Dual-Gated Microparticles for Switchable Antibody Release - ACS

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Dual-Gated Microparticles for Switchable Antibody Release Benedikt Ketterer, Huey Wen Ooi, Dominik Brekel, Vanessa Trouillet, Leonie Barner, Matthias Franzreb, and Christopher Barner-Kowollik ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16990 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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ACS Applied Materials & Interfaces

Dual-Gated Microparticles for Switchable Antibody Release Benedikt Ketterer,1,# Huey Wen Ooi,1,2,#, Dominik Brekel,1 Vanessa Trouillet,3 Leonie Barner,4,5* Matthias Franzreb,1,* Christopher Barner-Kowollik2,4,5* 1

Institute for Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann-

von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany 2

Macromolecular Architectures, Institute for Chemical Technology and Polymer Chemistry,

Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany 3

Institute for Applied Materials (IAM) and Karlsruhe Nano Micro Facility (KNMF), Karlsruhe

Institute

of

Technology

(KIT),

Hermann-von-Helmholtz-Platz

1,

76344

Eggenstein-

Leopoldshafen, Germany 4

Institute for Biological Interfaces (IBG), Karlsruhe Institute of Technology (KIT), Hermann-

von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany 5

School of Chemistry, Physics and Mechanical Engineering, Queensland University of

Technology (QUT), 2 George Street, QLD 4000, Brisbane, Australia #

Authors contributed equally.

*Corresponding

authors:

[email protected],

[email protected],

[email protected]

Keywords: Thermo-responsive polymer microparticles, pH responsive microparticles, polymer grafting, protein separation, column chromatography

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ABSTRACT We pioneer the design of dual-gated microparticles – both responsive to changes in temperature and pH – for stimuli-responsive chromatography targeted at the efficient separation of antibodies. Dual-gated microspheres were synthesized by introducing RAFT-based thiol-terminal block copolymers of poly(N-isopropylacrylamide-b-4-vinylpyridine) (P(NIPAM-b-4VP, 4 800 ≤ Mn / Da ≤ 10 000, featuring block length ratios of 29:7, 29:15, 29:30, respectively) by thiol-epoxy driven ligation to the surface of poly(glycidyl methacrylate) (PGMA) microparticles (10-12 µm), whereby the 4-vinylpyridine units within the lateral chain enable protein binding. The switchable protein release abilities of the resulting microparticle resins are demonstrated by adsorption of immunoglobulins at 40 °C and pH 8 and their release at 5 °C or pH 3, respectively. We demonstrate that P(NIPAM29-b-4VP30)-grafted PGMA particles show a maximum adsorption capacity for immunoglobulins of 18.9 mg mL-1 settled resin at 40 °C / pH 8, whereas the adsorption capacity decreased to 7.5 mg mL-1 settled resin at 5 °C while retaining the pH value, allowing the unloading of the chromatographic column by a facile temperature switch. Critically, regeneration of the dual-gated microspheres became possible by lowering the pH to 3.

INTRODUCTION Highly-crosslinked

polymeric

microspheres

are

powerful

base

materials

for

chromatographic applications due to their porosity and rigidity compared to soft resins such as agarose-based particles. Critically, polymer-based microparticles exhibit high chemical tolerance over a wide pH range and concomitantly provide substantial versatility in their surface chemistry.1,2 Through selection of the constituting polymer system, microspheres can be customized via post-functionalization processes to possess properties that are required for the separation of specific molecules.3 Poly(glycidyl methacrylate) (PGMA) particles are attractive


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due to their abundant surface functional epoxy groups, which are readily available for reactions with a wide range of functional groups to introduce different properties to the particles.4 For example, our team introduced cyclopentadienes via nucleophilic addition to the epoxy groups on PGMA microspheres.5,6 The cyclopentadienes were subsequently utilized to graft reversible addition fragmentation chain transfer (RAFT) synthesized polymers bearing either dithioesters or photoactive phenacyl sulfides through hetero Diels-Alder reactions to produce microparticles with tailored polymeric surfaces. Further, Nordborg and co-workers developed cation-exchange polymeric microparticles based on surface modified divinylbenzene particles and demonstrated the separation of proteins such as cytochrome C, lysozyme, myoglobin, and ribonuclease A through chromatography.7 The design of their microparticles entailed the conversion of the residual vinyl groups of divinylbenzene to epoxides to provide functional sites for grafting of thiol-terminated telomers and enhancing the hydrophilicity of the microparticles through the formation of diols from unreacted epoxy groups. In a further example, Wang et al. grafted polyethylene glycol to poly(glycidyl methacrylate-divinylbenzene) porous microspheres via the reaction between the epoxy groups of the particles to the hydroxyl groups of PEG to prepare protein fouling chromatographic materials.8 Stimuli-responsive chromatographic matrices are attractive as these materials have the ability to undergo changes in their protein adsorption properties based on small external changes in their environment. PNIPAM is the most reported thermo-responsive polymer to-date due to its lower critical solution temperature of 32 °C, which allows the polymer chains to undergo a phase transition from a coil to globule conformation close to physiological temperatures, rendering it an attractive material for biomolecule separation. The introduction of polymers onto microparticles’ surfaces can be achieved either through a ‘grafting-to’ or ‘grafting-from’ approach. The ‘grafting-from’ approach enables higher grafting



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densities,9 yet at the same time is experimentally more challenging due to the limited molecular characterization of the grafted polymer chains. In contrast, the ‘grafting-to’ approach limits the ability to achieve high grafting densities, yet allows in-depth characterization of the polymer chains before attachment to the microspheres’ surfaces.10 Precision polymers can be synthesized via

reversible-deactivation

radical

polymerization

(RDRP)

methods

such

as

RAFT

polymerization that allow the syntheses of polymers with predetermined molecular weight and narrow molar mass dispersity.11–14 Furthermore, RAFT polymerization provides flexibility in designing complex molecular architectures through end-group modification and chain extension reactions. For example, Paulus et al. synthesized dual-responsive magnetic particles via ‘graftingfrom’ RAFT polymerization of PNIPAM and utilized the living character of the RAFT polymers to grow a second ion-exchange block copolymer.15 The modified particles were successfully used for protein capture at ambient conditions and enhanced separation of proteins was performed in the presence of a magnetic field at a temperature above its phase transition temperature. Through the ‘grafting-to’ method, Goldmann et al. reported the use of thiol-ene chemistry to attach thiol end

group

functionalized

PNIPAM

synthesized

by

RAFT

polymerization

onto

polydivinylbenzene microspheres.16 The thiol-epoxy reaction has been long used in industry for adhesive, coatings, chromatography, biomedical, and biosynthetic applications, although it has not found as much popularity as its thiol-ene counterpart in polymer chemistry until recent years.17–19 In certain cases, the stringent click criteria20,21 have been associated with this reaction when the reaction conditions entailed solvent-free or aqueous conditions while rapidly achieving near quantitative yields and high regioselectivity.22 Oxirane groups undergo nucleophilic ring-opening by deprotonated thiol (thiolate anion), followed by protonation of the alkoxide anion to form a hydroxyl group, which can subsequently be exploited for further functionalization. For example,


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Harvison et al. modified RAFT-synthesized polystyrene and poly(N,N-diethylacrylamide) dithioester end-groups to prepare thiol-terminated polymers, which were successfully reacted with numerous epoxide-functionalized small molecules.17 Khan and co-workers have demonstrated the usefulness of the thiol-epoxy reaction for the formation of polymers with various architectures, spanning from functionalization of pendent epoxides on PGMA linear chains,23 formation of a polythioether-based hyperbranched polymer,24 to crosslinking of bifunctionalized-epoxy poly(ethylene glycol) with a tetrafunctionalized-thiol linker to form threedimensional hydrogels.25 However, in spite of the efficiency of the thiol-epoxy reactions reported, it is still debatable whether this reaction fulfills the click paradigm, as the reactions reported so far were performed under off-stoichiometric conditions. Poly(4-vinylpyridine) (P4VP) is an amphiphilic polymer with pendent ionizable pyridyl moieties that has the ability to protonate at pH 5 and below, rendering it hydrophilic and soluble in aqueous media. These pyridyl rings provide multiple interaction sites with proteins, which makes them highly attractive as building block for mixed-mode resins. P4VP has been previously applied in chromatography resins, biosensors, and anti-bacterial materials26 due to its ability to form hydrogen bonds, electrostatic interactions in its quarternized or protonated form, and strong affinity for metal ions.27,28 Sepharose® 6 FF resins functionalized with P4VP were prepared by Li et al. via quaternization of the pyridyl amines of P4VP to bromides on the surfaces of the resins.29 These sorbents exhibit strong adsorption for immunoglobulins and bovine serum albumin through electrostatic/hydrophobic interactions and quantitative protein recovery was possible when pH was lowered. The current work aims at the development of novel materials in the commercially important field of protein chromatography. Here, we used well-established microspheres as starting material due



to their robustness and high specific surfaces, resulting in superior adsorption capacities when compared to monolithic materials or membrane adsorbers. However, these microspheres are equipped with dual gated stimuli-responsive behavior in order to achieve several advantages: (i) The purification of immunoglobulins commonly requires the use of strong acids for elution.30 However, the elution under acidic conditions often leads to the formation of aggregates, which have to be removed in further purification steps, thus causing additional costs and reducing the final process yield.31 The possibility of temperature induced elution may overcome this hurdle of pH-sensitive immunoglobulins. (ii) An elution caused by external stimuli instead of changes in the mobile phase enables new strategies of process control. E.g., cooling of the entire column enables simultaneous elution at each point of the column, thus avoiding concentration peaks that occur during conventional elution in the liquid front of the elution buffer. Critically, avoidance of concentration peaks reduces the occurrence of unwanted contaminants as high concentrations during the elution are another major cause for the formation of protein aggregates.32 (iii) Besides its potentially negative effect on product stability, elution buffers are a substantial cost factor of the consumables for protein purification processes and often chemicals included in the elution buffers (e.g. high salt loads) must be removed before further purification steps. Therefore, elution by external stimuli will reduce costs and the number of process steps required. To enable the above aims, we herein introduce the synthesis and characterization of dualgated PGMA microspheres as an advanced chromatographic material capable of independent temperature and pH-triggered release of immunoglobulins. The block copolymer design entails a RAFT synthesized PNIPAM as the temperature responsive block copolymer, which was subsequently used as a macroRAFT chain transfer agent to grow the second pH responsive P4VP block as depicted in Scheme 1. The trithiocarbonate RAFT groups of the block copolymers were


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then reduced to thiols and grafted onto epoxy-rich PGMA microparticles via the thiol-epoxy reaction. The chemical strategy employed for the modification of the microparticles allows for the design of a dual gated microparticle system, where a high temperature trigger leads to the collapse of the outer PNIPAM block allowing proteins to interact with the inner P4VP segments. A low temperature trigger subsequently promotes the stretching of the PNIPAM block and therefore a gentle release of proteins. This temperature-triggered effect on PNIPAM grafted microparticles has been observed in our previous studies16,33,34 and has been attributed to the ability of the PNIPAM chains to undergo conformational changes above and below their phase transition temperature. Importantly, the desorption of proteins is also enabled by a reduction of the pH. The dual-gated properties of the PGMA-P(NIPAM-b-4VP) particles are explored through temperature/pH-induced adsorption/desorption in batch isotherms and chromatographic experiments using bovine γ-globulins as model immunoglobulins.

Scheme 1. Schematic (not to scale) overview of the synthetic approach employed for the preparation of thermo-responsive P(NIPAM-b-4VP) grafted PGMA microparticles. The design



strategy to capture and release immunoglobulins upon temperature and pH trigger is additionally depicted.

MATERIALS AND METHODS Materials Poly(glycidyl methacrylate) (PGMA) microparticles (porosity 1000 Å, size 10-12 µm, specific surface area 162 m² g-1) were synthesized by Polymer Standards Service (PSS) GmbH and characterization of these materials have been reported in a previous publication.5 Hexylamine (for synthesis, Merck Millipore), tetrahydrofuran (THF, EMSURE®, Merck), dimethylformamide (GPR RECTAPUR®, VWR Collection), diethyl ether (GPR RECTAPUR®, VWR Collection), lithium hydroxide (anhydrous 98%, Alfa Aesar), tris(2-carboxyethyl)phosphine hydrochloride (98%, Alfa Aesar, TCEP) and γ-globulins from bovine blood (99%, Sigma Aldrich) were used as received. 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.35 4-vinylpyridine (95 %, Aldrich, 4VP) and 1,4-dioxane (GPR RECTAPUR®, VWR Chemicals) were passed through a column of basic alumina before use. Methods RAFT polymerization of NIPAM (PNIPAM CTA): The employed general method was previously described.33 Briefly, NIPAM (29.9749 g, 2.65·10-1 mol), DDTTC (3.8938 g, 1.07·10-2 mol) and AIBN (0.1754 g, 1.07·10-3 mol) were weighed into a round bottom flask. 1,4-dioxane (67.5 mL) was added and the reaction solution was stirred until all solids were dissolved. The flask was then


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sealed with a rubber septum and the reaction solution was purged with nitrogen for 1 h. Subsequently, the polymerization was carried out at 60 °C for 5 h. Polymer was purified via dialysis in 1 kDa MWCO tubes for 3 d. Water was then removed using a freeze-dryer to yield PNIPAM. The polymer was analyzed by NMR spectroscopy and GPC (Table 1). General procedure for RAFT polymerization of 4-vinylpyridine with PNIPAM CTA: PNIPAM CTA (5.1286 g, 1.79·10-3 mol) and AIBN (0.0301 g, 1.83·10-4 mol) were weighed into a round bottom flask. 4VP (2.1500 g, 2.04·10-2 mol) and dimethylformamide (17.25 mL) were added to the flask and reaction mixture was stirred while deoxygenated with nitrogen for 30 min. Polymerization was carried out at 70 °C for 18 h. The polymers were purified via dialysis in 1 kDa MWCO tubes for 3 d. Subsequently, water was removed by freeze-drying. The polymers were re-dissolved in tetrahydrofuran and precipitated in diethyl ether twice to yield block copolymer (PB2). Polymerization was also repeated using ratios of PNIPAM CTA/AIBN/4VP = 1/0.1/4 and 1/0.1/28 to yield PB1 and PB3, respectively. Molecular weight and molar mass dispersity of polymers were measured via GPC and copolymer compositions were estimated by NMR spectroscopy (as shown in Table 1). RAFT polymerization of 4VP homopolymer. 4VP (9.9996 g, 9.75·10-2 mol), DDTTC (1.3114 g, 3.6·10-3 mol) and AIBN (0.058 g, 3.53·10-4 mol) were weighed into a round bottom flask. Dimethylformamide (22.5 mL) was added and the reaction solution was stirred until all solids were dissolved. Subsequently, the flask was sealed with a rubber septum and the reaction solution was purged with nitrogen for 1 h. Polymerization was carried out at 70 °C for 18 h. Next, the polymer was purified via precipitation in diethyl ether (3×). P4VP was obtained as a red powder and was analyzed by NMR spectroscopy and GPC (Table 1).



General procedure for aminolysis of RAFT end groups: PB1 (3900 Da (NMR), 3.0569 g, 7.64·10-4 mol) was weighed into a round bottom flask. A catalytic amount of TCEP was added to the flask, followed by addition of tetrahydrofuran (18 mL). The reaction solution was purged with nitrogen for approximately 30 min. Hexylamine (1.2141 g, 1.20·10-2 mol) was added via syringe and the solution was further purged for another 15 min before left to react for 2 h. Polymer was purified via precipitation in deoxygenated diethyl ether (2×). Thiol-functionalized polymer (PB1-thiol) was used immediately for the next reaction. General procedure for grafting thiol-functionalized polymers onto PGMA microparticles: PGMA microparticles (0.557 g) were weighed into a 50 mL Falcon tube. PB1-thiol (2 g, 5·10-4 mol) in tetrahydrofuran (20 mL) was prepared and added to the tube. LiOH (0.8358 g, 3.49·10-2 mol) was weighed into a glass vial and was dissolved with water (2 mL) before added to the microspheres. The reaction was left to react on a mixer for 18 h. Particles were washed with copious amounts of acetone, water, dichloromethane, tetrahydrofuran and isopropanol to remove unreacted compounds. A small amount of particles were set aside and dried under vacuum in a desiccator for FTIR spectroscopy and elemental analysis to confirm the success of the modification. Preparation of particle slurries. Synthesized particles were subjected to a washing procedure and made up to particle slurries of known concentrations before any protein adsorption experiments. Particles were transferred to 15 mL centrifuge tubes and known amounts of deionized water (four times settled bed volume of particles) were added. Particles were mixed thoroughly with a vortex mixer before centrifuged for 5 min at 3000 rpm. The supernatant was then removed and tubes were refilled with deionized water. These steps were repeated five times and the particles were transferred to a measuring cylinder. Specific amounts of deionized water were added to the


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settled particles in a measuring cylinder to prepare 25% v/v (e.g. 1 mL of settled particles in a total volume of 4 mL) and 50 % v/v particle slurries. Adsorption isotherms were recorded using the automated liquid handling system JANUSTM workstation from Perkin Elmer with VarispanTM 4-fixed tips arm. An automated protocol, which was optimized and established with commercial resins, was used for all experiments carried out using the liquid handling system. In case of aqueous solutions, Perkin Elmer states a mean pipetting accuracy better than ±3.64% in the range between 1 µL to 200 µL and better than ±1.5% in the range from 10 µL to 100 µL. Therefore, for the multi-step protocol used in the current work, we estimate an average error of 6.5% for the determined loadings, with a maximum error of up to 12% at the lowest concentration. Incubation and temperature control was conducted on an integrated Shaker/Cooler Thermoshake (Inheco) with an adapter for round bottom well plates. Eight concentrations of bovine γ-globulins (99%, Sigma Aldrich) – 15, 10, 7.5, 5, 3.75, 2.5, 1.25, 0.625 mg mL-1 – were prepared from a stock protein solution of 20 mg mL-1. Protein solutions were prepared in the particular buffer (75 mM HEPES, pH 8 and 75 mM MES, pH 6 with or without 150 mM NaCl). Subsequently, 100 µL of each protein concentration were transferred to four wells of a 96 round bottom well plate. 50 µL of this 25% particle slurry was added by hand to the wells with protein solution. The particle slurry was re-suspended prior to every transfer. As a result of the mixing particle slurry and protein solutions, the buffer and protein concentration were decreased by two thirds (e. g. 75 mM HEPES was diluted to a final concentration of 50 mM). Incubation was subsequently carried out at a specific temperature while shaking (1100 rpm) for 60 min and further 30 min without shaking for the particles to settle. During the first 60 min of incubation the shaker was switched on and off periodically to avoid settling of the



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particles. After incubation and settling, a sample of the supernatant was taken. In case of the four samples with initial protein concentrations of 5 mg mL-1 and higher, the samples were diluted with the particular buffer to be in the linear range of the UV/vis calibration. The samples were transferred to UV-Star® 96 well half-area microplates (Greiner). Protein concentration, c*, was determined by absorbance measurement at 280 nm in a microplate reader (Enspire®, Perkin Elmer). The initial protein concentration, c0, of the solutions was determined through measurements of two blank samples, where water was added to the wells instead of particle slurry. The equilibrium concentrations of adsorbed proteins (q*) at various concentrations of liquid phase protein (c*) were calculated using the mass balance equation 1. ∗ =

( ∗ )

(1)

Whereby Vliquid is the volume of the combined protein solution and particle slurry. The Langmuir equation for a single component was used to calculate the maximum loading qmax as well as the desorption constant Kd. ∗ =

∙ ∗ ∗

(2)

The isotherm parameters qmax and Kd were determined by fitting the data to the Langmuir model. This was done using the least squares method in SigmaPlot®11 (Systat Software Inc.).

General procedure for temperature-induced column chromatography. The column (Omnifit BenchmarkTM 100×6.6 mm, Diba Industries) was packed with the synthesized particles (PGMAPB3), resulting in a settled resin volume of 0.58 mL. First, the packed column was connected to a FPLC (ÄKTApurifierTM, GE Healthcare Life Sciences) system and placed in a temperature-


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controlled water bath (RC20, Lauda, ± 0.5 °C). The column was equilibrated prior to each run with at least five column volumes (CV) of loading buffer. Subsequent steps were performed using a Sample Pump P-960. The protein feed (2 mg mL-1) was loaded with a flow rate of 0.1 mL min-1 while the column was heated to 40 °C. The column was then washed by loading buffer at 1 mL min-1. The flow was stopped before placing the column into an ice bath (2 ± 0.5 °C) for 30 min. Chilled binding buffer was subsequently passed through the column at 0.2 mL min-1. Finally, the column was washed with 50 mM acetate buffer (pH 3) and with 50 mM HEPES (pH 8) with a flow rate of 1 mL min-1 at ambient temperature for regeneration. A schematic diagram illustrating the experimental setup (Figure S4) has been included in the Supporting Information (Section 6). To determine the amount of proteins eluted, the peak areas in the chromatogram at 280 nm were evaluated using Matlab R2016a (Mathworks). The trapezoidal rule was applied to calculate the integrals of the elution peaks.

Characterization Nuclear Magnetic Resonance (NMR) Spectroscopy: 1H and

13

C NMR spectroscopy were

performed on a Bruker AM 500 spectrometer (500 MHz). All compounds were dissolved in CDCl3 and the residual chloroform peak was employed for shift correction (7.26 ppm). Gel Permeation Chromatography (GPC). Polymer samples with concentrations of 1-2 g L-1 were analyzed on an Agilent Series 1200 HPLC system equipped with an isocratic pump (G1310A), an autosampler (G1329A), a thermostat controlled column compartment (G1316A) and a refractive index detector (G1362A). N,N-dimethylacetamide (DMAc) containing 10 g L-1 LiCl was used as the eluent at a flow rate of 0.5 mL min-1 (50 °C). Separation in this system was performed on a



SEC column (PSS, GRAM analytical, 300 × 8.00 mm, 10 µm particle size, 3000 Å porosity) and a precolumn (50 × 8.00 mm). Calibration was carried out with poly(methyl methacrylate) standards with molecular weights (Mp) ranging from 800 to 2.2 10-6 Da. Polymer samples were filtered through polytetrafluorethylene (PTFE) membranes with a pore size of 0.2 µm prior to injection. X-ray Photoelectron Spectroscopy (XPS): XPS measurements were performed on a K-Alpha+ XPS spectrometer (ThermoFisher Scientific, East Grinstead, UK). Data acquisition and processing using the Thermo Avantage software is described elsewhere.36 All microspheres were analyzed using a microfocused, monochromated Al Kα X-ray source (400 µm spot size). The kinetic energy of the electrons was measured by a 180° hemispherical energy analyzer operated in the constant analyzer energy mode (CAE) at 50 eV pass energy for elemental spectra. The KAlpha+ charge compensation system was employed during analysis, using electrons of 8 eV energy, and low-energy argon ions to prevent any localized charge build-up. The spectra were fitted with one or more Voigt profiles (BE uncertainty: +0.2 eV) and Scofield sensitivity factors were applied for quantification.37 All spectra were referenced to the C 1s peak (C-C, C-H) at 285.0 eV binding energy, controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, and Au, respectively. Scanning Electron Microscopy (SEM): An ethanol particle slurry was made up (approximately 25% v/v) and added dropwise to offcut silicon wafers. The silicon wafers were then left to dry at ambient conditions before being placed on sample holders. Samples were imaged with a Philips XL 30 FEG ESEM. 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.


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Elemental Analysis: The elemental composition of the microspheres was analyzed by 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.

Results and Discussion In the following, the synthesis of the block copolymers employed in the particle postmodification is described, before moving onto the microsphere functionalization and the demonstration of their dual gated release nature. Synthesis of Thermo-Responsive Block Copolymers PNIPAM (Mn 3600 Da) was synthesized using DDTTC, a trithiocarbonate RAFT agent, AIBN as initiator and 1,4-dioxane as solvent at 60 °C. The chosen RAFT polymerization conditions afforded PNIPAM of narrow molar mass dispersity while conserving end-group fidelity. The efficiency of this polymerization reaction has been reported in our previous work.33 PNIPAM was subsequently used as macroRAFT chain transfer agent to prepare P(NIPAM-b-4VP) block copolymers with different composition of 4-vinylpyridine, using AIBN as initiator and dimethylformamide as solvent at 70 °C (Scheme 2).

Scheme 2. General scheme of RAFT polymerization of PNIPAM macroCTA with 4vinylpyridine to form P(NIPAM-b-4VP) copolymers of variable monomer composition.



The molecular weights and polydispersity of the PNIPAM macroCTA and its 4VP block copolymers that were used to produce dual-gated microparticles were determined via NMR spectroscopy and GPC as presented in Table 1.


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Table 1. Molecular weight, polydispersity, and monomeric composition properties of P(NIPAMb-4VP) block copolymers prepared by RAFT polymerization. Mn,GPC Mn,NMR 1 1 nNIPAM n4VP Polymer Đ / Da / Da PNIPAM

29

0

3 600

3 200

1.2

PB1

29

7

4 800

3 900

1.4

PB2

29

15

5 800

4 800

1.4

PB3

29

30

10 000

6 400

1.4

P4VP

0

90

7 300

9 500

1.2

1

Number of monomer units determined by 1H NMR spectroscopy.

Figure 1 compares the 1H NMR spectra of the PNIPAM and P4VP homopolymers as well as the different P(NIPAM-b-4VP) block copolymers. The number of monomer units n of the homopolymer and block copolymers were determined by comparing the integrals of the methyl proton resonances of the Z group of the RAFT agent (0.88 ppm) with the resonances associated with the pyridyl protons of P4VP (8.32 ppm) and the NHCH- methine proton of PNIPAM (4.00 ppm) and are reported in Table 1. The molar mass distribution of the polymers were recorded via GPC and are depicted in Figure 2. The GPC traces clearly indicate that the block copolymers maintain a monomodal distribution behavior after chain extension reactions with 4-vinylpyridine, which is an indication of the success of the controlled block copolymerization. In addition, diffusion-ordered spectroscopy (DOSY) experiments were conducted on the polymer samples to provide further evidence that block copolymers were successful synthesized. Resonances corresponding to both the PNIPAM and P4VP blocks were selected and the diffusion coefficients for these peaks were measured. An example of the DOSY experiment for PB3 is included in the Supporting Information (Figure S2), indicating that the diffusion coefficients measured from the resonances derived from both block copolymers (D (8,35 ppm, Ar-H pyridine) = 3.23 × 10−10 m2



s−1 and D (4,01 ppm, CH-NIPAM) = 3.25 × 10−10 m2 s−1) were similar, thus providing evidence that the introduced 4-vinylpyridine was successfully grown from the dormant PNIPAM chain. Diffusion coefficients derived from resonances of both block copolymers for PB1 and PB2 are also included in the Supporting Information Section 2.

Figure 1. 1H NMR spectra of PNIPAM, PB1, PB2, PB3 and P4VP in CDCl3. Copolymer compositions and molecular weights were estimated from the integration of the pyridyl protons of P4VP (red) and the NHCH- methine proton of PNIPAM (green) relative to the methyl protons on the Z-group of the RAFT agent (blue), x denotes residual tetrahydrofuran.


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1,0

Normalized RI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PNIPAM PB1 PB2 PB3 P4VP

0,5

0,0 1000

10000

Molar mass (Da)

Figure 2. GPC traces of polymers (refer to Table 1) used for thiol-epoxy grafting onto PGMA microparticles.

Grafting-to of Dual-gated Block Copolymers onto PGMA Microspheres PGMA microspheres with epoxy surface groups were coupled to the thiol-functionalized block copolymers via ring-opening of oxirane rings by thiolate anions forming thioether bonds. Thioether bonds are known to be stable and are commonly utilized to introduce specific ligands onto chromatographic media.38 First, model reactions were performed by grafting a small molecule onto the epoxy surfaces of PGMA microspheres. 4-Mercaptoethylpyridine (MEP) was selected as a model compound as it also contains a pyridyl ring – a ligand commonly used for adsorption of antibodies.39–41 The reaction conditions employed to synthesize the PGMA-MEP microparticles are shown in Scheme 3A. The success of the thiol-epoxy reaction was proven via



FTIR spectroscopy by the presence of the characteristic vibration bands of pyridine rings from MEP at 1 635, 1 602, and 1 559 cm-1 (Figure S1 in Supporting Information).42,43 In addition, the detection of nitrogen and sulfur measured via elemental analysis (Table S1 in Supporting Information) also confirms the attachment of MEP on the microparticles. The loading capacity and grafting density of the PGMA-MEP microparticles based on equations 3 and 4 were estimated to be 2 000 µmol g-1 and 7.44 molecules per nm2. Subsequently, P(NIPAM-b-4VP) block copolymers were grafted onto PGMA microspheres. Initially, the P(NIPAM-b-4VP) block copolymers were hydrolyzed using hexylamine to afford polymers with thiol end groups (refer to Scheme 3B) and dodecane-1-thiol. Using the thiol-epoxy reaction, thiol terminated P(NIPAM-b-4VP) chains were subsequently grafted onto the PGMA microparticles. Compared to the reaction using MEP, a different base catalyst and solvent system comprising of LiOH in a mixture of THF and water was employed as the copolymers exhibited poor solubility in the phosphate buffer system and showed no presence of block copolymers on the microsphere surface after the grafting reactions.

Scheme 3. Preparation of (A) PGMA-MEP functional microparticles and (B) PGMA-P(NIPAMb-4VP) microparticles via the thiol-epoxy reaction using polymers from Table 1.


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Subsequently, the modified microparticles were characterized via FT-IR spectroscopy to determine the success of the grafting reactions (Figure 3). All spectra were normalized to the peak at 1 725 cm-1, which is attributed to the stretching vibration of the carbonyl group (C=O) of PGMA base microparticles.5 The initial non-modified PGMA microparticles (Figure 3e) also possess peaks corresponding to the ether C-O-C stretching (1 148 cm-1) vibration and epoxy groups (908 cm-1).44 After the grafting-to reactions, the N-H bending vibrations and C=O stretching bands from the PNIPAM block were observed at 1 550 cm-1 and 1 645 cm-1, respectively (Figure 3b-d). In Figures 3a–d, the characteristic vibration bands of the pendant pyridine rings from P4VP were observed at 1 602, 1 556, 1 496, and 1 417 cm-1 (peaks i to iv).42 The appearance of distinctive bands of the P(NIPAM-b-4VP) block copolymers (PB1–3) and P4VP homopolymers indicate the success of the grafting-to reactions. The intensities of these bands increase when longer P4VP chains were incorporated in the grafted polymer chains. The amount of 4VP units added to each block copolymer were estimated to be 7.6% (PGMA-PB1), 17.8% (PGMA-PB2), and 33.7% (PGMA-PB3) by integrating bands attributed to the P4VP block (peaks ii to iv) relative to the PNIPAM carbonyl band (peak i), These values were close to the results obtained via NMR spectroscopy as shown in Table 1.



(i) (ii)(iii)

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(iv)

a b c d e 2000

1800

1600

1400

1200

1000

800

-1

Wavenumber (cm )

Figure 3. FT-IR spectra of (a) PGMA-P4VP, (b) PGMA-PB3, (c) PGMA-PB2, (d) PGMA-PB1 and (e) unmodified PGMA microparticles. The vibration labelled (i) 1 645 cm-1 corresponds to the PNIPAM carbonyl bands while the band (ii) 1 602, (iii) 1 556 and (iv) 1 417 cm-1 represent characteristic vibration bands associated with the P4VP block. Importantly, XPS measurements were carried out to evidence the success of the grafting reactions. Figure 4 depicts the C 1s spectra of the unmodified PGMA microparticles as well as the modified particles with P4VP and PB3. The unmodified PGMA micoparticles showed – evidenced by deconvoluted peaks – the characteristic peaks of the O=C-O (288.9 eV) and C-O (286.6 eV) groups, which are associated with glycidyl methacrylate units.45 After the grafting reactions, both peaks shifted to lower binding energies attributed to O=C-N (288.1 eV)46 and C-N (286.4 eV), evidencing the successful attachment of the polymers. In addition, a shake-up satellite of weak intensity corresponding to photoelectron interactions with π-π* transitions due


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to the aromatic pyridine rings can also be observed at 292.0 eV for the P4VP modified particles (Figure 4B).47 Further evidence of the success of the grafting reactions is provided by the presence of nitrogen (8.5 at%) from the pyridine ring in the one repeat unit as well as from NIPAM repeat units and sulfur (0.3 at%) on the surface of PGMA-PB3 microspheres. The spectra of the unmodified PGMA microparticles do not feature any nitrogen or sulfur as expected. The N 1s and S 2p spectra of PGMA and PGMA-PB3 are included in the Supporting Information (refer to Figure S3).

C-C, C-H C-O, C-N O=C-O, O=C-N

A

Normalized Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B

Pi-Pi* C-O O

O=C-O

O

C 294

292

290

288

286

284

282

Binding energy [eV]

Figure 4. C 1s XPS spectra of (A) PGMA-PB3, (B) PGMA-P4VP and (C) PGMA. All spectra are normalized to the highest intensity. SEM images taken of the PGMA microparticles before and after the grafting reaction indicate no significant changes in the overall morphology of the resins. As shown in Figure 5, images of the



Page 24 of 47

PGMA-PB1 microparticles are used as an example. The preservation of the spherical shape and integrity of the microparticles after the grafting reaction indicates that the reaction conditions and designed setup were not destructive to the microparticles.

a

c

d

b

Figure 5. SEM images of the non-modified PGMA microparticles (a and b) and the PGMA-PB1 microparticles (c and d). Quantification of the grafted amount of polymer onto the modified microparticles was carried out by elemental analysis (refer to the Supporting Information Table S1). The nitrogen content derived by elemental analysis was used to calculate the amount of polymer chains (i.e. loading capacity) that were successfully attached onto the surfaces of the microparticles. The loading capacity (LC) in mol g-1 was subsequently calculated according to equation (3) using the weight


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of nitrogen per 1 g of microparticles, W(N), molecular weight of nitrogen, M(N), and number of nitrogen atoms, n(N), which is the number of nitrogen atoms associated with the pendant NIPAM and 4-VP units present on each polymer chain as determined by NMR spectroscopy (Table 1).48 The n(N) values for each polymer is taken as the sum of nNIPAM and n4VP as presented in Table 1, M(N) is 14 g mol-1. LC =

() ()∙!()

(3)

No sulfur and very low amounts of nitrogen were detected on the unmodified particles providing further evidence that the measured nitrogen and sulfur contents is associated with the modification reactions. It should also be noted that the amount of sulfur measured from the elemental analysis experiments was three to six times higher than expected (refer to Supporting Information Table S1). The excess sulfur measured is likely associated with the reaction of dodecane-1-thiol with epoxy groups on the microparticles. Traces of dodecane-1-thiol may still be present from the removal of the RAFT Z groups after the aminolysis of the polymers and therefore could have also participated in the thiol-epoxy reactions.



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Table 2. Comparison of the loading capacities (per gram) – calculated from elemental analysis data – and grafting densities (per surface area) obtained on the PGMA microparticles grafted with different copolymers and P4VP with the assumption that the microparticles are perfect spheres.

Sample

LC

Grafting density

Capacity of 4VP

Area per chain

(µmol g-1)

(chains per nm2)

(µmol g-1)

(nm2)

PGMA-PB1

45

0.17

9

6

PGMA-PB2

46

0.17

16

6

PGMA-PB3

36

0.14

18

7

PGMA-P4VP

24

0.09

24

11

The characterization of the employed PGMA microparticles used in the present work have been carried out and reported in a previous publication by our group.5 With the assumption that the pores are represented by cylindrical geometries, the specific surface area (SSA) was found to be 162 m² g-1 or 1.62⋅1020 nm2 g-1 as measured by inverse SEC.5 The grafting densities of each microparticle were then calculated using equation (4) by multiplying the loading capacities (LC) with the Avogadro’s number (NA, 6.022⋅1023 mol-1) to obtain the number of chains per gram. By dividing these values with the specific surface area (SSA), the number of chains per nm2 was estimated. Grafting density =

./∙01 223

(4)

The grafting densities of the modified particles are tabulated in Table 2. PB1 (3 900 Da) showed approximately a two-fold increase in grafting density (0.17 chains nm-2) to P4VP (9 500 Da, 0.09 chains nm-2). This is not unexpected as longer chains tend to encounter more significant steric


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hindrance effects during grafting-to hence affecting the efficiency of the grafting reactions. The grafting densities of PB1 and PB2 were similar, while the longer copolymer, PB3, showed a slight decrease in grafting efficiency. The distance between the chains could also be estimated from the area per chain by assuming the area conforms a square geometry and was found to be between 2 to 3 nm. Table 2 compares the LC, the grafting density, the capacity for 4VP and the area per polymer chain for PGMA samples before and after modification with the different stimuli-responsive polymers. The grafting density values were also compared with other studies, which have utilized the ‘grafting-to’ strategy. For example, our team grafted different polymers onto PGMA microparticles using both thermally and photoinduced hetero-Diels-Alder (HDA) chemistry and have reported similar grafting densities to the current work.5,49 In this study, the highest grafting density achieved via photoinduced HDA was 0.12 chains nm-2 (distance between chains of 2.9 nm) with poly(N,N-dimethylacrylamide), while thermally induced HDA produced particles with densities up to 0.16 chains nm-2 (distance between chains of 2.5 nm) with glycopolymers. Nordborg and co-workers utilized the thiol-epoxy reaction to graft poly(sulfopropyl methacrylate) onto epoxy functionalized divinylbenzene (DVB) microparticles.7 They reported a sulfur content of 0.6% measured via elemental analysis, which correspond to approximately 9 µmol g-1 of chains by taking into account the number of sulfur atoms per chain as 21. RAFT synthesized trimethoxysilane-functionalized block copolymers grafted to hydroxide groups on silica particle surfaces were reported to achieve grafting densities between 0.018 to 0.076 chains nm-2 for polymers of 7 500 to 24 000 g mol-1.50 Similar grafting densities were also measured, i.e. 0.017 to 0.085 chains nm-2, even when the coupling reaction was switched to the coppercatalyzed azide-alkyne cycloaddition.51 In comparison to their reported LC values, we can



achieve significantly higher chain densities, but using different reaction conditions and microparticles. The configurations of the grafted polymer chains were predicted based on the estimated distance between chains to the Flory radius (included in the Supporting Information Table S2). It should be noted that predicting the regime using the Flory radius is only a rough estimation as the bulky side groups of the polymer chains are not accounted for. Based on these estimations, the polymers are postulated to have brush-like structures. Previous reports of chromatographic microparticles grafted with PNIPAM have shown that densely packed polymer brushes undergo phase transition over a broad temperature range due to the less solvated polymer segments within the brush.52–54 In comparison, tethered PNIPAM chains with lower grafting densities collapsed close to the expected lower critical solution temperature of the polymer. While acknowledging that having high polymer density of grafted homopolymer PNIPAM may affect the temperature responsiveness of microparticles, we have placed the thermoresponsive block of our grafted copolymers as the outer layer of the brush. Above its phase transition temperature, the outer thermoresponsive PNIPAM block would collapse, therefore enabling interactions between proteins and the inner P4VP segments. Batch Isotherms and Column Chromatography Experiments Adsorption Isotherm Studies The PGMA microparticles grafted with the P(NIPAM-b-4VP) copolymers of different compositions were designed to have dual-gated bind and release properties for immunoglobulins. Above the LCST of the block copolymer, adsorption of immunoglobulins takes place while the immunoglobulins are released when the temperature is adjusted below the LCST. Besides


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temperature induced partial desorption, a full desorption of the immunoglobulins can be achieved by lowering the pH to pH 3, however, at these rather harsh desorption conditions partial inactivation of the released immunoglobulins may occur. The dual-gated adsorbent particles developed allow the gentle temperature controlled bind and release operation utilizing a fraction of the adsorption capacity and a complete pH controlled regeneration of the adsorbents when required for cleaning purposes. In order to determine the temperature controlled fraction of the adsorption capacity, initially a set of microparticles with different P(4VP-b-NIPAM) compositions were assessed using bovine immunoglobulins dissolved in HEPES pH 8 to determine the particle type (in terms of grafted polymer species) with the most efficient adsorption. At pH 8 immunoglobulins bind to the PGMA-P(4VP-b-NIPAM) microparticles, while the degree of binding is temperature dependent. Figure 6 compares the isotherms of immunoglobulins binding onto four PGMA-P(4VP-bNIPAM) particle types at 40°C. As expected, the binding capacity improves with increasing chain length of the 4VP block with the P4VP homopolymer yielding the highest qmax of approximately 29 mg mL-1 settled resin. Within the group of PGMA-P(4VP-b-NIPAM) microparticles, the particles grafted with the block copolymer constituted of approximately 50% 4VP (PB3) shows the highest qmax (18.9 mg mL-1 settled resin) followed by PB2 and finally PB1, where no significant adsorption was measured. These observations indicate that the adsorption of the immunoglobulins only occurs via interactions with the 4VP segment of the copolymer. These results support the assumption that NIPAM blocks display no protein binding capabilities under low salt conditions.55 Since PGMA-PB3 showed the best binding capacity among the assessed copolymer modified microspheres, it was subsequently used in further experiments to demonstrate the temperature-switchable release properties of the particles.



Figure 6. Adsorption isotherms of immunoglobulins with modified PGMA particles of different 4VP compositions in HEPES at pH 8 (40 °C). PGMA-PB3 (black circles), PGMA-PB2 (green squares), PGMA-PB1 (blue diamonds) and PGMA-P4VP (red triangles).

In order to investigate the influence of pH and salt onto immunoglobulin binding, buffers of pH 6 (MES) and pH 8 (HEPES) with and without NaCl (100 mM) were used to perform adsorption isotherms on the PGMA-P4VP and PGMA-PB3 microparticles. For the PGMA microparticles grafted with the homopolymer, adsorption (∆qmax ≈ 6 mg mL-1 settled resin) and binding affinity were shown to decrease slightly with the addition of salt at both pHs (Supporting Information Figure S6). The lower binding capacity and decreasing Kd values with the addition of salt was previously reported by Li et al.29 Their batch isotherm results of immunoglobulins in 20 mM


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Tris-HCl buffer at pH 8 and 9 showed a trend of decreasing binding capacity with increasing ionic strength through the addition of 0 to 500 M NaCl. The highest loadings on the copolymer grafted microparticles were achieved using 50 mM HEPES at pH 8 (Supporting Information Figure S5). A higher adsorption affinity (indicated by the higher initial slope of the graph) was also observed at pH 8 compared to pH 6. In 50 mM MES at pH 6, the immunoglobulin solution appeared to be slightly turbid due to partial precipitation, which is not surprising as the pI of immunoglobulins has been reported to be between 6.5 to 9.5.56,57 At pH 6, the immunoglobulins should be positively charged and the pyridyl groups of the copolymer are closer to their pKa, which explains the lower loadings attributed to electrostatic repulsion between the protein and ligand. At pH 8, the pyridyl groups are uncharged, leading to higher adsorption and adsorption affinity. The solubility of the proteins was found to improve significantly with the addition of 100 mM NaCl to MES buffer. The higher binding capacities observed in the MES buffer with 100 mM NaCl is likely attributed to the better solubility of immunoglobulins at pH 6. However, the addition of 100 mM NaCl to the HEPES buffer at pH 8 decreased the binding capacities of the copolymers significantly and no adsorption was measured. Having identified optimal binding conditions, the adsorption of immunoglobulins on PGMA-PB3 and PGMA-P4VP was also performed at 5 °C, 50 mM HEPES, pH 8 to determine the temperature dependence of binding. As shown in Figure 7, the binding capacity on PGMA-PB3 at 5 °C was found to be 7.5 mg mL-1 settled resin, which is approx. 2.5 times lower compared to the qmax (18.9 mg mL-1 settled resin) at 40 °C, resulting in a ∆qmax of 11.3 mg mL-1 settled resin. A plausible explanation for the temperature responsive nature of these particles is that at 40 °C the exterior PNIPAM segment of the copolymer chains are in a collapsed state, which allows



more space for proteins to interact with the inner P4VP block. In contrast, the PNIPAM segments are fully extended at 5 °C, resulting in less space for the proteins for adsorption. The fitted Kd values of adsorption at 5 °C coincide with the expectations of a lower binding affinity at temperatures below the LCST of PNIPAM. It is interesting to note that no significant changes in the qmax value were observed for the PGMA-P4VP particles with temperature, demonstrating that the temperature effect observed for PGMA-PB3 particles is not simply caused by less pronounced hydrophobic interactions at lower temperatures.

Figure 7. Adsorption isotherms of immunoglobulins with PGMA-PB3 (circles) and PGMAP4VP (triangles) in 50 mM HEPES (pH 8) at 40 °C (black) and 5 °C (red).


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Release Studies Based on Dual-gated Microparticles Other than the possibility to adsorb proteins at different temperatures with strongly differing affinities, it is also necessary for the dual-gated microparticles to be able to desorb bound proteins as a result of a temperature or pH switch. The suitability of the PGMA-PB3 resins as potential dual-gated stationary phase for chromatography was examined in the following column experiments applying a protein concentration of 2 mg mL-1. Figure 8a illustrates the chromatogram of a column experiment in 50 mM HEPES buffer at pH 8. As shown in the chromatogram, there is a steep increase in the immunoglobulin concentration starting at 3 mL attributed to the fact that the adsorption capacity of the column is reached and the protein starts to appear in the effluent. This protein breakthrough during loading reaches a plateau of 1.9 mg mL1

. The increase in immunoglobulins concentration was significantly faster in the first 5 mL of the

loading phase in comparison to the remaining 15 mL, where the immunoglobulin concentration was only increased by 0.6 mg mL-1. Only a part of the immunoglobulins in the feed solution was bound to the stationary phase while the rest was in the flow-through. The aim of this intensive loading phase was to reach almost equilibrium conditions between the loading of the PGMA-PB3 resin and the concentration of immunoglobulins in the feed. After the loading phase, a washing phase was commenced at 40 °C marked as stage (II) in the chromatogram. Partial immunoglobulin desorption was then carried out by lowering the temperature of the column to 2 °C, leading to the elution of immunoglobulins (stage (III)). Subsequently, remaining immunoglobulins were fully desorbed with acetate buffer at pH 3 as depicted in Figure 8a as stage (IV). Summing up, the immunoglobulins amounts desorbed by cooling and by lowering the pH, a minimum of 5.6 mg immunoglobulins mL-1 settled resin were bound to the dual-gated particles during the loading phase. Of this amount approx. 22% could be desorbed solely by



Page 34 of 47

cooling the column once and the remaining 78% by lowering the pH. It can be assumed that the percentage of temperature induced desorption could be increased by a repeated temperature switch between above and below the LCST, as it is reported e.g. for lactoferrin bound to thermoresponsive cation-exchange resins.58 However, this mode of operation was not tested as we aimed to determine the working capacity of our resin for regular operation using only one elution step. In contrast to the described behavior of a column filled with PGMA-PB3 resin, column experiments with PGMA-P4VP resin (Figure 8b) using the same setup do not show an elution peak in region III where the column was cooled. This leads to the conclusion that temperature controlled elution is a specific feature of the PGMA-PB3 resin, which results from the

combination

of

4-vinylpyridine

with

the

thermo-responsive

polymer

poly(N-

isopropylacrylamide).

Figure 8. Chromatogram for adsorption of γ-globulins on a) PGMA-PB3 and b) PGMA-P4VP in a column experiment. The loading (I) and the washing phase (II) took place at 40 °C. For the elution phase III the column was placed in an ice bath. The flow was paused between region II and III for 30 min to cool the column. The regeneration phase IV was conducted by changing the feed buffer to acetate buffer (pH 3) at ambient temperature. Flow rates were 0.1 mL min-1 for


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loading (I), 1 mL min-1 for washing (II), 0.2 mL min-1 for temperature induced elution (III) and 1 mL min-1 for pH induced regeneration (IV).

A summary of all experiments with a column packed with PGMA-PB3 microparticles carried out in various buffer systems with and without addition of salt (NaCl) is included in the Supporting Information (Table S3). The experiments followed the same procedure as described in Figure 8. In the first run with 50 mM HEPES at pH 8, the largest amount of protein was ad- and desorbed, corresponding to a total elution (the sum of the proteins amounts desorbed by temperature and pH) of 10.4 mg immunoglobulins mL-1 settled resin. In subsequent experiments with 50 mM HEPES buffer pH 8 (runs 2 and 6 in Table S3) total elution amounts of 5.6 and 4.5 mg immunoglobulins mL-1 settled resin were found. Thereof, the amount eluted due to a temperature shift made up 1.6, 1.2 and 0.7 mg immunoglobulins mL-1 settled resin (runs 1, 2 and 6 in Table S3). One possible explanation for the observed decline in the total elution amount as well as the temperature induced elution amount is an irreversible adsorption that may lead to a blocking of binding sites. Furthermore, small particles or particle debris caused by mechanical stress might have been flushed out, reducing the available amount of adsorbent. The addition of 100 mM NaCl in HEPES buffer showed a strongly negative influence on immunoglobulins loading (run 7 in Table S3). This supports the batch adsorption isotherm results shown earlier, which show no binding when 100 mM of NaCl was added at pH 8. Further, experiments at pH 4 and pH 6 using acetate and MES buffer (runs 3 and 5 in Table S3) showed no significant loading and no temperature-induced elution. To outline the applicability of the dual-gated microspheres in the field of preparative chromatography, the principle of combining 4-vinylpyridine with the thermo-responsive polymer poly(N-isopropylacrylamide) to provide dual-gated microspheres could be successfully



transferred to agarose particles (Epoxy-Activated Sepharose 6B from GE Healthcare Life Science), which are well established in preparative chromatography of proteins. These results have been included in the Supporting Information Section 7. Conclusions PGMA-P(NIPAM-b-4VP) microparticles showing temperature as well as pH switchable binding and release behavior for the adsorption of immunoglobulins have been successfully synthesized. This new class of dual-gated adsorbents offers advanced process options for protein chromatography applying gentle temperature induced desorption for product elution and more rigorous pH dependent desorption for column regeneration and cleaning purposes. Initially, block copolymers of P(NIPAM-b-4VP) of different compositions were successfully synthesized using RAFT polymerization. The block copolymers were endgroup-modified to form thiol moieties, which were subsequently reacted with epoxide groups on the surfaces of PGMA microparticles to form dual-gated resins. The block copolymers as well as the final microparticles were thoroughly characterized by different analytical techniques verifying their chemical composition. Subsequently, temperaturedependent adsorption of immunoglobulins was demonstrated by batch isotherms at 40 and 5 °C for PGMA-P(NIPAM-b-4VP) microparticles. Finally, temperature induced desorption as well as pH controlled regeneration was applied in chromatographic experiments, demonstrating the dualresponsiveness of the modified microparticles. The application of these dual-gated resins in purification processes of immunoglobulins offers several beneficial properties. First, harsh and potentially product damaging conditions like high acidity typically encountered in the eluate can be avoided. Second, elution with external physical stimuli eliminates the need to remove


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components of the elution buffer from the product. Therefore, the eluate may be directly used in subsequent purification steps without the need for dilution or buffer exchange often required in classical purification procedures. Third, an elution caused by external stimuli instead of changing the mobile phase enables new strategies of process control, e.g. by controlling the speed of elution independent from the flow of the mobile phase within the column.58

Associated Content Supporting Information. Experimental details for grafting of MEP onto PGMA microspheres, DOSY NMR experiments, XPS, elemental analysis, column setup for temperature-induced elution and results, dual gated agarose particles and protein G analytics for a temperature eluted peak.

Acknowledgements L.B. and C.B.-K. acknowledge continuous financial support from the Karlsruhe Institute of Technology in the context of the Helmholtz program Biointerfaces in Technology and Medicine (BIFTM) and key financial support from the Queensland University of Technology (QUT) as well as the Australian Research Council (ARC) in the form of a Laureate Fellowship (to C.B.-K.) and an ARC Linkage Grant (to C.B.-K. and L.B.). The authors thank Moritz Ebeler (IFG) for recording the SEM images, Dr Maria Schneider for the GPC measurements and Dr Eva Blasco for the DOSY experiments. The XPS K-Alpha+ instrument was financially supported by the



Federal Ministry of Economics and Technology on the basis of a decision by the German Bundestag.

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