Impact of Polymer Bioconjugation on Protein Stability and Activity

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Impact of Polymer Bioconjugation on Protein Stability and Activity Investigated with Discrete Conjugates: Alternatives to PEGylation Josefine Morgenstern, Gabriela Gil Alvaradejo, Nicolai Bluthardt, Ana Beloqui, Guillaume Delaittre, and Jürgen Hubbuch Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01020 • Publication Date (Web): 17 Sep 2018 Downloaded from http://pubs.acs.org on September 18, 2018

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Impact of Polymer Bioconjugation on Protein Stability and Activity Investigated with Discrete Conjugates: Alternatives to PEGylation Josefine Morgenstern,‡,§ Gabriela Gil Alvaradejo,‡,ǁ,◊ Nicolai Bluthardt, § Ana Beloqui,ǁ,◊,† Guillaume Delaittre,*, ǁ,◊ Jürgen Hubbuch*,§ §

Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering,

Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, 76131 Karlsruhe, Karlsruhe, Germany ǁ

Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology (KIT), Hermann-

von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. ◊

Macromolecular Architectures, Institute for Chemical Technology and Polymer Chemistry

(ITCP), Karlsruhe Institute of Technology (KIT), 76128 Karlsruhe, Germany.

KEYWORDS enzyme; polymer conjugation; stability; activity; PEGylation

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ABSTRACT Covalent attachment of synthetic polymers to proteins, known as protein–polymer conjugation, is currently one of the main approaches for improving the physicochemical properties of these biomolecules. The most commonly employed polymer is polyethylene glycol (PEG), as evidenced by extensive research and clinical track records for its use in biopharmaceuticals. However, the occurrence of allergic reactions or hypersensitivity and the discovery of PEG antibodies, on the one hand, and the rise of controlled polymerization techniques and novel monomers, on the other hand, have been driving the search for alternative polymers for bioconjugation. The present study describes the synthesis, purification, and properties of conjugates of lysozyme with poly(N-acryloylmorpholine) (PNAM) and poly(oligoethylene glycol methyl ether methacrylate) (POEGMA). Particularly, conjugate species with distinct conjugation degrees are investigated for their residual activity, aggregation behavior, and solubility, by using a high-throughput screening approach. Our study showcases the importance of evaluating conjugates obtained by nonsite-specific modification through isolated species with discrete degrees of conjugation rather than on the batch level. Monovalent conjugates with relatively low molar mass polymers displayed equal or even higher activity than the native protein, while all conjugates showed an improved protein solubility. To achieve a comparable effect on solubility as with PEG, the conjugates PNAM and POEGMA of higher molar masses.

INTRODUCTION The use of engineered enzymes in the biotechnological field is rapidly increasing due to the wide range of applications available, from biocatalysis to textile, food, and pharmaceutical industries. Particularly, the pharmaceutical market has benefited from protein- or peptide-based drugs since these bring specificity for molecular targets in the body, while


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exhibiting reduced side effects.1–3 Particularly, the complex molecular structure of proteins provides the specificity required from a drug with minimal side effects. However, it also brings a series of challenges with respect to purification, formulation, storage, and delivery.4,5 Problems that need to be addressed include limited stability, low solubility, short in vivo circulation times, and unwanted immunogenicity.4,6,7 A strategy to improve the properties of these protein-based active pharmaceutical ingredients is the generation of protein–polymer conjugates by covalent attachment of a polymer to the protein of interest.7–12 This strategy offers the opportunity to combine the structural and functional features of proteins (i.e., a folded, compact structure with catalytic activity or recognition ability) with the versatility of synthetic polymers and thereby to develop novel high-performance biomaterials. Required features of the potential polymer to be grafted include high solubility in aqueous environments, biocompatibility, as well as the presence of a functional group available for conjugation to the protein.13 Notably, so-called “stealth” polymers are valuable because they may enhance the physicochemical properties of the protein, while also providing improved pharmacokinetics – particularly blood circulation time – by regulating interactions with the immune system. Polyethylene glycol (PEG) is the most commonly used FDA-approved hydrophilic polymer in biomedical applications. PEGylated proteins possess increased stability,14 solubility,15 and circulation time.16 However, the large exposure to PEG over the last decades has led to the occurrence of allergic reactions or hypersensitivity and the formation of antibodies against PEG conjugates,17–22 which results in an increased clearance rate through the immune system,23 defeating the purpose of conjugation. In addition, depending on the PEG hydrodynamic size, larger conjugates cannot be excreted from the body and accumulate as vacuoles in the liver, the kidneys, or the spleen.24,25 Such findings have increased the interest for alternatives to PEG, and several polymers have recently been


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proposed.26–28 Notably, the recent advances in controlled polymerization techniques such as ringopening polymerization29 or reversible-deactivation radical polymerization30 (RDRP) enable the development of a wide range of macromolecular architectures with defined narrow distributions, and the possibility to add functional groups at specific positions, particularly at the chain ends. In addition, by varying the monomer structure, the physicochemical properties of the resulting hybrid systems can be readily modulated.31 The biological and physicochemical properties of protein–polymer conjugates depend on the careful selection of the polymer structure and architecture, its molar mass, the number of chains attached to the protein, and the position of the conjugation sites. Fine tuning of these parameters is crucial to optimize activity and stability attributes of the conjugates.32,33 However, the underlying physicochemical interactions between polymer and protein, as well as the effect of these interactions on the conjugate properties, are still not fully understood. The development and processing of conjugates with specific features hence require the establishment of standardized methods to quantify the influence of the aforementioned parameters.34 In this study, poly(N-acryloylmorpholine) (PNAM) and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) of distinct molar masses were synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization and conjugated to lysozyme as a model protein in a nonsite-specific manner. The heterogeneous mixtures of conjugates were then fractionated by cation-exchange (CEX) chromatography to isolate “discrete” conjugates, i.e., lysozyme with a defined number of grafted polymer chains (Scheme 1). PNAM and POEGMA are interesting choices to explore as PEG alternatives in a therapeutic context. They have been shown to be biocompatible in many cases but are not (yet) FDA-approved. Nevertheless, they present interesting features. PNAM is a water-soluble acrylamide derivative that has been


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synthesized by RAFT polymerization and modified for selective attachment to surfaces, as well as to enzymes to reduce immunogenicity.35 For instance, synthesis of drug–PNAM conjugates has been reported for doxorubicin and amoxicillin, albeit without further evaluation.35 In addition, PNAM conjugated to a lipase resulted in hybrids with a higher solubility in organic solvents and higher remaining activity when compared to its reported PEG analogue.35 POEGMA, on the other hand, is a hydrophilic, PEG-based comb-like polymer, that has been extensively studied for potential biomedical applications. Particularly, in comparison to linear polymers, POEGMA may adopt peculiar conformations in solution, as well as at the surface of the proteins, where it has been observed to impart a molecular sieving character.36,37 POEGMA can be synthesized by various RDRP techniques and modified to introduce specific functionalities to the polymeric chain ends depending on the initiator or transfer agent used.38–40 POEGMA has been conjugated to various proteins using grafting onto or grafting from approaches, in a random fashion or site-selectively.41–44 Despite its PEG-like chemical composition, it is possible to quantitatively eliminate PEG antigenicity by careful structural design.45 In addition, in vivo studies showed an increased blood circulation time of 15–50 times, as well as an increased tumor accumulation when compared to the native protein.43,46 We present an automated high-throughput approach to assess the influence of polymer molar mass and protein conjugation degree on the functional and colloidal stability of PNAM- and POEGMA-based protein–polymer conjugates. The solubility, aggregation (phase) behavior, and residual in vitro activity are evaluated. To this end, lysozyme was conjugated to Nhydroxysuccinimidyl (NHS) ester-functionalized PNAM and POEGMA in a random fashion, and the discrete conjugates were isolated by CEX chromatography. Subsequently, protein phase diagrams were generated using an automated liquid handling station. To do so, 96-batch systems


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with varying concentrations of conjugate and precipitant were prepared on a microtiter plate and examined for phase behavior and solubility. The residual enzyme activity after conjugation was assessed and compared to the native protein, as well as to analogue PEG conjugates, using the lysozyme-specific activity assay based on the substrate Micrococcus lysodeikticus.

Scheme 1. Graphical illustration of the process employed in the current study to produce lysozyme–polymer conjugates with discrete degrees of conjugation.

EXPERIMENTAL PART Materials 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester (NHS-ACPDB; Sigma Aldrich), 1,4-dioxane (Sigma Aldrich, 99%), acetonitrile (anhydrous, 99.8%, Sigma Aldrich), dichloromethane (DCM; ≥ 99.5%, VWR), diethyl ether (99.5%, Roth), sinapic acid (98%, Sigma Aldrich), and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB; ≥ 99.0%, Sigma Aldrich) were used as received. N-acryloylmorpholine (NAM; 97%, Sigma Aldrich) was distilled from calcium hydride prior to use. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA; Mn = 300 g mol–1, Sigma Aldrich) was passed through a basic alumina


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column prior to polymerization. 2,2’-Azobis(2-methylpropionitrile) (AIBN; 98%, Aldrich) was recrystallized from methanol. The buffered solutions were prepared using sodium citrate tribasic dihydrate (Sigma Aldrich) for pH 3, sodium acetate trihydrate (Sigma Aldrich) for pH 5, and sodium phosphate monobasic dihydrate (Sigma Aldrich) and disodium hydrogen phosphate dihydrate (Merck) for pH 7 and pH 7.2.

Instrumentation 1

H nuclear magnetic resonance (NMR) spectroscopy

Measurements were performed on a Bruker AM 500 spectrometer (500 MHz). All compounds were dissolved in CDCl3 and the residual solvent peak was used for shift correction (7.26 ppm).

Matrix-assisted laser desorption ionization coupled to time-of-flight (MALDI-ToF) mass spectrometry Mass spectra were acquired with a 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) in positive ion linear mode with a mass range of 1000 to 40000 Da. The laser intensity was set to 4800. The spectra obtained represent the average of laser shots taken by an automatic scheme measuring spectra over the whole spot. The matrices used were sinapic acid for conjugated proteins and DCTB for the polymer samples. Peak lists were generated using Data Explorer Software 4.0 (Applied Biosystems).

Size-exclusion chromatography (SEC) for polymers The measurements were performed on a TOSOH EcoSEC HLC-8320 GPC System, comprising an autosampler, a SDV 5 µm bead size guard column (50 × 8 mm, PSS) followed by three SDV


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5 µmm columns (300 × 7.5 mm, subsequently 100 Å, 1000 Å, and 105 Å pore size, (PSS)), and a differential refractive index (DRI) detector using THF as eluent at 30 °C with a flow rate of 1 mL min–1. The SEC system was calibrated using linear polystyrene standards ranging from 266 to 252 × 106 g mol–1. Before injection, the samples were filtered using a 0.2 µm filter.

High-throughput capillary gel electrophoresis (HT-CGE) for proteins and conjugates To assess the purity of the POEGMA conjugates, automated capillary gel electrophoresis was performed according to our previous work.47 Briefly summarized, the Caliper LabChip® GX II with an HT Protein Express LabChip® and an HT Protein Express Reagent Kit (all Perkin Elmer, USA) were used. Experiments were carried out using the HT Protein Express 200 assay in the LabChip® GX 3.1 software. The LabChip® GX II installation, sample preparation, and analysis were performed according to the manufacturer standard protocol. Sample preparation was performed in skirted 96-well polypropylene twin.tec® PCR plates from Eppendorf (Germany). Molecular weight determination was performed according to protein standards from the HT Protein Express Reagent Kit. An apparent increase in molecular weight of conjugate species, was taken into account according to our previous work.15,47

Polymer synthesis RAFT polymerization of N-acryloylmorpholine In a 250 mL round-bottom flask, NHS-ACPDB (1.0 eq.), AIBN (0.3 eq.), and NAM (34 or 68 equivalents) were successively introduced. Trioxane was added as reference. The mixture was then dissolved in 1,4-dioxane to reach a final monomer concentration of 1.6 M, before being deoxygenated by nitrogen bubbling for 30 minutes. The polymerization mixture was heated to 70


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°C and monitored by 1H NMR spectroscopy until the target conversion value (70%) was reached. The polymerization was stopped by cooling the reaction flask. After evaporation of the solvent under reduced pressure and dissolving the residue in DCM, the polymer was precipitated twice in diethyl ether, centrifuged, separated, and dried under vacuum. The dry product was analyzed by SEC, MALDI-ToF, and 1H NMR spectroscopy.

RAFT polymerization of oligo(ethylene glycol) methyl ether methacrylate In a 250 mL round-bottom flask, NHS-ACPDB (1.0 eq.), AIBN (0.2 eq.), and OEGMA (20 or 40 equivalents) were successively introduced. The mixture was dissolved in acetonitrile to reach a final RAFT agent concentration of 16 mM, before being deoxygenated by nitrogen bubbling for 30 minutes. The polymerization mixture was then heated to 70 °C and monitored by 1H NMR spectroscopy until the target conversion value (50%) was reached. The polymerization was stopped by cooling the reaction flask. After evaporation of the solvent under reduced pressure and dissolving the residue in DCM, the polymer was precipitated twice in a 1:1 v/v mixture of petroleum ether:diethyl ether. The resulting polymer was a pink viscous liquid that was separated by decantation of the supernatant and dried under vacuum. The dry product was analyzed by SEC, MALDI-ToF, and 1H NMR spectroscopy.

Protein conjugation Conjugation experiments were performed batch-wise in 50 mL Falcon tubes (BD Biosciences). For conjugation, lysozyme and the respective polymer were dissolved in 25 mM sodium phosphate buffer at pH 7.2. The concentration of lysozyme was set to 0.28 mM. PNAM and POEGMA were added based on a molar polymer-to-protein ratio of 2. For the reaction, the tube


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was continuously shaken in an overhead shaker (LabincoLD79, Labinco BV) for 1 h at room temperature. Purification of protein–polymer conjugates The preparative separation of protein and protein–polymer conjugates with varying numbers of attached polymer molecules was performed on an ÄKTA® purifier system equipped with a Fraction Collector Frac-950 (GE Healthcare). A 5 mL prepacked Toyopearl GigaCap S-650M cation-exchange (column (TOSOH Bioscience) was used as stationary phase. For column loading, the system was equilibrated in 10 mM sodium acetate buffer (pH 5). In order to reduce the influence of unbound polymer on the binding behavior of the conjugates to the CEX resin, the conjugation batch was diluted 1:6 in 10 mM sodium acetate buffer (pH 5). Injection of the diluted batch was performed using a 50 mL super loop (GE Healthcare). Elution was performed by applying an NaCl step gradient with an elution buffer containing 1 M NaCl in 10 mM sodium acetate buffer (pH 5). The NaCl concentrations used for each step of the elution of the different conjugates are displayed in Table 1. NaCl concentrations (in mM) used during CEX chromatography for the step elution of lysozyme and lysozyme–polymer conjugates at pH 5.. The flow rate for equilibration, binding, and elution was set to 1 mL min–1. For process monitoring, absorbance values at 280 nm (for protein) and 320 nm (for polymer), as well as conductivity, were employed in continuous mode. From each chromatographic separation, fractions of 2 mL were collected in a 96-well deep well plate (VWR). To obtain sufficient sample for stability assessment of the different conjugate species the corresponding fractions of multiple chromatographic runs were pooled. Peak allocation to the different conjugate species was performed by MALDI-ToF. Additionally, the


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different conjugate species were characterized by DLS and SEC-MALS in order to determine size and molecular weight of the hybrid molecules.

Table 1. NaCl concentrations (in mM) used during CEX chromatography for the step elution of lysozyme and lysozyme–polymer conjugates at pH 5. Native lysozyme

Monoconjugated lysozyme

Diconjugated lysozyme

PNAM3.4kDa

1000

360

230

PNAM6.9kDa

1000

400

175

POEGMA3.6kDa

1000

370

190

POEGMA7.5kDa

1000

240

120

Conditioning and quantification of protein–polymer conjugates The separation of protein–polymer conjugates with different NaCl molarities led to fractions with different salt concentrations. Characterizing the isolated conjugates with regard to activity and phase behavior thus required a sample reconditioning consisting of buffer exchange (resp. pH change) and concentration increase. Buffer exchange was carried out using Slide-A-Lyzer™ dialysis cassettes (Thermo Fisher Scientific) with a molecular weight cut-off of 2 kDa. Concentrating of protein samples was performed by evaporation using a vacuum concentration unit RVC 2-33CDplus (Martin Christ Gefriertrocknungsanlagen GmbH) operated at 24 mbar. Concentrations of unmodified lysozyme were determined by measuring the absorbance at 280 nm using a NanoDrop 2000c UV–Vis spectrophotometer (Thermo Fisher Scientific, USA) and an extinction coefficient of εlysozyme,280nm1% = 22.00.48 To convert mass concentrations into molar concentrations, a value of 14.3 kDa for the molecular weight of lysozyme was used.49


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Since the two polymers PNAM and POEGMA also absorb at 280 nm (Figure S5), the total absorbance at 280 nm (A280nm) is described by Equation 1 for protein–polymer conjugates. Equation 1 = , + , In order to quantify the molar concentration of the protein–polymer conjugates, measurements solely at 280 nm are therefore not sufficient. However, the two polymers absorb at 320 nm due to the RAFT group,50 while lysozyme does not (Figure S5). Thus, by measuring the absorbance of discrete conjugate species at 320 nm as well as at 280 nm, it is possible to quantify the concentration of lysozyme and thereby the concentration of the conjugate species. The parameter A280nm,polymer has to be substituted by a polymer specific factor which is independent of the overall A280nm spectra. By introducing K as the following intensity ratio for a given polymer concentration C Equation 2 () =

, ,

it is possible to rearrange Equation 1 and calculate the absorbance A280nm,lysozyme as follows: Equation 3 , = − ∗ , with K being the mean value of K(C) measured for 10 polymer concentrations. For polymer calibration, absorption spectra between 240 nm and 340 nm were recorded for polymer solutions (in conjugation buffer) at 10 concentrations between 0.1 and 1.0 mg mL–1 using the NanoDrop 2000c UV–Vis spectrophotometer. Using the respective molar mass of the polymer, the mass concentrations were converted to molar concentrations. From the recorded polymer spectra, the


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absorbance values were extracted for 280 nm and 320 nm. In order to convert the calculated absorption A280nm,lysozyme into a concentration, the extinction coefficient of ε280nm,lysozyme1% = 22.00 was used as for unmodified lysozyme.47 All concentration measurements were performed as triplicates. A potential aminolysis of the RAFT end group during incubation with lysozyme – due to the presence of free lysines – was ruled out by monitoring the UV absorption at 320 nm for 24 hours. As shown in Figure S6, for none of the investigated polymers a decrease in A320nm was observed, which validates the present concentration determination method.

SEC-(UV-MALS(QELS)-RI) analysis of proteins and conjugates The SEC-(UV-MALS(QELS)-RI) setup consisted of on ÄKTA™ purifier system equipped with a UV detector (UV-900, 10 mm optical path length, GE Healthcare, Sweden) in line with a multiangle light-scattering (MALS) detector (DAWN-HELEOS, Wyatt Technology Corporation, USA) operating at a wavelength of 659 nm, a quasi-elastic light scattering (QELS) module embedded at angle of 99° of the MALS detector within the DAWN-HELEOS instrument (Wyatt Technology Corporation, USA), and a differential refractive index (RI) detector operating at a wavelength of 658 nm (Optilab rEX, Wyatt Technology, USA). Prior to measurements, normalization of the MALS detector was performed using bovine serum albumin (BSA) (Aldrich) as isotropic scatterer (

Impact of Polymer Bioconjugation on Protein Stability and Activity

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