L-histidinepH 6.0, and (2) 20 mM glycine, 20 mM L-glutamic acid, 20mM L-histidine pH 4.0. Samples were prepared in triplicate.The samples were vortexed and then placed on an orbital mixerat 10 rpm for 4 h. The sample tubes were then centrifuged at3400 RCF for 15 min to pelletize the insoluble material, andthe supernatant was diluted 1:1 in buffer prior to UVabsorbance measurement at 280 and 320 nm (the lattermeasurement made for correction for any light scattering dueto the presence of insoluble matter). The sample concentrationwas calculated using the extinction coefficient for lipase-CLECdrug substance at 280 nm, 1.121 (mg/mL)−1 cm−1. At the timeof the solubility analysis, the exact same samples were alsotested by Karl Fischer titration for water content. Thecalculated lipase-CLEC concentration was divided by thetheoretical concentration, based on percent water-correctedweight, to determine the percent solubility.

USP Apparatus 4 Dissolution Testing. Dissolutionmedia consisted of the citrate/phosphate (McIlvaine) buffersystem13 with a total combined concentration of citrate andphosphate of 10 mM. Media were prepared at pH 4.00 (stage1) and pH 5.00, 5.50, 6.00, 6.25, and 6.50 (stage 2) by adjustingthe ratio of citric acid and dibasic sodium phosphate to achievethe desired pH ± 0.1 pH units. The pH of the buffer was notfurther adjusted with acid or base, in order to maintainconsistency of the buffer preparations. Dissolution media weredeaerated via purging with nitrogen. A USP Apparatus 4dissolution apparatus14 (Sotax CP7) was prepared byassembling 22.6 mm flow-through cells and placing a 5 mmruby bead at the bottom of each flow-through cell, and adding 1mm glass beads to the “elbow” of each cell.14 One liprotamasecapsule was placed into each flow cell (n = 6 capsules at eachpH for medium screen, n = 3 capsules of each drug product lotfor the cross-linking study) and two glass filters (0.7 μm



Whatman Glass Microfibre Filter GF/F and 2.7 μm WhatmanGlass Microfibre Filter GF/D) were inserted into the flow cellcap such that when the system was operated the flowingmedium reached the 2.7 μm filter prior to the 0.7 μm filter.Two filters were used in order to minimize back pressurecaused by particulates clogging the filter membranes. Theseventh flow cell did not contain a liprotamase capsule andserved as a “blank” for the analysis. Note that capsule or tabletholders were not used for these studies, and, upon initiation offlow, the capsules floated to the top of the flow cell and restedagainst the upper filter until the capsule shell disintegrated.Future studies may be warranted to evaluate any potentialbenefit of employing a capsule or tablet holder which isavailable from the manufacturer of the Apparatus 4 dissolutionsystem. For the proof-of-concept experiment (data presented inFigure 7), stage 1 consisted of pH 4.00 for 60 min at 4 mL/minand pH 6.00 for the following 80 min at 4 mL/min, and releaseof each enzyme was monitored via the HPLC method detailedbelow, with results normalized to final total release [i.e., theHPLC peak area of lipase-CLEC at a given time point wasdivided by the HPLC peak area at the final time point (140min, assumed to represent complete release) and multiplied by100 to provide % release]. For the medium screen (datapresented in Figure 8), flow was initiated and the system wasoperated at 12 mL/min for 30 min with stage 1 (medium pH4.00) and 32 min with stage 2 with medium pH of 5.00, 5.50,6.00, or 6.50 (note that a separate experiment was conductedfor each different pH of the medium screen, each experimentconsisting of stage 1 pH 4.00 for 30 min and stage 2 for 32min). Fractions were collected every 4 min. A follow-upmedium screen was performed at pH 6.25 with a flow rate of 16mL/min with 3 min fractions. Unless otherwise stated in thetext, all subsequent analyses were performed at a flow rate of 16mL/min with 48 mL fractions collected every 3 min. For themedium screen experiments and analyses of the cross-linkedsample drug product capsules, quantitation was performed byUV absorbance measurements as detailed below.HPLC Analysis for Lipase-CLEC. For HPLC analysis of

dissolution samples, an Agilent 1100 HPLC was equipped witha Zorbax Poroshell 300SB-C8 column, 2.1 μm internaldiameter × 75 mm length, 5 μm particle size (Agilent). Mobilephase A consisted of 0.05% trifluoroacetic acid (TFA) indeionized water, and mobile phase B consisted of 0.05% TFA inacetonitrile (ACN). The autosampler of the HPLC wasmaintained at 5 °C, and the column was set to a temperatureof 60 °C. A 20 μL injection volume was used, and UV detectionwas employed at 214 nm. The flow rate was set to 2.0 mL/min,and the gradient presented in Table 1 was performed over a 30min run time. The combination of the nonporous stationaryphase, high column temperature, and gradient cycling wasnecessary to minimize sample carryover and provide accuratequantitation.UV Analysis for Lipase-CLEC. An Agilent Chemstation UV/

vis spectrophotometer was employed, and medium from theseventh flow-through cell of the USP Apparatus 4 instrument,which did not contain a capsule, was used as a blank for the UVmeasurements. Samples and standard solution were transferredto a 1 cm path length quartz cuvette, and the absorbance wasmeasured at 280 nm with background correction at 380 nm.The concentration of lipase-CLEC in samples was calculated vsa one-point calibration curve prepared with a lipase-CLECstandard at approximately 0.2 mg/mL.

USP Apparatus 2 Dissolution Testing. Dissolutionmedia consisted of the citrate/phosphate (McIlvaine) buffersystem13 with a total combined concentration of citrate andphosphate of 15 mM. Media were prepared at both pH 5.0 and6.5 by adjusting the ratio of citric acid and dibasic sodiumphosphate to achieve the desired pH ± 0.1 pH units; asdescribed above, no pH adjustments were made. Thedissolution media were degassed via vacuum filtration througha 0.45 μm nylon filter, and 500 mL of medium was added toUSP Apparatus 2 dissolution vessels14 and equilibrated to 37 ±0.5 °C. Liprotamase capsules (n = 3) were placed into JPsinkers,15 the Apparatus 2 paddles were started at 50 rpm, andthe capsules were introduced into the dissolution vessels. At 10min, 40 mL of the solution was withdrawn from each vessel andfiltered through a 0.45 μm 25 mm PES syringe filter (Whatman,Puradisc), and the filtrate was stored on ice until analyzed viathe enzyme activity method described below. At 30 min, thepaddle rotation speed was increased to 250 rpm for 15 min andan additional 40 mL was sampled, filtered, and stored on ice asdescribed above. Samples (n = 3 capsules) were analyzed forlipase activity via the method described in Lipase Activity Assay.

Lipase Activity Assay. Lipase-CLEC samples with varyingdegrees of cross-linking, as well as samples from the Apparatus2 testing described above, were analyzed for lipase enzymaticactivity based on the method described in the USP monographfor pancrelipase.16 Briefly, a solution of acacia was prepared byadding 50 g of acacia to a 1 L beaker and adding 450 mL ofdeionized water, stirring for 1 h, and then centrifuging for 15min at 3000 rpm. The clear top layer was collected for furtheruse. Substrate solution was prepared by combining 20 mL ofNF-grade olive oil, 165 mL of acacia solution, and 15 mL ofdeionized water in an electric blender and cooling to 2−8 °C.The mixture was blended in 5 min increments, with 10−15 minof cooling on ice between blending, for 3 cycles. Suitability ofthe substrate was confirmed vs the USP monograph require-ments. Tris buffer was prepared by adding approximately 120mg of tris(hydroxymethyl)aminomethane and 468 mg ofsodium chloride to a 200 mL volumetric flask and dilutingwith deionized water. The bile salt solution was prepared byadding approximately 4.0 g of dried bile salts to a 50 mLvolumetric flask and diluting to volume with deionized water.Samples and standards were analyzed by mixing 10.0 mL of

Table 1. Gradient Profile Used for HPLC Analysis ofAmylase, Protease, and Lipase-CLEC

% mobile phase

time (min) A B

0.0 75 258.0 40 6010.0 5 9511.0 65 3513.0 5 9514.0 65 3516.0 5 9517.0 95 519.0 5 9520.0 65 3522.0 5 9523.0 65 3525.0 5 9525.5 75 2530.0 75 25



olive oil substrate, 8.0 mL of Tris buffer solution, 2.0 mL of bilesalt solution, and 9.0 mL of deionized water in a jacketed glasstitration vessel. The temperature of the vessel was maintainedat 37 °C with a water bath and pump. An automatic titrator(Radiometer Analytical, TM 854) was used to pretitrate thesolution to pH 9.20 with 0.1 N NaOH solution. Once thesolution had been pretitrated to pH 9.20, 1.0 mL of samplesolution was pipetted into the titration vessel. The autotitratorwas set to maintain a pH of 9.00 by titrating 0.1 N NaOH intothe vessel to balance the enzymatic release of acid from theolive oil substrate by the lipase. The rate of NaOH addition wasmonitored for 5 min, and lipase activity was determined bycomparing the rate of NaOH addition required for a sample tothe rate of NaOH addition required for a standard.

■ RESULTS AND DISCUSSIONCharacterization of Cross-Linked Lipase-CLEC Sam-

ples. A summary of the data from the evaluation of the lipase-CLEC drug substance samples is displayed in Table 2. Detaileddiscussions of the results from each of the analyses are includedin the following sections.CE-SDS−PAGE. An example CE-SDS−PAGE profile (sample

C) is displayed in Figure 2. The 10 kDa internal standard

marker migrated at approximately 19 min, and the lipasemonomer migrated at 24 min. The lipase monomer has a MWof 33,127 Da (330 amino acids) with a single disulfide bridgeand a pI of 4.6. The identity of the monomer was confirmedthrough direct comparison to the profiles of a lipase standardand a mixture of protein markers of known MW. It is importantto note that the CE-SDS−PAGE separation provided effectiveresolution between components differing in the number ofenzyme subunits, but did not discriminate between differentcross-linking configurations. As previously described, compo-nents of up to n = 11 lipase units were integrated and included

in the calculations of degree of cross-linking (DoC), althoughspecies representing up to n = 16 were visible in the baseline ofthe electropherogram. The DoC values were observed toincrease in response to increases in the BS3:lipase ratio duringthe cross-linking reaction. Incomplete solubility in the samplebuffer hindered the analysis of sample D (representing thehighest BS3:lipase ratio and theoretically the highest DoC), andtherefore the DoC could not be calculated for this sample.These observations aligned with the solubility results observedat pH 6.0 for sample D (see Solubility Determination).

Size Exclusion Chromatography. Size exclusion chromatog-raphy analyses of the cross-linked lipase samples clearlydemonstrated the effects of BS3:lipase molar ratio upon thesize distribution of the enzyme components (see Figure 3A−E). Under these chromatographic conditions, the lipasemonomer eluted at a retention time of 11.5 min. Higher levelsof cross-linking resulted in a shift toward reduced retentiontime as the size of the cross-linked species was increased. The %high MW peak data based on integrated peak area between 6and 8 min is listed in Table 2 and reflects the growth in higherorder enzyme configurations at the higher BS3:lipase molarratios. A qualitative assessment of peak area recoveries acrossthe sample set suggests that the components of this samplewere successfully solubilized through the sample preparation inthe pH 9.0 buffer containing 6 M urea and solubility wassubsequently maintained in the pH 7.4 mobile phase.

Solubility Determination as a Function of Cross-Linking.The lipase enzymes produced by B. cepacia are highly soluble atpH 4.0 and pH 6.0.17−19 In contrast, as listed in Table 2, allcross-linked lipase-CLEC samples demonstrated limitedsolubility at pH 4.0, and only the lowest BS3:lipase molarratio of 2.0 resulted in a solubility >1.0% (most likely due to thepresence of a measurable amount of monomer). At pH 6.0,solubility appeared to reach a maximum at recovery levels ofapproximately 90.0%, with 10% remaining insoluble. Underthese conditions (pH 6.0), the degree of cross-linking facilitatedby the 6.6 BS3:lipase molar ratio resulted in a significantlydecreased solubility of 50%. Plots of % solubility at pH values of4.0 and 6.0 were generated for the lipase-CLEC samples acrossthe range of % high MW (see Figure 4). To provide additionalclarity toward understanding the effects of solution pH uponsolubility, the solubility test was performed in 20 mM glycine,20 mM L-glutamic acid, 20 mM L-histidine pH 5.0 for thenominal cross-linked lipase-CLEC. The result is plotted inFigure 5 together with the pH 4.0 and pH 6.0 data for thissample and demonstrates the dependence of solubility uponpH.

Enzymatic Activity as a Function of Cross-Linking.Likewise, the specific enzymatic activity of each lot correlatedinversely with the BS3:lipase molar ratio employed duringcross-linking. The sample with the lowest BS3:lipase molar

Table 2. Results from the Analyses of Lipase-CLEC by Solubility Testing (Average ± SD), CE-SDS−PAGE, SEC, and SpecificEnzyme Activity Testing

% solubility

sampleBS3:lipase molar ratio during cross-linking

reaction at pH 4.0 at pH 6.0average DoC by CE-SDS−

PAGE% high MW peak by

SECenzyme activity (USP

U/mg)

A 2.0 5.0 ± 0.5 88.9 ± 0.6 2.7 15 3096B 3.0 0.8 ± 0.5 89.4 ± 2.4 3.5 34 2378C 4.0 0.5 ± 0.5 90.0 ± 1.0 4.0 59 1865D 6.6 0.5 ± 0.5 50.4 ± 1.5 a 73 1125

aSample was not effectively solubilized in the SDS-MW Sample Buffer.

Figure 2. CE-SDS−PAGE analysis of sample C, obtained using theparameters described in the Experimental Section. In addition to the10 kDa migration time marker, the identities of the individualcomponents in the profile are labeled with the number of lipasesubunits that are represented.



ratio (A) was characterized with the greatest enzymatic activity(3096 USP U/mg) while the sample with the greatestBS3:lipase molar ratio (D) was characterized with the lowestenzymatic activity (1125 USP U/mg). The data presented inTable 2 indicate that the specific activity of each lot is directlyimpacted by degree of cross-linking, and differences in activityare not simply a function of changes in solubility. For example,the solubility of lots A, B, and C are all roughly equivalent atpH 6.0 (i.e., all are approximately 90%). However, for thosesame samples, specific activity ranges from 3096 USP U/mg forlot A to 1865 USP U/mg for lot C. The enzyme activity test isconducted at pH 9.0; thus, the differences in specific activity aremost likely due to differences in degree of cross-linking

imparted based on the ratio of BS3 as opposed to differences insolubility. The correlation between extent of cross-linking (asmeasured by % high MW peak) and enzyme activity isdemonstrated in Figure 6, where the decrease in enzymeactivity may be due to decreased access of the substrate to the

Figure 3. Size exclusion chromatography profiles of cross-linkedsamples A, B, C, and D and a lipase standard (E) obtained using theparameters described in the Experimental Section. The monomer andhigh MW peak are labeled.

Figure 4. Impact of cross-linking (as measured by % high MW peak bySEC) on % solubility at pH 4.00 (◆) and pH 6.00 (■).

Figure 5. Solubility of nominally cross-linked lipase-CLEC as afunction of pH.

Figure 6. Impact of cross-linking (as measured by % high MW peak bySEC) on enzyme activity as measured by the USP lipase activity test.



active site caused by inhibition of protein structural flexibility asDoC (and subsequently % high MW peak) increased. Thisrelationship between enzyme activity and cross-linking has beenpreviously observed.20,21 Importantly, subsequent dissolutionevaluations with enzyme activity end points were normalized tothe total lipase activity content of each capsule based on thespecific activity of each lot and the mass of lipase-CLEC in eachcapsule.Dissolution Characterization and Product Perform-

ance. Other PERTs rely on enteric coating of lipase to conferstability through the acidic environment of the stomach andmaintain activity until reaching the duodenum.31 Dissolutioncharacterization of these products is based on the USPmonograph for Pancrelipase Delayed-Release Capsules, whichdetails a two-stage dissolution test.22 The test is conducted witha USP 1 dissolution apparatus (baskets)14 with the first (acidic)stage designed to challenge the enteric coating to demonstrateintegrity through the low-pH stomach environment. Thesample is then exposed to a higher-pH second stage tosimulate the environment of the duodenum where the entericcoating dissolves, thereby releasing lipase. Specifically, thedissolution test for delayed-release pancrelipase capsulesinvolves transferring capsule contents to a USP Apparatus 1basket, then performing a 60 min challenge with 800 mL ofUSP simulated gastric fluid which has a pH of approximately1.2.23 The contents of the baskets are then removed andtransferred to a vessel with 800 mL of pH 6.0 phosphate bufferand agitated with Apparatus 2 paddles at 100 rpm. A sample iscollected after 30 min of exposure to the second stage (pH 6.0),and lipase activity is measured via an enzymatic lipase activitytest. Amylase and protease activity are not measured as part ofthe USP dissolution test for delayed-release pancrelipase.The Apparatus 1 delayed-release pancrelipase dissolution

method described above is not suitable for characterizing lipase-CLEC release from liprotamase capsules due to the particle sizeof the lipase-CLEC crystals. Whereas the size of enteric coatedbeads is such that they are retained within the 0.36−0.44 mmwire openings of the Apparatus 1 basket,14 the particle size oflipase-CLEC crystals is smaller and they are not retained by theApparatus 1 basket. The possibility of a dual-stage dissolutiontest in a single Apparatus 1 or 2 vessel was explored forliprotamase by adjusting pH upward within a single dissolutionrun; however, the presence of the protease in solution withlipase-CLEC (leading to rapid enzymatic degradation of thedrug substance in a substrate-limited environment) and theionic strength of the buffer required to adjust the pH in arobust manner led to inconsistent dissolution results (data notshown).To bypass these challenges and enable a dual-stage

dissolution test, a method employing the USP Apparatus 4flow-through system was evaluated. The design, operationalprinciples, and applications of Apparatus 4 have been reviewedin the literature.24−26 The Apparatus 4 system is designed tocontain the dosage form in a flow cell through whichdissolution medium is passed at a controlled flow rate.Sampling can be performed at fixed time points by collectingfractions of the medium that had passed through the cell, orcontinuous monitoring can be performed via online UVdetection. Apparatus 4 methods have proven useful forsustained release products as well as dosage forms for whichApparatus 1 and 2 methods may not be suitable, such assuppositories, implants, powders, and granules.22 A furthersignificant benefit of a flow-through system is that dissolution

media with different pH values can be employed in a single runwithout needing to isolate the contents of the dosage form tomove to a separate vessel for a second stage.22 This representsan important advantage especially applicable to the lipase-CLEC technology used to confer acid stability to lipase inliprotamase capsules.The principle of procedure for the Apparatus 4 method for

dissolution testing of liprotamase capsules with nominal levelsof lipase-CLEC cross-linking is illustrated in Figure 7, which

shows results from an initial feasibility experiment demonstrat-ing release of all three enzymes. Individual capsules were placedin 22.6 mm Apparatus 4 flow-through cells, and pH 4.00citrate/phosphate buffer was introduced into the cell at 4 mL/min. After 60 min, the medium was switched to pH 6.00citrate/phosphate buffer. Fractions were collected every 10 minthroughout the entire 140 min run, resulting in 14 totalfractions. Release of each enzyme was monitored via reversed-phase HPLC. Figure 7 shows that amylase and protease rapidlydissolve in the pH 4.00 medium and are observed in fractions1−4. As expected, no lipase-CLEC dissolved in any of thefractions generated with pH 4.00 medium, demonstrating thesolubility properties conferred by cross-linking. After 60 min,the medium was changed to pH 6.00 citrate/phosphate bufferand a further 8 fractions were collected over 80 min. Nosignificant amylase or protease release was observed in fractionscollected at pH 6.00, but as shown in Figure 7, significant levelsof lipase-CLEC were observed in the first several fractionscollected with pH 6.00 medium.Two primary advantages of the Apparatus 4 method are

apparent from the data presented in Figure 7. The first centerson product performance of the dosage form. The dual-stage testwith physiologically relevant pH conditions demonstrates theintended performance of the most critical component of the

Figure 7. Release profiles for lipase-CLEC (◇), amylase (○), andprotease (+) demonstrating proof-of-concept for the dual-stageApparatus 4 dissolution test. Stage one (0−60 min) consisted of 10mM pH 4.00 citrate/phosphate buffer, and stage two (60−140 min)consisted of 10 mM pH 6.00 citrate/phosphate buffer. Release resultswere generated with n = 1 sample and are normalized to final totalrelease.



drug product (i.e., lipase) in a dual-stage test. Several reportsexist in the literature detailing fed-state gastric pH values.27−29

For example, a study from Dressman and colleaguesdemonstrated that, within the first hour after ingesting ameal, the average gastric pH across 24 subjects ranged fromapproximately pH 3 to pH 5.28 Thus, the first stage of theApparatus 4 test at pH 4.0 is a reasonable mimic of the gastricpH environment that would be expected when liprotamasecapsules are taken with food. Note that liprotamase is notintended to be taken with any one specific type of meal (such ashigh fat/high calorie). It is known that different meal types canresult in different postprandial gastric pH values,30 andconsideration should be given to stage 1 pH selection forother products intended to be taken with certain types of meals.Postprandial duodenal pH has been reported to vary fromapproximately pH 5 to pH 7,31−34 and other cofactors such aslipids and bile salts are present in the duodenum that can aid indissolution.27 The purpose of the dissolution test is notnecessarily to mimic the stomach or duodenum exactly butrather serve as a control point for key attributes that may affectproduct performance (e.g., degree of cross-linking) withcorrespondence to physiological pH being an added benefitto model potential in vivo performance. Thus, the second stageshould have a relevant pH but not necessarily exactly match thepH of the duodenum in the fed state. Indeed, reports in theliterature describe a wide range of fed-state intestinal pHvalues.28−31

The second significant advantage of the Apparatus 4 methodis the ease of execution. It was known from previous work thatwhen lipase-CLEC and protease are in solution together,proteolytic degradation of lipase-CLEC resulted in a UVresponse factor change for lipase-CLEC (data not shown),preventing accurate quantitation via HPLC analysis. Byexploiting the differential solubility between protease andlipase-CLEC with the Apparatus 4 test, the two enzymes arenever in solution together, preventing degradation andpermitting accurate quantitation of lipase-CLEC when it isreleased in the second stage at higher pH. Moreover, byseparating lipase-CLEC from amylase and protease during thedissolution portion of the test, chromatography is not necessaryto detect lipase-CLEC release during the second stage, and amore rapid and simple UV measurement (280 nm) can be usedto monitor lipase-CLEC release as opposed to requiring HPLCanalysis. These advantages discussed above are unique to themultistage Apparatus 4 test and would not be feasible with asingle-stage Apparatus 1 or 2 test. All subsequent Apparatus 4lipase-CLEC release data were generated via direct UV analysis.After proof-of-concept experiments were performed (Figure

7) and initial method conditions were determined, a pHmedium screen was conducted with the second stage (i.e.,higher pH) to better understand the influence of pH on lipase-CLEC release from liprotamase capsules. Note that after proof-of-concept data were generated for Apparatus 4 testing, flowrates were increased as described in the Experimental Section toenhance the speed of analysis. For the medium screen,liprotamase capsules filled with nominally cross-linked lipase-CLEC were used. The purpose of the medium screen was toidentify conditions where complete release (>90%) of lipase-CLEC was observed, but where release was not so rapid as toobscure any potential discrimination of the method. For themedium screen, the McIlvaine citrate/phosphate buffersystem13 was selected so various pH media could be evaluatedwithout necessitating a change in the identity of buffer

components or concentrations. This is especially importantfor measuring rate of release of protein therapeutics due to theHofmeister effect of charge and ion identity on proteinsolubility.35 For the medium screen, the first stage was keptconstant at pH 4.00 citrate/phosphate buffer to sweep amylaseand protease from the dissolution cell prior to initiating lipase-CLEC release in the second stage with a higher-pH buffer, andenabling the direct UV end point to monitor lipase-CLECrelease. Specificity of the method for lipase-CLEC wasmonitored by analyzing the first fraction collected in thesecond stage by HPLC to confirm that all amylase and proteasewere removed from the dissolution cell during the first stage atpH 4.00.The results of the medium screen are shown in Figure 8. The

initial media screen was conducted with medium at pH 5.00,

pH 5.50, pH 6.00, and pH 6.50. Those results show, asexpected, very slow release of lipase-CLEC at pH 5.00 and avery rapid release at pH 6.50, with intermediate rates of releaseat pH 5.50 and pH 6.00. These results were expected based onthe known pH-dependence of lipase-CLEC solubility (seeCharacterization of Cross-Linked Lipase-CLEC Samples:Solubility Determination as a Function of Cross-Linking) andthe fact that dissolution rate is directly proportional tosolubility.36 Since one goal of the medium screen was toidentify a medium that achieved >90% release of lipase, pH5.00, 5.50, and 6.00 were eliminated from further consideration.The pH 6.50 medium demonstrated complete release of lipase;however, the release profile was very rapid, with 100% releasebeing achieved within the first three fractions collected (withinvariability of the UV method). A dissolution method with suchrapid release may not be sensitive to parameters that mayimpact product performance (e.g., degree of cross-linking), so afifth medium at pH 6.25 was also evaluated. As expected, thismedium resulted in a slightly lower overall total release than thepH 6.50 medium. This may suggest that there is a small portionof the lipase-CLEC material, presumably with the greatest

Figure 8. Release profiles of lipase-CLEC from liprotamase capsules atpH 5.0 (◆), pH 5.5 (■), pH 6.0 (▲), pH 6.25 ( × ), and pH 6.5 (●)generated during the stage 2 medium screen with USP Apparatus 4.The x-axis represents time after initiation of stage 2. Stage 1 was pH4.00. Error bars represent ±1 standard deviation (n = 6 capsules).



degree of cross-linking, which may not be completely soluble atpH 6.25 yet is soluble at pH 6.50. Nevertheless, due to itsgreater likelihood of discriminating between critical parameterssuch as degree of cross-linking, pH 6.25 citrate/phosphatebuffer was selected as the second stage of the Apparatus 4 testfor all further characterization of lipase-CLEC release fromliprotamase capsules. Method performance was characterizedwith respect to specificity of separation of lipase-CLEC fromamylase and protease between stages 1 and 2, linearity,accuracy, precision, limit of detection for lipase-CLEC instage 2 fractions, and sample and standard stability (data notshown).After an appropriate stage 2 medium had been selected and

the Apparatus 4 method was characterized, the conditions wereused to determine the impact of degree of lipase-CLEC cross-linking on release from the drug product. Lipase-CLEC releasewas evaluated for liprotamase capsules generated with lipase-CLEC with different degrees of cross-linking (see Table 2). Asshown in Figure 9, the dual-stage Apparatus 4 dissolution

method with pH 6.25 for stage 2 effectively discriminatedagainst degree of cross-linking at early time points. Specifically,at the 3 min time point, lipase-CLEC release values of 63%,62%, 44%, and 26% were observed for samples A, B, C, and D,respectively. Note that this is in the order of lowest to highestlevels of cross-linking based on BS3:lipase molar ratio usedduring the lipase-CLEC cross-linking reaction (Table 2). Theseresults demonstrate the impact of cross-linking on rate ofrelease in the higher pH environment that would be expected inthe small intestine. Importantly, the rate of release alsocorrelates well with other direct (e.g., CE) and indirect (e.g.,% high MW peak by SEC, solubility) measures of degree ofcross-linking, as shown in Table 2. For example, sample Ademonstrated the lowest degree of cross-linking (2.7) by CEanalysis and, correspondingly, had the fastest initial rate ofrelease. In contrast, sample C had the greatest measured degreeof cross-linking by CE (4.0) and had a significantly lower initial

rate of release compared to sample A. In a similar fashion, theinitial release rates are inversely proportional to the % high MWpeak as measured by SEC (Table 2). This is consistent with theunderstanding that greater degrees of cross-linking, and thusincreased % high MW peak as measured by SEC, result in lowerinitial rates of release. A plot of % release at 3 min vs % highMW peak by SEC (a proxy for degree of cross-linking) isdisplayed in Figure 10.

The differences in overall levels of release (i.e., final resultachieved at 30 min) are also likely due to the differences incross-linking obtained across the samples. The two samplesgenerated at the extremes of cross-linking (A, the least cross-linked sample, and D, the most cross-linked sample) resulted inlower overall release (approximately 80%) as measured at thefinal time point (Figure 9). It is likely that, for sample A, theunder-cross-linking resulted in a portion of the lipase-CLECthat was soluble in the first stage (pH 4.00) of the dissolutiontest. Indeed, as shown in Figure 3, SEC data confirm thepresence of monomer in sample A. The solubility properties ofthe monomer differ from those of the cross-linked material,with the monomer demonstrating greater solubility at lowerpH. It is likely that the un-cross-linked monomer and minimallycross-linked components were soluble in stage 1 medium (pH4.00), which would result in the decreased overall releaseobserved in stage 2, as shown in Figure 9. This understandingmay also explain the <100% overall total release for sample B.Indeed, the SEC results presented in Figure 3 demonstratelower amounts of high MW peak and more later-elutingspecies, presumably with less cross-linking, for sample B thanfor C or D. Like sample A, this may result in a portion of thelipase-CLEC being soluble in stage 1, resulting in lowerapparent release in stage 2. In contrast, the lower overall releaseobserved for sample D is likely due to the lower solubility of theover-cross-linked material, even at the higher pH of stage 2. Forexample, the solubility study (Table 2) indicates that sample Dhas only 50% solubility at pH 6.0, while samples A, B, and Chave approximately 90% solubility at the same pH. These datademonstrate that there is a population of highly cross-linkedmaterial that is not soluble at approximately pH 6.0, leading tothe decreased release/recovery from sample D in stage 2, asshown in Figure 9.

Figure 9. Stage 2 (pH 6.25) release profiles of lipase-CLEC fromliprotamase drug product generated with lots A (●), B (■), C (◆),and D ( × ) of variably cross-linked lipase-CLEC. The x-axisrepresents time after initiation of stage 2. Stage 1 was pH 4.00. Errorbars represent ±1 standard deviation (n = 3 capsules).

Figure 10. Correlation between % released at 3 min (Apparatus 4stage 2, pH 6.25) and cross-linking as measured by % high MW peakby SEC.



These results serve as an important risk assessment of theimpact of over- or under-cross-linking of lipase-CLEC withBS3. These analyses reveal that a possible risk with under-cross-linked material may be that it would not remain insoluble in thelow-pH gastric environment, and thus not achieve full release atthe higher pH values in the small intestine where it is intendedto act. Conversely, over-cross-linked material may not have thedesired solubility properties to release even at the elevated pHof the small intestine. However, the Apparatus 4 data presentedin Figure 9 show that for both under- and over-cross-linkedsamples, as well as samples with a nominal level of cross-linking, ≥80% lipase-CLEC release was observed in the secondstage of the test (i.e., at pH 6.25). These results indicate thatover- or under-cross-linked material within the ranges examinedhere largely maintained insolubility through the low-pH stage 1and subsequent release in the higher-pH stage 2.In addition to characterizing lipase-CLEC release with the

dual-stage Apparatus 4 method with a UV end point, it was alsodesirable to understand lipase-CLEC release from an enzymeactivity standpoint to ensure that the lipase-CLEC remainedactive and capable of acting on fatty acid substrates in the pHconditions investigated in this study. The data presented inFigure 11 show lipase-CLEC release from liprotamase capsulesin pH 5.00 (Figure 11A) and pH 6.50 (Figure 11B) 10 mMcitrate/phosphate buffer. The capsules tested were those thatwere generated with lipase-CLEC with different degrees ofcross-linking described above, and the % released wasnormalized to the total theoretical lipase activity per capsulebased on the specific activity of each lot used (Table 2) and themass of lipase-CLEC in each capsule. These single-stage datawere generated with the USP Apparatus 2 (paddle) dissolutionapparatus and the USP lipase enzyme activity test.16 Within thevariability of the Apparatus 2 test, it was experimentallyconfirmed that no difference in release rate was observedbetween 10 mM (used for Apparatus 4 testing described above)and 15 mM (used for Apparatus 2 testing) citrate/phosphatebuffer.Figure 11A shows that, at pH 5.00, there is a strong impact of

degree of cross-linking on rate of lipase-CLEC release. Forexample, the rate of release from 0 to 10 min from the samplewith the lowest degree of cross-linking (A) was >2.5× fasterthan the rate of release from the sample with the greatestdegree of cross-linking (D). The final time point from the“infinity” spin essentially measures the solubility of lipase-CLEC in that medium. Thus, the differences in rate of releaseare very likely due to differences in the solubility of the variousdegrees of cross-linking of the lipase-CLEC at pH 5.00. Incontrast, Figure 11B shows the rate of release of the variablycross-linked material at pH 6.50. There is much lessdiscrimination between the two extremes of cross-linking atpH 6.50 than at pH 5.00. At pH 6.50, the rate of release from 0to 10 min from the sample with the lowest degree of cross-linking (A) was only 1.3× faster than the rate of release fromthe sample with the greatest degree of cross-linking (D). It islikely that the differences in solubility of the cross-linkedmaterial also drive the differences in rate of release at pH 6.50.For example, with the exception of sample B, the “infinity” timepoint results at pH 6.50 correlate as expected with the degree ofcross-linking (i.e., greater levels of cross-linking, as measured byBS3:lipase molar ratio, CE, and SEC, result in lower overall %released). It is important to note that the method used tomeasure enzyme activity has an inherently higher level ofvariability (up to 10% RSD) compared to HPLC or UV

methods, and this variability most likely was the source of therelatively lower recovery of activity for sample B at the finaltime point. The activity-based end point tests are important,however, to demonstrate that the lipase-CLEC remainsenzymatically active after release from the drug product capsule.The data presented in Figure 11 can be useful to develop an

understanding of product performance. For example, asobserved from the Apparatus 4 work described previously, atelevated pH values (6.25 and 6.50), capsules manufactured withlipase-CLEC across all levels of cross-linking achieved 75% orgreater release within the time frame of the dissolution test.This demonstrates that while cross-linking plays an importantrole in the rate of drug product release, even the extremes ofcross-linking examined here result in essentially completerelease at pH values ≥6.25. In contrast, however, lower pHvalues (e.g., pH 5.00 shown in Figure 11A) result in muchgreater discrimination between amounts released as a function

Figure 11. Release profiles of lipase-CLEC from liprotamase drugproduct generated with lots A (◆), B (■), C (▲), and D ( × ) ofvariably cross-linked lipase-CLEC at pH 5.00 (A) and pH 6.50 (B)with USP 2 (paddles) dissolution apparatus and enzyme activity-basedend point. Error bars represent ±1 standard deviation (n = 3 capsules).



of cross-linking. This is an important attribute to understandsince patients with cystic fibrosis have been characterized ashaving a lower duodenal pH than the overall population.31 Thisknowledge of the specific patient population reinforces theneed for robust control over the degree of cross-linking duringthe manufacturing process since the patients for whichliprotamase is intended may have duodenal pH values thatmay result in variable release rates and levels as a function oflipase-CLEC degree of cross-linking.

■ CONCLUSIONSCross-linking of lipase monomer to generate lipase-CLECrepresents a novel method of conferring low-pH stability to thecritical component of liprotamase, and ensures its activity ismaintained until reaching the duodenum where it is intended toact on food substrates. The degree of cross-linking of lipase-CLEC was characterized by capillary electrophoresis and sizeexclusion chromatography, and the impact of cross-linking onthe Critical Quality Attribute of dissolution was determined.The product performance tests demonstrated that lipase-CLECpossessed the intended attributes of insolubility at lower pHvalues with subsequent solubility and release at the higher pHvalues that would be expected in the duodenum. As expected,degree of cross-linking was found to play a critical role in bothsolubility and dissolution of lipase-CLEC. A novel dual-stageApparatus 4 dissolution method was developed to demonstrateproduct performance in pH environments representative of thehuman gastrointestinal tract. Compared to the compendialApparatus 1 dissolution method for delayed-release pancreli-pase, the Apparatus 4 method described here has severaladvantages and may prove useful for understanding the pH-dependence of release from other drug products as well.Collectively, these studies confirmed that degree of cross-linking is a critical parameter that may impact in vivo release oflipase-CLEC, and also provided a risk assessment tool forunderstanding the potential impact of under- and over-cross-linked material. Similar studies could be employed tounderstand solubility and rate of release in pH environmentsunique to certain patient populations, such as those with cysticfibrosis.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors wish to acknowledge contributions from JamesZabrecky, Anne McCasland-Keller, Cynthia Brown, JeffreySherman, Richard Berglund, Gregory Beck, BenjaminMcLaughlin, David Hollowell, Bart Connor, Andrew Cleaver,Suzanne Stehr, Kelly Valenti, and Jeff Buehrer.

■ REFERENCES(1) Borowitz, D.; Stevens, C.; Brettman, L. R.; Campion, M.;Chatfield, B.; Cipolli, M. International Phase III Trial of LiprotamaseEfficacy and Safety in Pancreatic-Insufficient Cystic Fibrosis Patients. J.Cystic Fibrosis 2011, 10, 443−452.(2) Borowitz, D.; Stevens, C.; Brettman, L. R.; Campion, M.;Wilschanski, M.; Thompson, H. Liprotamase Long-Term Safety andSupport of Nutritional Status in Pancreatic-Insufficient Cystic Fibrosis.Clin. Trials 2012, 54, 248−257.

(3) Livesey, G. A Perspective on Food Energy Standards forNutrition Labelling. Br. J. Nutr. 2001, 85, 271−287.(4) St. Clair, N. L.; Navia, M. A. Cross-Linked Enzyme Crystals asRobust Biocatalysts. J. Am. Chem. Soc. 1992, 114, 7314−7316.(5) DeSantis, G.; Jones, J. B. Chemical Modification of Enzymes forEnhanced Functionality. Curr. Opin. Biotechnol. 1999, 10, 324−330.(6) Margolin, A. L. Novel Crystalline Catalysts. Trends Biotechnol.1996, 14, 223−230.(7) Lee, T. S.; Vaghjiani, J. D.; Lye, G. J.; Turner, M. K. A SystematicApproach to the Large-Scale Production of Protein Crystals. EnzymeMicrob. Technol. 2000, 26, 582−592.(8) Quiocho, F. A.; Richards, F. M. Intermolecular Cross Linking of aProtein in the Crystalline State: Carboxypeptidase-A. Proc. Natl. Acad.Sci. U.S.A. 1964, 52, 833−839.(9) Margolin, A. L.; Navia, M. A. Protein Crystals as Novel CatalyticMaterials. Angew. Chem., Int. Ed. 2001, 40, 2204−2222.(10) Staros, J. V. N-Hydroxysulfosuccinimide Active Esters: Bis(N-hydroxysulfosuccinimide) Esters of Two Dicarboxylic Acids AreHydrophilic, Membrane-Impermeant, Protein Cross-Linkers. Biochem-istry 1982, 21, 3950−3955.(11) Kim, K. K.; Song, H. K.; Shin, D. H.; Hwang, K. Y.; Suh, S. W.The Crystal Structure of a Triacylglycerol Lipase from Pseudomonascepacia Reveals a Highly Open Conformation in the Absence of aBound Inhibitor. Structure 1997, 5, 173−185.(12) International Conference on Harmonization section Q8(R2)Pharmaceutical Development.(13) Elving, P. J.; Markowitz, J. M.; Rosenthal, I. Preparation ofBuffer Systems of Constant Ionic Strength. Anal. Chem. 1956, 28,1179−1180.(14) United States Pharmacopeia <711> Dissolution, USP 36, 2013,307−313.(15) Japanese Pharmacopeia 6.10 Dissolution Test, JP 16, 137−141.(16) United States Pharmacopeia Official Monograph for Pancreli-pase, USP 36, 2013, 4675−4676.(17) Rathi, P.; Saxena, R. K.; Gupta, R. A Novel Alkaline Lipase fromBurkholderia cepacia for Detergent Formulation. Proc. Biochem. 2001,37, 187−192.(18) Rathi, P.; Gradoo, S.; Saxena, R. K.; Gupta, R. A Hyper-Thermostable, Alkaline Lipase from Pseudomonas sp. with the Propertyof Thermal Activation. Biotechnol. Lett. 2000, 22, 495−498.(19) Gupta, R.; Gupta, N.; Rathi, P. Bacterial Lipases: An Overviewof Production, Purification and Biochemical Properties. Appl. Micro-biol. Biotechnol. 2004, 64, 763−781.(20) Tsou, C. Review: The Role of Active Site Flexibility in EnzymeCatalysis. Biochemistry (Moscow) 1998, 63, 253−300.(21) Cui, J. D.; Sun, L. M.; Li, L. L. A Simple Technique of PreparingStable CLEAs of Phenylalanine Ammonia Lyase Using Co-aggregationwith Starch and Bovine Serum Albumin. Appl. Biochem. Biotechnol.2013, 170, 1827−1837.(22) United States Pharmacopeia Official Monograph for PancrelipaseDelayed-Release Capsules, USP 36, 2013, 4677.(23) United States Pharmacopeia Test Solutions, USP 36, 2013,1210−1218.(24) Looney, T. J. USP Apparatus 4 (Flow-Through Method)Primer. Dissolution Technol. 1996, 3, 10−12.(25) Brown, W. USP Apparatus 4 Flow Through Cell: SomeThoughts on Operational Characteristics. Dissolution Technol. 2005,12, 28−30.(26) Fotaki, N. Flow-Through Cell Apparatus (USP Apparatus 4):Operation and Features. Dissolution Technol. 2011, 18, 46−49.(27) Malagelada, J. R.; Longstreth, G. F.; Summerskill, W. H.; Go, V.L. Measurement of Gastric Functions During Digestion of OrdinarySolid Meals in Man. Gastroenterology 1976, 70, 203−210.(28) Dressman, J. B.; Berardi, R. R.; Dermentzoglou, L. C.; Ressell,T. L.; Schmaltz, S. P.; Barnett, J. L.; Jarvenpaa, K. M. UpperGastrointestinal (GI) pH in Young, Healthy Men and Women. Pharm.Res. 1990, 7, 756−751.(29) Charman, W. N.; Porter, C. J. H.; Mithani, S.; Dressman, J. B.Physiochemical and Physiological Mechanisms for the Effects of Food



mailto:[email protected]

on Drug Absorption: The Role of Lipids and pH. J. Pharm. Sci. 1997,86, 269−281.(30) Simonian, H. P.; Vo, L.; Doma, S.; Fisher, R. S.; Parkman, H. P.Regional Postprandial Differences in pH Within the Stomach andGastroesophageal Junction. Dig. Dis. Sci. 2005, 50, 2276−2285.(31) Youngberg, C. A.; Berardi, R. R.; Howatt, W. F.; Hyneck, M. L.;Amidon, G. L.; Meyer, J. H.; Dressman, J. B. Comparison ofGastrointestinal pH in Cystic Fibrosis and Healthy Subjects. Dig. Dis.Sci. 1987, 32, 472−480.(32) Geus, W. P.; Eddes, E. H.; Gielkens, H. A. J.; Gan, K. H.;Lamers, C. B. H. W.; Masclee, A. A. M. Post-Prandial Intragastric andDuodenal Acidity are Increased in Patients with Chronic Pancreatitis.Aliment. Pharmacol. Ther. 1999, 13, 937−943.(33) Zentler-Munro, P. L.; Assoufi, B. A.; Balasubramanian, K.;Cornell, S.; Benoliel, D.; Northfield, T. C.; Hodson, M. E. TherapeuticPotential and Clinical Efficacy of Acid-Resistant Fungal Lipase in theTreatment of Pancreatic Steatorrhoea due to Cystic Fibrosis. Pancreas1992, 7, 311−319.(34) Gregory, P. C. Gastrointestinal pH, Motility/Transit andPermeability in Cystic Fibrosis. J. Pediatr. Gastroenterol. Nutr. 1996, 23,513−523.(35) Zhang, Y.; Cremer, P. S. Interactions between Macromoleculesand Ions: The Hofmeister Series. Curr. Opin. Chem. Biol. 2006, 658−663.(36) Hanson, W. A. Theoretical Concepts. In Handbook of DissolutionTesting, 2nd ed.; Aster Publishing Corporation: Eugene, OR, 1991; pp13−23.



characterization of a novel cross-linked lipase: impact of cross-linking on solubility and release...

Documents