Thermoresponsive Agarose Based Microparticles for Antibody

Article pubs.acs.org/Biomac

Thermoresponsive Agarose Based Microparticles for Antibody Separation Huey Wen Ooi,†,∥ Benedikt Ketterer,† Vanessa Trouillet,‡ Matthias Franzreb,*,† and Christopher Barner-Kowollik*,§,∥ †

Institute for Functional Interfaces (IFG), ‡Institut für Angewandte Materialien (IAM) and Karlsruhe Nano Micro Facility (KNMF), and §Institut für Biologische Grenzflächen (IBG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ∥ Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany S Supporting Information *

ABSTRACT: We report the development of thermoresponsive 4mercaptoethylpyridine (MEP)-based chromatographic microsphere based resins for antibody separation that show switchable release abilities by adsorbing immunoglobulins at 40 °C and releasing the proteins at 5 °C. The thermoswitchable release properties were introduced to the porous resins by the grafting of linear poly(Nisopropylacrylamide) (PNIPAM) chains synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization, which were modified to possess MEP end functionalities. Adsorption of γglobulins as a model antibody on the shortest PNIPAM-MEP (3 kDa) grafted microparticles display binding capacities of up to 20 g L−1 at 40 °C and a significant decrease in binding capacity to less than 2.5 g L−1 at 5 °C. By switching the temperature to 5 °C, the release of bound γglobulins is shown to be as high as 90%. The effects of polymer chain length on the binding capacity are studied in detail and found to be critical as they influence the density of MEP functionalities on the particle surfaces.

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INTRODUCTION The development of biopharmaceuticals is a key research field, which requires purification and recovery processes to obtain high purity products.1−3 In general, the purification of biologics involves process schemes with often eight or more unit operations. The main purification process typically requires several liquid chromatography steps. There exist variable types of chromatography systems available today, and they are all based on different physicochemical principles. However, today’s systems are associated with specific disadvantages, which include high expenses, high consumption of hazardous solvents and chemicals for the elution, as well as the need to remove the elution chemicals through the incorporation of additional process steps. In the process of antibody purification, Protein A (Staphylococcus aureus) chromatography is a commonly employed chromatographic step for process-scale purification of monoclonal antibodies (mAbs). This bioaffinity chromatography functions by exploiting the specific interactions that occur between the Fc region of mAbs and the immobilized Protein A.4 Adsorption of antibodies occurs at near neutrality and physiological ionic strength, while elution is performed at a low pH. One of the limitations of the Protein A resins is the high cost of the materials. In addition, as a protein is utilized as © 2015 American Chemical Society

ligand in this mode of chromatography, the ligand is prone to proteolysis and the cleaved products can require additional separation steps for removal.5,6 Additional limitations are associated with choosing suitable conditions for storage and cleaning. Other than Protein A, there are alternative types of synthetic ligands that are utilized for the same purpose. One example is thiophilic chromatography, where immobilized structures that contain sulfur and nitrogen atoms have also shown good selectivity for antibodies.7 The mechanism is similar to hydrophobic-based chromatography with the adsorption occurring at high salt concentrations and elution occurring when the salt concentrations are lowered. Thiophilic sorbents also showed higher selectivity for immunoglobulins (IgGs) compared to hydrophobic chromatography, which only adsorbs hydrophobic proteins. An increasingly popular alternative to Protein A chromatography is hydrophobic charge-induction chromatography (HCIC). HCIC utilizes pH-dependent ligands to combine thiophilic, hydrophobic, and an ionizable pyridine ring. The adsorption of antibodies occurs via hydrophobic interactions Received: October 15, 2015 Revised: November 28, 2015 Published: December 1, 2015 280

DOI: 10.1021/acs.biomac.5b01391 Biomacromolecules 2016, 17, 280−290

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Biomacromolecules

Scheme 1. Schematic Overview of the Synthetic Steps for the Grafting of the PNIPAM-MEP Polymers onto Agarose-Based Microparticles (Superose) Reported in the Current Work

temperature-modulated separation of angiotensin peptide subtypes.15,16 In general, polymer chains can be attached onto microparticle surfaces by either a “grafting-to” or “grafting-from” approach.17−19 The “grafting-to” approach is advantageous as it allows the in-depth characterization of the precusor polymer. However, there may be limitations in achieving high grafting densities due to steric hindrance. In contrast, higher grafting densities can be achieved with the “grafting-from” approach, which is experimentally more challenging due to the complex determination of the grafted chain length on the surface. As opposed to the commonly employed free radical polymerization processes, which lead to irreversibly terminated polymeric material, reversible-deactivation radical polymerization (RDRP) techniques, such as reversible addition− fragmentation chain-transfer (RAFT) polymerization, allow for the syntheses of polymers with predetermined molecular weight and narrow molar mass dispersity.20,21 These techniques additionally provide the opportunity to control the molecular architecture to afford complex molecular entities via either end group modification or chain extension reactions. Goldmann et al. have shown the effects of imparting poly(divinylbenzene) microspheres with thermoresponsive properties via the “grafting-to” approach, using a radical thiol−ene reaction to attach RAFT-synthesized PNIPAM.22 Similarly, Ohno and coworkers prepared RAFT-agent functionalized silica particles and successfully performed the polymerization of different monomers via the “grafting-from” approach with good control over the molecular weight and molar mass dispersity.23 In the current study, we present the design and development of thermoresponsive cross-linked agarose-based chromatographic microsphere based resins capable of temperatureinduced release of immunoglobulins. At temperatures above the LCST of PNIPAM, immunoglobulins were adsorbed onto the

and via a change in the pH of the mobile phase, bound antibodies take a net ionic charge similar to the stationary phase, hence promoting elution.8 4-Mercaptoethylpyridine (MEP), which possesses a pyridine ring, has been attached to hydrophilic resins via hydrophobic spacers and has shown comparable selectivity for antibodies such as Protein A.9 MEP appears to be noncharged at neutral conditions and becomes positively charged when the pH is less than 4.8. This characteristic of MEP is utilized to promote the adsorption of antibodies at almost neutral conditions and to induce desorption at low pH conditions. Although MEP-based chromatography has shown specific benefits when compared to classical methods,10 there is still a need to use special elution buffers, which have to be removed at later stages.11 The use of stimuli-responsive polymer matrices for chromatography is highly attractive as these materials are capable of undergoing conformational changes based on small external changes in their environment.12 Poly(N-isopropylacrylamide) (PNIPAM) is the most widely used thermoresponsive polymer with a lower critical solution temperature (LCST) of approximately 32 °C.13 Its ability to undergo phase transition from a coil to globule conformation at physiological temperature renders it attractive for the development of materials that are used for biomolecule separation. For example, Maharjan and co-workers developed a temperature-responsive ionexchange agarose based resin by grafting cross-linked poly(NIPAM-co-acrylic acid-co-N-tert-butylacrylamide-co-N,N′methylenebis(acrylamide)).14 Lactoferrin, which is a cationic protein, was found to adsorb three times higher at 50 °C than at 20 °C, and desorption of the proteins could be induced when temperatures were lowered. Similary, by using silica beads, Kobayashi et al. grafted poly(NIPAM-co-acrylic acid-co-N-tertbutylacrylamide) hydrogels as sorbents for anionic-exchange thermoresponsive chromatography and have shown successful 281


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Aminolysis/Michael Addition of PNIPAM with N,N′-Methylenebis(acrylamide). PNIPAM (2600 Da, 1.3767 g, 5.22 × 10−4 mol) and TCEP (6.2 mg, 2.16 × 10−5 mol) were weighed into a round-bottom flask. Tetrahydrofuran (4 mL) was added, and the yellow solution was purged with nitrogen for approximately 30 min. 1-Butylamine (0.3832 g, 5.24 × 10−3 mol) was added via a syringe and the solution was further purged for an additional 15 min before being left to react for 1 h. The yellow solution turned colorless. A further round-bottom flask was loaded with N,N′-methylenebis(acrylamide) (1.8092 g, 1.17 × 10−2 mol) and purged with nitrogen. The aminolyzed polymer solution was subseqeuntly transferred to this round-bottom flask via a syringe. The reaction was left to stir 16 h. THF was removed under vacuum, and the polymer was dissolved in water before being dialyzed in a 1 kDa MWCO tube overnight. The water was removed using a freeze-dryer to yield PNIPAM-BA. The polymers were analyzed by NMR and GPC (Table S2). The 3000 g mol−1 PNIPAM was also analyzed by ESI-MS (Table S3). Michael Addition of PNIPAM-BA and MEP. MEP (1.0594 g, 3.67 × 10−3 mol) was weighed into a vial and dissolved in deionized water (20 mL). The pH of the solution was adjusted to approximately 10−11 with NaOH as indicated by pH paper. PNIPAM-BA (0.9269 g, 3.45 × 10−4 mol) was weighed into a round-bottom flask, and the MEP solution was transferred therein. Hexylamine (34.9 mg, 3.45 × 10−4 mol) was added and the reaction was left to stir for 16 h. The reaction solution was transferred to a 1 kDa MWCO tube and dialyzed overnight. The water was removed under reduced pressure to yield PNIPAM-MEP and the polymers were analyzed by NMR and GPC (Table S2). The 3000 g mol−1 PNIPAM was also analyzed by ESI-MS (Table S3). Grafting of PNIPAM-MEP onto Superose. The Superose particles were washed and suction-drained with copious amounts of water, acetone and dichloromethane. The particles were subsequently dried in a vacuum desiccator overnight before use. Superose (0.1218 g) was weighed into a 10 mL round-bottom flask, followed by addition of PNIPAM-MEP (0.1013 g, 3.59 × 10−5 mol), EDC (0.0372 g, 1.94 × 10−4 mol), DMAP (0.0237 g, 1.94 × 10−4 mol), and DPTS (0.0563 g, 1.91 × 10−4 mol) before sealing the flask with a septum. The flask was deoxygenated via five cycles of purging with nitrogen and vacuum before being kept under a constant nitrogen flow. Anhydrous dichloromethane (4.5 mL) was added to the flask and the reaction was left to react for 18 h while shaking at 700 rpm. The SuperosePNIPAM-MEP microparticles were subsequently washed with copious amounts of dichloromethane, acetone, water, and acetone before being left in a vacuum desiccator to dry. Grafting of MEP onto Superose. Superose particles (2 mL) were suction-drained with copious amounts of water before being transferred to a 15 mL falcon tube. Allyl glycidyl ether (0.5707 g, 5.00 × 10−3 mol) was added to 2 mL of 3 M NaOH in a glass vial. The solution was added to the falcon tube and the reaction was left to shake at 700 rpm for 18 h. The microparticles were washed with copious amounts of water and used immediately for the following step. The suction-drained allyl-activated Superose particles were transferred to a falcon tube, which contained N-bromosuccinimide (0.1484 g, 8.34 × 10−4 mol). Next, 50% acetone (2 mL) was added to the tube. The reaction was left to shake at 700 rpm for 1 h at 30 °C. Subsequently, the microparticles were washed with copious amounts of water and acetone before being used for the next step. Here, MEP (0.1480 g, 5.13 × 10−4 mol) and NaBH4 (2.5 mg, 8.67 × 10−6 mol) were weighed into a glass vial. Then 2 mL of 1 M carbonate buffer was added to the vial and the pH was adjusted to approximately 10. Suction-drained brominated Superose was transferred to a falcon tube, and the MEP solution was added. The reaction was left to react at 700 rpm at 30 °C for 18 h. Subsequently, the microparticles were washed with water and acetone before being left in a vacuum desiccator to dry. Protein Adsorption Studies. The microparticles were transferred to a falcon tube before being swelled in 15 mL of 1 M carbonate buffer, (pH 10). The particles were subsequently centrifuged for 10 min at 3000 rpm, and the supernatant was discarded. Next, 15 mL of the carbonate buffer was added to the tube and mixed with a vortex mixer. The particles were centrifuged, and the supernatant was discarded.

MEP functional microparticles and the release of proteins was induced when the temperature was lowered below the LCST of the polymers. Different chain length poly(N-isopropylacrylamide) telechelic polymers of narrow molar mass dispersities were synthesized via RAFT polymerization in solution and subsequently end group modified with MEP, which is known to show high selectivity for antibodies. The PNIPAM-MEP polymers were subsequenly utilized to decorate cross-linked agarose microparticles via the “grafting-to” approach. The synthetic design is presented in Scheme 1. γ-Globulins were employed as model immunoglobulins to demonstrate the temperature-switchable release abilities of the synthesized microparticles.

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MATERIALS AND METHODS

Materials. Agarose-based microparticles (Superose 6 Prep grade, GE Healthcare, 30−40 μm), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%, Alfa Aesar), 4-mercaptoethylpyridine HCl (MEP, 97%, Carbosynth), tetrahydrofuran (THF, EMSURE, Merck), diethyl ether (AnalaR Normapur, VWR Chemicals), hexylamine (for synthesis, Merck), 1-butylamine (99%, Alfa Aesar), N,N′methylenebis(acrylamide) (99%, Sigma-Aldrich), N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, 98+ %, Alfa-Aesar), 4-(dimethylamino)pyridine (DMAP, for synthesis, Merck), dichloromethane (DCM, anhydrous, VWR Prolabo), allyl glycidyl ether (for synthesis, Merck-Schuchardt), N-bromosuccinimide (for synthesis, Merck-Schuchardt), sodium borohydride (98%, AlfaAesar), and γ-globulins (Fraction II, Bovine Blood, Affymetrix (USB Products)). N-Isopropylacrylamide (97%, Aldrich, NIPAM) and 2,2′azobis(2-methylpropionitrile) (≥98% GC, Fluka Analytical, AIBN) were recrystallized in n-hexane and methanol, respectively, before use. S-1-Dodecyl-S′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DDTTC) was synthesized according to Lai et al.24 4- (N,NDimethylamino)pyridinium-4-toluenesulfonate (DPTS) was synthesized as reported.25 Description for the syntheses of DDTC and DPTS are included in the Supporting Information. 1,4-Dioxane (GPR RECTAPUR, VWR Chemicals) was passed through basic alumina before use. Methods. RAFT Polymerization of NIPAM. NIPAM (7.8865 g, 6.97 × 10−2 mol), DDTTC (0.2726 g, 7.48 × 10−4 mol), and AIBN (0.013 g, 7.92 × 10−5 mol) were weighed into a glass vial. 1,4-Dioxane (12.44 mL) was added, and the reaction solution was stirred until all solids were dissolved. Next, 50 μL of the solution was removed and stored for NMR analysis (t = 0 h). Then 2 mL of the reaction solution was added into five individual glass tubes. The tubes were sealed with rubber septa before being purged with nitrogen for approximately 40 min. The tubes were subsequently placed in a 60 °C heating block and left to react. The polymerization was stopped at specific times by immersing the tubes in an ice bath. Then 50 μL was removed from each tube for NMR analysis (t = 1, 2, 3, 4, and 21 h). The remaining reaction solutions were purified via dialysis in 1 kDa MWCO tubes for 3 days. The water was subsequently removed using a freeze-dryer to yield PNIPAM. The Mn of the polymers was determined via 1H NMR and GPC (Table 1). The 3000 g mol−1 PNIPAM was also analyzed by ESI-MS (Table S3).

Table 1. Molecular Weight Properties of the Different Telechelic PNIPAM Chain Lengths Used in This Work That Were Prepared by RAFT Polymerization Using DDTTC as CTA polymer

Mn,GPC (g mol−1)

Mn,NMR (g mol−1)

ĐM

PNIPAM 3 kDa PNIPAM 6 kDa PNIPAM 20 kDa

2500 5900 19 300

3000 6300 17 000

1.2 1.2 1.1 282


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Scheme 2. Synthetic Strategy for the Generation of the PNIPAM-MEP Terminal Polymers of Variable Molecular Weight

The washing step was repeated five times. The particles were subsequently transferred to a 5 mL measuring cylinder and left to settle. The carbonate buffer was added to obtain a 25% slurry (e.g., 1 mL of settled particles in a total volume of 4 mL). The slurry was subsequently transferred to a falcon tube for storage until further use. All protein adsorption studies were carried out using a Janus Automated Liquid Handling station. First, the absorbance of γ-globulin solutions of known concentrations (between 2.8 and 0.25 g L−1) was measured to calculate the extinction coefficient of the protein in carbonate buffer, followed by the addition of 50 μL of particle slurry to 100 μL of protein solution of different concentrations. Incubation was subsequently carried out at a specific temperature while shaking at 1100 rpm. The particles were left to settle for 30 min before the absorbance of the supernatant was measured at 280 nm to determine the concentration of the bound proteins. Protein Desorption Using Low pH Buffer. The microparticles that have been used for protein adsorption experiments were collected in 15 mL falcon tubes. The particles were centrifuged and the supernatant was discarded. Here, 15 mL of 0.1 M sodium acetate buffer (pH 3) was added to approximately 3 mL of particle slurry in falcon tubes. The particles were mixed with a vortex mixer and centrifuged before discarding the supernatant. The washing step was repeated three times. Subsequently, 6 mL of the same buffer was added to the particles, and the slurry was mixed at 25 °C for 30 min (750 rpm). The particles were subsequently washed five times with water and 1 M carbonate buffer (pH 10) via the washing steps noted above. The efficiency of the desorption process was evidenced via repeated protein adsorption experiments as described previously. Protein Desorption Studies via Temperature Change. A γglobulin solution of 10 g L−1 was prepared in 1 M carbonate buffer (pH 10) and used for protein adsorption with a 25% particle slurry. To this end, 50 μL of particle slurry was added into an Eppendorf tube, followed by 100 μL of the γ-globulin solution. Incubation was carried out for 1 h at 40 °C while shaking at 1100 rpm, after which the solution was left to settle for 30 min. Then 70 μL of the supernatant was removed and the absorbance was measured at 280 nm. The concentration of the proteins in the supernatant before and after adsorption were determined in order to calculate the concentration of bound proteins on the particle surface (q at 40 °C). The desorption step was initiated by addition of 70 μL of carbonate buffer to the tubes before incubating them for 1 h at 5 °C, after which the solution was left to settle for 30 min. Subsequently, 70 μL of the supernatant was removed to measure the absorbance of the supernatant at 280 nm. The concentration of proteins in the supernatant before and after adsorption was determined in order to calculate the concentration of proteins remained bound on the particle surface (q at 5 °C). Gravimetric Analysis of Superose Microparticles. A known settled bed volume of Superose particle slurry in water was measured by using a measuring cylinder. The microparticles were subsequently suctiondrained and washed with acetone before being dried in a 70 °C oven

until constant mass. Measurements for three batches of Superose slurry were carried out. Characterization. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H and 13C NMR spectroscopy were performed on a Bruker AM 500 spectrometer (500 MHz). All compounds were dissolved in CDCl3, and the residual solvent peak was employed for shift correction (7.26 ppm). Gel Permeation Chromatography (GPC). GPC with N,Ndimethylacetamide (DMAc) containing 0.03 wt % LiBr as eluent was performed for PNIPAM with a sample concentration of 2 g L−1 on a Polymer Laboratories PL-GPC 50 Plus Integrated system comprising an autosampler, a PLgel 2 5 μm bead-size guard column (50 × 7.5 mm) followed by three PLgel 5 μm MixedC columns (300 × 7.5 mm), and a refractive index detector at 50 °C with a flow rate of 1 mL min−1. The GPC system was calibrated against linear poly(methyl methacrylate) standards with molecular weights ranging from 700 to 2 × 106 Da. The samples were filtered through polytetrafluorethylene (PTFE) membranes with a pore size of 0.2 μm prior to injection. Electro Spray Ionization Mass Spectrometry (ESI-MS). ESI-MS Spectra were recorded on an LXQ mass spectrometer (Thermo-Fisher Scientific, San Jose, CA) equipped with an atmospheric pressure ionization source operating in the nebulizer assisted electrospray mode. The instrument was calibrated in the m/z range 195−1822 using a standard containing caffeine, Met-Arg-Phe-Ala acetate (MRFA), and a mixture of fluorinated phosphazenes (Ultramark 1621) (all from Aldrich). A constant spray voltage of 4.5 kV was used, and nitrogen at a dimensionless sweep gas flow rate of 2 (approximately 3 L min−1) and a dimensionless sheath gas flow rate of 5 (approximately 0.5 L min−1) were applied. The capillary voltage, the tube lens offset voltage, and the capillary temperatures were set to 34 V, 90 V, and 275 °C, respectively. The samples were dissolved with a concentration of 0.1 mg mL−1 in a mixture of THF and MeOH (3:2) containing sodium trifluoroacetic acid at a concentration of 0.14 μg L−1. Turbidimetric Studies. UV−vis spectroscopy was conducted on a Varian Cary 300 Bio spectrophotometer. Turbidity points were measured on a Cary 300 Bio UV−vis spectrophotometer (Varian) at 600 nm in the temperature range from 20 to 40 °C at a heating rate of 0.1 °C min−1. Particle slurries of 1 wt % in 1 M carbonate buffer (pH 10) were prepared and used for the measurements. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were performed on a K-Alpha spectrometer (Thermo Fisher Scientific, East Grinstead, U.K.) using a microfocused, monochromated Al Kα X-ray source with a spot size of 400 μm. For elemental spectra, a 180° hemispherical energy analyzer operated in the constant analyzer energy (CAE) mode at 50 eV pass energy was employed to measure the kinetic energy of the electrons. Detection of the photoelectrons was carried out at an emission angle of 0° with respect to the normal of the sample surface. The K-Alpha charge compensation system was utilized during analysis with electrons of 8 eV energy and low-energy 283


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Biomacromolecules argon ions to avoid buildup of localized charge. Acquisition and processing of data were carried out using the Thermo Avantage software. Spectra were fitted with one or more Voigt profiles (BE uncertainty: ±0.2 eV). All spectra were referenced to the C 1s peak of hydrocarbon at 285.0 eV binding energy, controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, and Au, respectively. Scanning Electron Micropscopy (SEM). The microspheres were imaged on a Zeiss Supra 55 instrument. All samples were sputtercoated with 30 nm of gold before measurements. Fourier Transform Infrared (FTIR) Spectroscopy. Infrared measurements were performed via attenuated total reflectance (ATR) using a Bruker research spectrometer VERTEX 80. Spectra were collected at a resolution of 4 cm−1 for a total of 32 scans. Elemental Analysis. The elemental composition of the microspheres was analyzed using a Vario MICRO cube by Elementar in the CHNS mode. The combustion temperature applied was 1150 °C with helium 5.0 as the carrier gas and oxygen as the combustion gas.

via Michael addition reactions to introduce the MEP moiety as shown in Scheme 2. The effect of aromatic/heteroaromatic ligand structures and different hydrophobic spacers on the degree of antibody selectivity has been reported.26 Nonheteroaromatic ligands have shown a lower specificity toward antibodies while heteroaromatic ligands including MEP demonstrated a similar ability to adsorb IgG.9,27 MEP functionalized resins have been utilized as sorbents for the separation of variable mixtures and showed limited nonspecific adsorption to other proteins.28,29 Further, the MEP ligand has been reported to show better binding capacity for IgGs in the presence of sulfur in the spacer arm.9 Thus, a thiol-functionalized MEP was employed in the synthetic design to introduce MEP moieties onto the polymer end groups (Scheme 2). One of the most efficient reactions involving thiols is the base-catalyzed Michael addition, where thiols take the role of the nucleophile and react with activated olefins.30 Thus, in an initial step, vinyl functional groups were introduced into the polymer end groups. As depicted in Scheme 2, the first end group modification step involves the introduction of vinyl functions at the polymer chain terminus by a one-pot aminolysis/Michael addition reaction of the terminal trithiocarbonate groups of PNIPAM with a difunctionalized vinyl compound, that is, methylene bis(acrylamide) (BA). In the one pot reaction, the trithiocarbonate groups were aminolyzed to form thiols in the presence of a reducing agent, and subsequently reacted with BA to introduce the vinyl moieties (refer to Scheme 2). The use of an excess amount of the difunctional BA minimizes the formation of coupled polymer species. The success of the reaction and confirmation of the presence of the desired vinyl end groups can be evidenced by inspection of the 1H NMR spectrum of the reaction product (PNIPAM-BA) depicted in Figure 2. The

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RESULTS AND DISCUSSION Preparation of MEP-Functionalized PNIPAM. Initially, PNIPAM was synthesized via RAFT polymerization using a trithiocarbonate chain transfer agent, DDTTC, and AIBN as the initiator in 1,4-dioxane at 60 °C (Scheme 2). The conversion of NIPAM was monitored by 1H NMR spectroscopy by comparing the integrals of the resonances associated with the monomer vinyl protons (5.60, 6.15, and 6.26 ppm) and the NHCH- methine proton of the polymer (4.00 ppm) with the methyl proton resonances of the Z group of the RAFT agent. The RAFT polymerization reactions showed good control and produced polymers of narrow molar mass dispersity (ĐM) as indicated in Figure 1. The number-average

Figure 1. Mn/ĐM vs conversion data for the polymerization of NIPAM at 60 °C with AIBN as initiator and DDTTC as RAFT agent ([NIPAM]0/[DDTC]0 = 93, [DDTC]0/[AIBN]0 = 0.1). The plot shows Mn,NMR (■), Mn,GPC (○), ĐM (▼), and Mn,theo (dashed line). The respective data is tabulated in the Supporting Information (Table S1).

Figure 2. 1H NMR spectra after each end group modification step: (A) PNIPAM 3 kDa, (B) PNIPAM-BA 3 kDa, and (C) PNIPAMMEP 3 kDa in CDCl3. The end group fidelity was monitored via the determination of the Mn of the polymer by integration of the resonance signals associated with the end group protons to the methine proton of the polymer (4.00 ppm) after each reaction step.

molecular masses determined via both GPC and NMR showed good agreement with each other and both sets of values were relatively close to the theoretical molecular weights. In addition, the molar mass dispersity of the polymers was kept below 1.3 up to conversions as high as 98%. The conversion over time evolution, 1H NMR spectra and GPC traces of these polymers are included in the Supporting Information (refer to Figures S2 and S3). PNIPAMs of three molecular weights were synthesized (Table 1) and subsequently end group modified

appearance of resonances at 4.72 ppm attributed to the methylene (−NCH2N−), and methine peaks from the vinyl groups at 5.68 and 6.29 ppm demonstrates the successful end group conversion. Via integration of the resonances at 5.68 ppm relative to the polymer methine proton resonance at 4.00 ppm, an Mn of 3000 Da was deduced, in good agreement with the expected Mn (3000 Da), indicating near quantitative conversion of the end groups. In Figure 3B, the mass spectrum 284


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Figure 3. ESI mass spectra of (A) PNIPAM 3 kDa, (B) PNIPAM-BA 3 kDa, and (C) PNIPAM-MEP 3 kDa. Vertical lines pass through the lightest isotope of (A) P2, (B) PB2, and (C) PM2.

of PNIPAM-BA is depicted where a decrease of 90.00 in m/z is observed (vertical lines from P2 to PB2), which is in excellent agreement with the expected theoretical decrease of 90.05. The ion structure of the two major species is associated with the incorporation of one (PB1) and two sodium (PB2) cations. The deprotonation of the secondary amide groups of PNIPAM in methanol to incorporate the second sodium atom has been reported and is not surprising.31 In addition, the GPC trace of PNIPAM-BA depicted in Figure 4 shows a monomodal distribution, thus indicating that an undesired coupling reaction between the two polymer chains did not occur.

showed the major species (PM1 and PM2) to be the desired PNIPAM-MEP. The increment in m/z of 139.36 from PB2 to PM2 is in excellent agreement with the expected calculated m/z of 139.04. All theoretical and experimental m/z values can be found in the Supporting Information (Table S3). The end group fidelities (approximately 70%) for the higher molecular weight PNIPAM-MEP chains (6 and 20 kDa) were analyzed by NMR (Table S2). The post-polymerization syntheses performed are as shown in Scheme 2. Grafting-To of Thermoresponsive Polymers onto Microparticles. Superose, which consists of highly crosslinked agarose, was selected as the resin due to its high hydrophilicity, thus rendering it a matrix with protein repelling properties. In addition, it is decorated with hydroxide groups that can serve as reaction sites for the grafting of polymers. PNIPAM-MEPs of different molecular weights (3, 6, and 20 kDa) were grafted onto Superose microparticles via Steglich esterification in the presence of EDC with DPTS and DMAP as catalyst. The addition of DPTS is important for the reaction to succeed as prior experiments without DPTS showed little to no grafting of the polymers onto the microparticles. DPTS, which was synthesized from an equimolar mixture of DMAP and ptoluenesulfonic acid, suppresses the formation of unwanted Nacylurea therefore leading to high yields of ester.33 The reaction path is as shown in Scheme 3A. In addition to grafting the MEP functional polymers, MEP ligands were immobilized onto Superose micorparticles using established protocols by Burton et al.32 First, allyl groups were introduced onto the microparticles by reacting the hydroxide moieties with allyl glycidyl ether. Their surfaces were subsequently rendered more reactive through the formation of bromohydrins by an addition reaction with N-bromosuccinimide. Thiol-functionalized MEP was subsequently reacted via substitution with the halohydrins to form MEP-functionalized Superose microparticles. The synthetic route to prepare the Superose-MEP matrices is as shown in Scheme 3B. FT-IR analysis was employed to determine the success of the grafting reactions. All spectra were normalized to the peak at 928 cm−1, which is the characteristic vibration for the 3,6anhydro moiety of the agarose based matrices.34 As shown in Figure 5a, the spectrum of the initial unmodified Superose microparticles possesses peaks corresponding to methylene bending (between 1370 and 1462 cm−1) and ether C−O−C stretching (1049 cm−1) vibration, which are typical of cross-

Figure 4. GPC traces recorded in DMAc for PNIPAM (), PNIPAM-BA (· · ·), and PNIPAM-MEP (− −).

Subsequently, a second Michael addition was carried out in basic aqueous solution with hexylamine as catalyst to react the vinyl groups with thiol functionalized MEP. It is vital that this reaction is carried out between pH 10 to 11, where the mercaptide ions are formed and the pyridine groups are uncharged, as a side reaction may occur from the substitution of the pyridyl instead of the thiol.32 The successful addition of MEP moieties onto the polymer end groups was confirmed via 1 H NMR (Figure 2). The presence of the pyridine rings of the MEP was ascertained through resonances at 7.15 and 8.50 ppm. The Mn of the polymer chain obtained based on the integration of the resonances at 8.50 and 4.00 ppm was similar to its Mn prior to the modification reactions, indicating retention of end group fidelity. In addition, an electrospray ionization mass spectrum (Figure 3C) of the polymer was also recorded and 285


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Scheme 3. Grafting Reactions of (A) PNIPAM-MEP of Variable Molecular Weight and (B) a Small Molecule MEP onto Superose Microparticles

the appearance of N−H bending vibrations and CO stretching bands was evident at 1550 and 1670 cm−1, respectively (Figure 5c−e). The spectrum of linear PNIPAMMEP chains was also included in Figure 5b as a comparison. In addition, XPS measurements were carried out to evidence the success of the grafting reactions. As depicted in Figure 6, the spectra of nonmodified Superose do not feature any nitrogen or sulfur, whereas the presence of both atoms is clearly observed after the grafting reaction. In the N 1s XP spectrum, the peak at 400.0 eV is assigned to the amide bonds of the polymer and a small peak at 402.0 eV could also be attributed to partial protonation of the pyridine nitrogen of the MEP end groups.36 Signals observed at 163.4 eV from S 2p3/2 can be attributed to the two S−C bonds in the polymer end group.37 Imaging techniques such as SEM were also employed to assess the integrity of the microparticles after the grafting reaction (on the example of the Superose-PNIPAM-MEP (Mn = 3000 g mol−1) microparticles). The SEM images in Figure 7 evidence that the spherical geometry and porosity of the Superose particles before and after the esterification reaction were preserved. There was also no significant damage incurred to the microparticles with the employed reaction setup. Quantitative analysis of the loading capacity of the modified microparticles was obtained by elemental analysis. As each

Figure 5. FT-IR spectra of (a) unmodified Superose, (b) PNIPAMMEP linear chains (Mn = 3000 g mol−1), (c) Superose-PNIPAM-MEP (Mn = 3000 g mol−1), (d) Superose-PNIPAM-MEP (Mn = 6000 g mol−1), and (e) Superose-PNIPAM-MEP (Mn = 20 000 g mol−1).

linked agarose.35 A broad peak at 1650 cm−1 is also observed, which is attributed to the H−O−H bands of agarose as well as to atmospheric water due to its hygroscopic nature. After the grafting-to reactions with polymers of three molecular weights,

Figure 6. N 1s and S 2p XPS spectra of (A) unmodified Superose and (B) Superose-PNIPAM-MEP (Mn = 3000 g mol−1). 286


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Figure 7. SEM images of (A) unmodified Superose microparticles and (B) Superose-PNIPAM-MEP (Mn = 3000 g mol−1) microparticles.

ponsive polymers on the surfaces of the particles, the hydrophilicity of the substrate is expected to be addressable via temperature variations. Thus, initially, cloud point measurements of the linear PNIPAM-MEP in solution were also carried out and have been found to be dependent on the chain length (refer to the Supporting Information, Figure S6). The cloud points of PNIPAM-MEP in solution have been found to increase with increasing chain length. This phenomenon is expected as the phase transition of the shorter chains is more likely to be influenced by the nature of their end group polarity, while the longer polymer chains show a much less pronounced end group effect and the cloud point becomes more dependent on the molecular weight of the polymer.40 The aggregation behavior of the modified microparticles was also assessed via turbidimetric studies employing Superose-PNIPAM-MEP 3 kDa in the temperature range between 20 and 35 °C as shown in Figure 8. The modified particle slurry appeared turbid at

PNIPAM-MEP chain entails two sulfur atoms, the sulfur content can be used to determine the number of polymer chains that were successfully grafted onto the microparticles’ surface. Table S4 in the Supporting Information collates the elemental analysis results for Superose samples before and after modification with different PNIPAM-MEP and MEP entities. The loading capacity (LC) can be calculated using eq 1.38 In eq 1, the LC is obtained in mol g−1 based on the weight of sulfur per 1 g of microparticles, W(S), number of sulfur atoms, n(S), and molecular weight of sulfur, M(S). LC =

W (S) n(S) M(S)

(1)

Based on the elemental analysis results, there are no nitrogen and sulfur detected on the unmodified Superose particles. Based on the sulfur content, the loading capacity of the 3 and 6 kDa polymer species are relatively similar and close to 40 μmol g−1, whereas a half-fold decrease was observed (20 μmol g−1) when grafting was performed with PNIPAM-MEP featuring a molecular weight of 20 kDa. Such results are not unexpected as the longer 20 kDa polymer chains encounter significantly more steric hindrance when reacting with the hydroxide groups on the particles’ surface compared to the shorter 3 and 6 kDa polymers. Furthermore, it is also likely that physical factors such as diffusion rates and chain entanglements of longer polymer chains play a role in the efficiency of the grafting reaction as free polymers have to diffuse through existing grafted chains to reach the reactive sites.18,39 The loading capacity of Superose-MEP microparticles (grafting of the small molecule MEP thiol) was also determined and found to be close to 730 μmol g−1. Via gravimetric analysis techniques, Superose particles of known settled bed volumes were dried to constant mass, and it was found that 1 g of Superose microparticles has a volume of 3.5 mL when fully swollen in water. Thus, the Superose-MEP microparticles have a loading capacity of approximately 200 μmol mL−1, which is comparable to the loading capacities reported for MEP-modified Sepharose CL-6B microparticles.28 The grafting densities of the different molecular weight PNIPAM-MEP chains grafted onto Superose microparticles have been estimated to be approximately 0.1 chains per nm2 on average. However, this estimation rests on specific assumptions and should thus be treated with care. Further calculations and discussions on the grafting density and the regime of the polymer chains can be found in the Supporting Information. The nonmodified Superose microparticles are decorated with hydroxide groups on their surface, which render the microparticles hydrophilic. By grafting the synthesized thermores-

Figure 8. Aggregation behavior of Superose-PNIPAM-MEP (Mn = 3000 g mol−1) through turbidity measurements at 500 nm.

temperatures below its phase transition temperature, because the PNIPAM-MEP chains are extended at these temperatures and promote hydrophilicity of the matrix through the formation of caged water structures around its hydrophobic isopropyl domains.41 As the temperature is increased above its phase transition temperature, a slight increase in transparency was observed. Above its phase transition temperature, the grafted PNIPAM-MEP chains collapse as the caged water structures disintegrate and the hydrophobic segments aggregate, thus rendering the matrix more hydrophobic. Therefore, particle− particle interaction is favored, which leads to the observed 287


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Biomacromolecules aggregation behavior of the modified particles at temperatures above its phase transition temperature. Adsorption/Desorption Studies Using γ-Globulin as Model Antibody. The Superose microparticles that were grafted with temperature-responsive PNIPAM-MEP of variable chain lengths were designed to have temperature-induced switchable release properties for immunoglobulins. The adsorption of proteins is triggered at temperatures above the LCST of PNIPAM and released when temperatures were lowered to below the LCST of the polymers. The adsorption and desorption capabilities of the prepared Superose-PNIPAMMEP microparticles were subsequently assessed using γglobulins as the model antibody. γ-Globulins were chosen because they consist mostly of immunoglobulins (IgGs). The amount of proteins successfully bound onto the microparticles was calculated from the difference between the initial concentration of γ-globulins and the supernatant after adsorption. From these measurements, the equilibrium concentrations of adsorbed proteins (q*) at various concentrations of liquid phase protein (C*) were determined using the Langmuir eq (eq 2). q* =

qmax C* Kd + C *

(2)

Buffers at different pH values were assessed to determine the most suitable pH for the adsorption of γ-globulins. The most efficient adsorption was observed at pH 10 in 1 M carbonate buffer. The buffer was subsequently used for all the presented adsorption/desorption data herein. Figure 9 compares the effect of temperature on the adsorption of γ-globulins for the three different PNIPAM-MEP resins. Superose-PNIPAM-MEP 3 kDa showed the best adsorption properties with γ-globulins yielding a qmax of 15 g L−1 at 25 °C and an increment in binding capacity up to 20 g L−1 at 40 °C (Figure 9A). When the molecular weight of grafted polymers was doubled (SuperosePNIPAM-MEP 6 kDa), the binding capacity of γ-globulins decreased to 5 g L−1 at 25 °C and 15 g L−1 at 40 °C (Figure 9B). As expected, the lowest binding capacity of γ-globulins was achieved with Superose-PNIPAM-MEP 20 kDa, which was grafted with the longest polymer chains (4 g L−1 at 25 °C and 10 g L−1 at 40 °C) as shown in Figure 9C. These results support the grafting density measurements from elemental analysis whereby the amount of polymers grafted on the Superose-PNIPAM-MEP 3 kDa was approximately double of the polymers on the Superose-PNIPAM-MEP 20 kDa particles. In addition, it is also possible that MEP end groups may be tangled within the longer polymer chains therefore displaying a decrease in their ability to capture proteins. The observed temperature effect on the binding capacity of γ-globulin can be explained by the fact that above the phase transition temperature of PNIPAM the polymer chains are collapsed, thus increasing the density per unit of surface area by bringing the MEP end groups within closer vicinity of one another, which will in turn allow more efficient binding of proteins onto particles. The binding of IgG has been reported to be dependent on the density of MEP and adsorption can only occur when a minimum critical number of hydrophobic ligands are available on the resins.9,28 At 5 °C, Superose-PNIPAMMEP 3 kDa showed a binding capacity lower than 5 g L−1, while Superose-PNIPAM-MEP 6 kDa and Superose-PNIPAMMEP 20 kDa did not show any protein adsorption. The PNIPAM chains are expected to be fully extended at this

Figure 9. Adsorption isotherm of (A) Superose-PNIPAM-MEP 3 kDa, (B) Superose-PNIPAM-MEP 6 kDa, and (C) Superose-PNIPAMMEP 20 kDa at 5 °C (purple squares), 25 °C (green squares), and 40 °C (red squares).

temperature, thus decreasing the density of MEP end groups per unit surface. The slight adsorption observed at 5 °C with the shortest PNIPAM-MEP 3 kDa could also be explained by the higher density per unit surface of these microparticles due to the shorter polymer chains as compared to the other longer PNIPAM-MEP chains. The adsorption of γ-globulin on the Superose base particles and the Superose-MEP particles were also performed at 5, 25, and 40 °C. The isotherm curves for these experiments are included in the Supporting Information section (refer to Figures S7 and S8). No adsorption was observed for the Superose nonmodified microparticles at all three temperatures, even when high concentrations of γ-globulin solutions were employed, indicating that the base particles are inert and incapable of binding any IgGs in the selected temperature range. As for the Superose-MEP microparticles, a qmax of 45 g L−1 was obtained at all three temperatures. In comparison to the particles with PNIPAM spacers, the higher qmax observed for Superose-MEP is attributed to the higher MEP density on 288


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denaturation of the proteins is unlikely to occur. At 40 °C, the polymer chains are in a collapsed state with the MEP end groups in closer vicinity to one another, promoting the adsorption of the IgGs. As shown by the adsorption results in the previous section, the binding capacity of the resins increases with decreasing PNIPAM chain length. These observations are attributed to the variability in the grafting density of the different chain length PNIPAM-MEP, where the efficiency of the “grafting-to” process is significantly affected by steric hindrance. The amount of γ-globulins bound to the microparticles at 40 °C is as shown in Figure 11. At the same time,

these particles. As the same qmax was achieved in the temperature range tested, it was evident that there was no temperature effect on the adsorption properties of the Superose-MEP particles. Other than having the ability to possess variable binding capacities at different temperatures, the resins should also be able to desorb the bound proteins via a trigger as chromatographic sorbents. Two approaches were employed to demonstrate the protein desorption capabilities of the synthesized microparticles. The first technique was the conventional desorption method used for HCIC sorbents, which utilizes changes in pH while the second technique was the new proposed approach of desorbing the proteins by triggering a temperature change. Superose-PNIPAM-MEP 3 kDa microparticles were succumbed to adsorption and the bound proteins were removed by washing the resins in acetic acid buffer at pH 3. At low pH, the microparticle surfaces are positively charged as well as the proteins, promoting repulsion between similarly charged species and thus successfully removing proteins from the particles’ surface. Figure 10

Figure 11. Adsorption of Superose microparticles at 40 °C with 10 g L−1 γ-globulin solutions and desorption at 5 °C.

the Superose and Superose-MEP microparticles were also included in the adsorption experiments. As expected, the Superose base particles showed no adsorption while the Superose-MEP, which has a much higher MEP density compared to the Superose-PNIPAM-MEP particles, exhibited high binding capacities at 40 °C. The same batch of particles was subsequently incubated at 5 °C to induce the desorption process. At 5 °C, all three Superose-PNIPAM-MEP microparticles displayed a temperature-triggered release of γglobulins whereas the Superose and the Superose-MEP particles showed no temperature responsive properties. The temperature-triggered release behavior of the thermoresponsive polymers can be explained by the fact that the polymers are fully extended below their LCSTs, thus increasing the distance between the MEP end groups while initiating the release of the bound proteins. The amount of remaining proteins left adsorbed on the particles’ surfaces were determined and is presented in Figure 11. Superose base microparticles showed no adsorption/desorption behavior, while the Superose-MEP further adsorbed more proteins at 5 °C. The results evidence that the Superose-PNIPAM-MEP microparticles have clear temperature-switchable release properties, which can desorb between 80 and 90% of adsorbed IgGs.

Figure 10. Adsorption isotherm of γ-globulins after the first cycle of adsorption (black squares) and second cycle of adsorption (gray squares) at 25 °C with Superose-PNIPAM-MEP 3 kDa.

shows the adsorption isotherm curves of a batch of SuperosePNIPAM-MEP 3 kDa microparticles that were used for two cycles of protein adsorption. After the first adsorption cycle, the particles were washed thoroughly in a low pH buffer to induce repulsion of bound proteins off the same charged resins. These resins were subsequently reused for a second cycle of adsorption to determine if the majority of the bound proteins were desorbed by comparing the qmax from the first cycle with the qmax from the second cycle. The isotherm curve for the first cycle appears to achieve qmax (12 g L−1) at lower concentrations whereas in the second cycle qmax (10 g L−1) is shown to occur in higher protein concentrations. Although the qmax values for both cycles are relatively close, the differences in the steepness of the isothermal slope may indicate a small portion of incomplete desorption of bound proteins. The antibody binding capacity of other MEP agarose-based resins such as MEP-Sepharose has been reported to decrease over repeated usage yet is still comparable to Protein A sorbents.28 The second method of desorption makes use of the temperature-switchable properties of the grafted thermoresponsive polymers. The synthesized Superose-PNIPAM-MEP microparticles of variable chain lengths were incubated in γglobulin solutions of 10 g L−1 at 40 °C. This temperature was chosen because it was above the LCST of the polymers (Figure S6) and was still within the working temperature range where

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CONCLUSIONS We successfully synthesized PNIPAM chains of different molecular weights with narrow molar mass dispersities using RAFT polymerization. These polymers were subsequently end group modified via Michael addition reactions to introduce MEP ligands. By utilizing a simple Steglich esterification reaction, the PNIPAM-MEP chains were grafted onto Superose microparticles to render them thermoresponsive. SuperosePNIPAM-MEP microparticles decorated with polymers of different molecular weights were employed for adsorption and desorption assays using γ-globulins as a model antibody. γGlobulins showed the highest binding capacity onto the 289


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Biomacromolecules microparticles at 40 °C and little or no binding at 5 °C, demonstrating the temperature dependent protein binding characteristics of the synthesized Superose-PNIPAM-MEP microparticles. Desorption was also shown effective via a temperature change, which in turn changes the conformation of PNIPAM and the binding capacity of the microparticles. The desorption capability of the three Superose-PNIPAM-MEP particles with different polymer chain lengths were compared and the microparticles grafted with the shortest PNIPAM-MEP chains showed the highest desorption by a temperature change. Thus, the Superose-PNIPAM-MEP microparticles show promising potential as sorbent materials for thermoresponsive chromatography systems. The ability of these materials to desorb bound proteins by changes induced by an external stimulus such as temperature is of significant interest for the development of chromatographic materials that do not require special buffers for elution.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01391. Experimental details and protocols, grafting densities, additional characterization data of the different PNIPAM-MEP chains and synthesized microparticles as well as isotherms of Superose and Superose-MEP (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.B.-K. acknowledges long term funding from the Helmholtz association via the STN and BIFTM programs. The authors thank Udo Geckle (KIT) for the SEM images.

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REFERENCES

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