Evidence, Manipulation, and Termination of pH 'Nanobuffering' for

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Evidence, Manipulation, and Termination of pH ‘Nano-Buffering‘ for Quantitative Homogenous Scavenging of Monoclonal Antibodies Anees Palapuravan, Yi Zhao, Andrea A Greschner, Thomas R. Congdon, Hendrick W de Haan, Nicolas Cottenye, and Marc A. Gauthier ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07202 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Evidence, Manipulation, and Termination of pH ‘Nano-Buffering‘ for Quantitative Homogenous Scavenging of Monoclonal Antibodies Palapuravan Anees, † Yi Zhao, † Andrea A Greschner, † Thomas R Congdon, † Hendrick W. de Haan‡ Nicolas Cottenye,⊥ and Marc A Gauthier*,† †Institut

National de la Recherche Scientifique (INRS), EMT Research Center, Varennes, Quebec

J3X 1S2, Canada ‡University

of Ontario Institute of Technology, Faculty of Science, Oshawa, Ontario, L1H 7K4,

Canada ⊥BioAstra

Technologies Inc., Montreal, Quebec H4P 2R2, Canada

ACS Paragon Plus Environment

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ABSTRACT: This study demonstrates that pH-responsive polymers have a very high buffering capacity in their immediate vicinity, a phenomenon termed ‘nano-buffering’. This can be exploited to dissociate local nanoscale pH from bulk solution pH. Herein, a series of pH-responsive polymers were conjugated to Protein-A to rationally manipulate the latter’s binding affinity towards antibodies via nano-buffering (i.e., this interaction is pH-dependent), independently of bulk solution pH. Moreover, the nano-buffering effect could be terminated using low concentrations of strong ion-pairing salts, to achieve quantitative release of the antibodies from the bio-conjugate. These complementary discoveries are showcased in the context of the development of a homogenous affinity precipitation agent (i.e., a scavenger) for the purification of polyclonal immunoglobulin G and two monoclonal antibodies from cell culture supernatant. Indeed, while bulk solution pH was used to induce precipitation of the scavenger, maintaining local nanoscale pH via nano-buffering maximized binding interaction with the antibodies. A 2:1 binding stoichiometry was observed, which was similar to that observed for native protein. The scavenger could be recycled multiple times, and the purification protocol circumvented lengthy/tedious physical purification processes typically associated with mAb manufacturing. Overall, this study provides perspectives on the local nanoscale pH near pH-responsive polymers, and establishes lines of thought for predictably manipulating or even terminating nano-buffering, to control the activity of proteins.

KEYWORDS: monoclonal antibody, immunoglobulin, pH-responsive polymer, nano-buffering, perchlorate, affinity precipitation


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Polymers with pH-titratable functional groups (i.e., pH-responsive polymers) exist in either an ionic hydrophilic state or a neutral hydrophobic one, depending on solution pH. Such polymers have found many biotechnological applications, from biosensing to purification,1-3 and have been extensively used as building blocks for nanoscale therapeutic systems such as protein–polymer conjugates (bio-conjugates), polymer micelles, polyplexes, polymersomes, nanoparticles, etc.4-9 In the latter cases, pH-responsiveness is most often exploited as trigger to induce dramatic changes to the physical properties of the system, for instance to provoke precipitation or to release an entrapped cargo.10-12 Alternatively, in their ionic state, pH-responsive polymers can be exploited to favorably interface with oppositely-charged biological molecules and structures. For example, polycations can interact with mucin13, 14 or the cell surface15 to promote bio-adhesion or uptake, respectively. Interestingly, pH-responsive polymers can also behave as ‘micro-buffering’ agents that locally control the bulk pH of very small compartments. This phenomenon has been extensively explored to prevent the acidification of sub-cellular compartments such as endosomes and lysosomes, via the so-called ‘proton sponge effect’.16 Wang et al., have recently reported an interesting adaptation of this strategy in which the pH of endocytic organelles in HeLa cells was buffered to specific pH values between 4 and 7.4.17 This was achieved using a series of ten pHresponsive copolymers with incrementally-increasing pKa values. Considering the ability of pHresponsive polymers to buffer the bulk pH of small compartments, it is reasonable to hypothesize that such polymers may buffer the local pH around nanoscale systems of which they are comprised (i.e., ‘nano-buffering’), and this value may be different from bulk pH. This effect may in turn influence the activity, conformation, or stability of other constituents, such as drug molecules, degradable linkers, biomolecules (e.g., therapeutic proteins or nucleic acids), or the interaction of


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appended ligands towards their binding targets (e.g., antibody–antigen interaction). Thus, the applications and implications of such a phenomenon are far reaching. While it is generally known that enzymes immobilized onto charged supports possess different pH–activity profiles than in solution,18 to the extent of our knowledge only one study has investigated comparable effects on the nanoscale. Zhang et al.,19 have grafted poly(methacrylic acid) to cytochrome C and have shown that the polymer extended the enzyme’s optimal range pH for activity ~3 pH units away from its maximal value, due to an altered local environment. While of significant conceptual interest, this study does not investigate the nanoscale range of the effect, its tunability, or methods to terminate it. Moreover, studies that investigate an effect of local environment on other forms of protein function, such as protein–protein binding interactions, do not exist. Framed in the context of the development of a homogenous affinity precipitation agent (i.e., a scavenger) for the purification of antibodies from cell culture supernatant, this study addresses these three challenges. Results indicate that nano-buffering effects are confined to the immediate vicinity of pH-responsive polymers (i.e., contact required via covalent, electrostatic interaction, or other), can be deliberately controlled to maximize the binding of antibodies to the scavenger independently of bulk pH (i.e., this interaction is pH-dependent), and terminated with small quantities of ion-pairing salts to quantitatively release the antibodies. RESULTS Homogenous scavenger design and preparation As illustrated in Scheme 1, the scavenger is composed of a pH-responsive (co)polymer and Protein-A, which primarily binds to the constant region of antibodies of the immunoglobulin G family (IgG) at near-neutral pH. The recombinant Protein-A used to prepare the scavenger is


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composed of five homologous IgG-binding domains and a single reduced cysteine residue at its C-terminus. The cell wall-, cell membrane-, and albumin-binding regions are absent in this protein, to ensure maximum specific IgG binding (full sequence in Fig. S9). Isothermal titration calorimetry revealed that each molecule of Protein-A bound two IgG molecules, in accordance with the literature (the remaining binding sites become inaccessible due to steric hindrance).20-22 Modification of the cysteine residue with a hetero-bifunctional linker yielded a macro-initiator for atom transfer radical polymerization (ATRP) that maintained similar IgG-binding properties to the native protein (Supplementary Table S3). From this macro-initiator was polymerized alkylated aminoethyl methacrylates. More specifically, homo- or co-polymers of 2-diisopropylaminoethyl methacrylate

(DIPAEMA),

2-diethylaminoethyl

methacrylate

(DEAEMA),

and

tert-

butylaminoethyl methacrylate (TBAEMA) were prepared (Scheme 1). Variation of polymer composition yielded a library of six scavengers (1–6) with sharp and incrementally-different pKa values between 6.3 and 7.8 (Fig. 1a, Supplementary Fig. S2). Higher and lower pKas were avoided because of potential protein deamidation (both of the scavenger and mAbs to be purified) and low affinity of Protein-A for IgG, respectively.23-25 Each scavenger narrowly buffered bulk pH within ±0.1 pH units around its pKa (titration curves in Supplementary Fig. S2) and the molecular weight of scavengers ranged between 280–400 kDa (Supplementary Table S1, Fig. S1).


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Scheme 1. Homogenous scavenger for mAb purification designed to nano-buffer local pH, while responding to bulk pH. At near-neutral pH, the scavenger binds up to 2 eq. of mAb. Basification above the pKa of the scavenger induces precipitation, enabling the scavenger–mAb complex to be isolated from cell culture supernatant. The pellet is dissolved in acidic medium, and a strong ionpairing agent (X–) added. The latter terminates nano-buffering and induces precipitation of the scavenger, leaving the mAb in solution. Dissolution of the scavenger at neutral pH enables recycling of the scavenger, so that the process can be repeated. Note: ‘i, j, k’ are the mole fractions of each monomer within the statistical (co)polymer; Protein-A is illustrated as a repetitive structure composed of five homologous binding domains (pdb 2spz).


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By design, basification of a solution containing mAb and scavenger above its pKa induces precipitation, which enables recovery of the scavenger (alongside any bound mAbs) from the supernatant. One important challenge to be addressed by nano-buffering is that binding between Protein-A and IgG is pH-dependent. It is strongest between pH 7.4–7.8 and substantially weaker outside this window (Fig. 1b, Supplementary Table S3).25, 26 Indeed, at acidic pH, poor affinity mainly results from charge repulsion involving the solvent-exposed residues H137, R146, and K154 on Protein-A and IgG (an illustration of Protein-A domain B bound to IgG1 is shown as an inset in Fig. 1b).27 Above pH 8, loss of affinity occurs due to neutralization of IgG (isoelectric point, pI = ~8.6 for IgG1, the major component of IgG).28 Thus, while important changes to bulk solution pH are required for precipitation of the scavenger, this is detrimental to mAb binding. As such, this is an exciting system with which to study nano-buffering effects, because local nanoscale pH should be buffered to attain maximum mAb–Protein-A binding affinity, despite important changes to bulk solution pH, required for precipitation.


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Figure 1. Buffering properties of scavengers and effect of bulk pH on IgG–Protein-A interactions. (a) Buffering capacity () of scavenger library (1.58 µM) in 1.5 mM NaCl (Mean + SD, n = 3). The pKa of each scavenger is defined as the bulk pH at which  is maximal. (b) pH-dependent binding properties of Protein-A to IgG by isothermal titration calorimetry (linear fit between pH 5–7.4, n = 1). Inset shows residues on a Protein-A sub-domain C (green) responsible for strong electrostatic repulsion with IgG (purple) at acidic pH (pdb 4wwi).


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Manipulation of nano-buffering to maximize IgG binding stoichiometry To begin investigations, the binding stoichiometry achieved by affinity precipitation of polyclonal human IgG was examined using Scavengers 1–6. As illustrated in Fig. 2a, binding stoichiometry strongly depended on both scavenger pKa and bulk pH. Two zones of divergent behavior were observed. Below pH ~8, each scavenger possessed different binding stoichiometry towards IgG and were independent of bulk pH (even up to ~2 pH units in the most extreme case examined). Remarkably, the stoichiometry values obtained in this region scaled linearly the pKa of the scavenger (Fig. 2b), reflecting the linear dependence of binding constant vs. pH observed by isothermal titration calorimetry (Fig. 1b). The insensitivity to bulk pH and linear scaling with scavenger pKa both reinforce the idea that the (co)polymer is buffering the local nanoscale pH of the scavenger, in particular (presumably) residues H137, R146, and K154 that are responsible for loss of affinity at acidic pH. Note that in the absence of a nano-buffering phenomenon, binding stoichiometry should be highest at a bulk pH of ~7.8 for all scavengers (independently of their pKa), because IgG–Protein-A interactions are strongest at this pH (Fig. 1b). Most notably, precipitation of the positively-charged polymer alone does not induce the co-precipitation of either IgG, a neutral antibody (infliximab, pI 7.4), or human serum albumin (pI 4.7; Supplementary Fig. S7). This suggests the absence of non-specific (e.g., electrostatic) interactions between the polymer and proteins of varying charge in solution, possibly because the polymer loses its charge concurrent with the precipitation process. It is remarkable that major differences in binding stoichiometry were observed amongst the scavengers despite relatively small changes of polymer structure, indicating that steric hindrance is not a determinant factor. Indeed, we have extensively considered the possibility that steric hindrance from the polymer might be influencing accessibility


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of the binding sites on Protein-A, as an alternative mechanism to explain the reported trends. However, this scenario does not fit all the data. All values of stoichiometry in Fig. 2 were obtained for collapsed (i.e., precipitated) polymers and the molecular weights and side-chain characteristics of all polymers were similar, which collectively removes the influence of this parameter. In fact, analogs of Scavenger 6 with shorter (130 kDa) and longer (430 kDa) polymers possessed identical binding stoichiometry to Scavenger 6 (Supplementary Fig. S3). Scavenger 6 (pKa 7.7 ± 0.1) displayed a stoichiometry of 2.02 ± 0.05 when precipitated at pH 7.8, which was statistically equivalent to that observed for native recombinant Protein-A by isothermal titration calorimetry. Above pH ~8, stoichiometry decreased in a similar manner for all scavengers, and eventually reached the same value (above pH ~8.5). This inflection point in the trend coincided with the isoelectric point of IgG1 (pH ~8.6), and suggested that the antibody itself continued to remain sensitive to bulk pH, even if its binding interaction to the scavenger should not be (based on data from above). This observation can be reconciled by the fact that charge neutralization of IgG (and reversal) occurs over its entire surface, rather than only in a small region near its binding site to the scavenger (located in the hydrophobic cleft between the 2nd and 3rd constant domains of the heavy chain). Considering that the hydrodynamic radius of IgG is ~6 nm, nano-buffering therefore appears to be localized to well below this distance from the polymer, and is truly a nanoscale phenomenon. The partial loss of binding stoichiometry above pH 8 thus appeared to reflect global changes to the conformation of IgG at basic pH that change its complementarity to Protein-A. Indeed, Jøssang et al.,29 have noted that the hydrodynamic radius of IgG increased by 3.5% when basified from pH 7.6 to pH 8.9, reinforcing this argument. Overall, this section demonstrates that nano-buffering is a programmable parameter that can be exploited to disconnect


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local nanoscale pH from bulk pH, which in the present case would enable the precipitation of the IgG–scavenger complex while maintaining strong interactions between the IgG and the scavenger.

Figure 2. Binding stoichiometry of scavengers. (a) Affinity precipitation of IgG at different bulk pH. Stoichiometry was independent of bulk pH below pH < 8 due to nano-buffering of the IgG– Protein-A interaction (note: the lowest pH examined for each scavenger was dictated by its pKa, as lower pH did not induce precipitation). Above pH 8, charge reversal of IgG (isoelectric point pH ~8.6) occurred at sites distant from the binding site, which were not buffered by the scavenger. Partial loss of binding affinity reflects conformational changes to IgG. (b) Relationship between


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pulldown stoichiometry (at pH 7.8) with scavenger pKa. Experiments were performed using 1.58 µM scavenger with 8.0 µM IgG in 1 mM phosphate buffer. Data presented as mean + SD, n = 3.

Recovery and release of IgG from the scavenger Typically, mAbs are released from Protein-A affinity resins by acidifying the medium to weaken mAb–Protein-A interactions (Fig. 1b). This approach, however, is not possible for the reported scavenger because nano-buffering maintained a local nanoscale pH that was ideal for binding. Indeed, all efforts to separate IgG from the scavenger at pH 3.5 by tangential flow filtration or centrifugal dialysis completely failed (Supplementary Fig. S4). As such, approaches to terminate nano-buffering became necessary. To achieve this, it was rationalized that salts could be added to attenuate the buffering capacity of the polymer by ion-pair formation with its ammonium groups. Strong ion-pair formation would reduce buffering capacity by restricting the ability of the proton to undergo acid–base equilibria with the solvent. In fact, the addition of 60 mM perchlorate essentially eliminated the ability of the scavengers to buffer bulk solutions (Fig. 3a), in support of this rationale. Perchlorate is known from the literature to a form strong ion-pair with ammonium groups.30 Moreover, because perchlorate is a hydrophobic ion that is a common proteinprecipitation agent, it can in principle influence the solubility of the scavenger or IgG. Remarkably, Scavenger 6 precipitated with ~40–60 mM ClO4– (Na+ and NH4+counter salts; Supplementary Fig. S5), while IgG solubility was unaffected (even > 300 mM ClO4–). Therefore, in addition to disrupting scavenger–IgG binding at acidic bulk pH (by terminating the nano-buffering effect), these small concentrations of perchlorate enabled the concurrent removal of the scavenger from solution. This is convenient for by-passing tedious or time-consuming steps such as tangential flow


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filtration by replacing them with e.g., continuous centrifugation. Indeed, while common, the former technique can be a process bottleneck for antibody manufacturing.31, 32 Interestingly, of the fifteen other salts examined for an equivalent effect, perchlorate was the only to induce scavenger precipitation at such low concentrations (Fig. 3b), highlighting its particularity in the present circumstance. With these data in hand, the use of perchlorate to isolate IgG from an IgG–scavenger complex was examined at pH 4. Addition of 60 mM perchlorate and removal of precipitated scavenger by centrifugation led to the release of 84% of the bound IgG into the supernatant (ca. ~1.7 molar eq. IgG vs scavenger). Non-quantitative release of IgG could be explained by the complexity of the composition of polyclonal IgG, which contains different sub-classes of variable binding affinity to Protein-A at acidic pH. To validate this, the quantitative (i.e., 100%) purification of two different mAbs, MEDI-573 (pI = 8.31) and RTX (pI = 8.68) (in two different media), from Chinese Hamster Ovary cell culture supernatant under different conditions (Supplementary Table S2) was achieved using Scavenger 6 (Fig. 3c). This reinforces the concept that the scavenger can not only be used to retrieve mAbs with high stoichiometry from complex crude media, but also quantitatively release them with a small amount of perchlorate. Moreover, as an illustrative example, SDS-PAGE analysis revealed that the recovered MEDI-573 solutions (after scavenger removal) were devoid of host cell proteins that were present in the initial crude mAb solution. As depicted in Fig. 3d, the crude solution contained a small amount of MEDI-573 (band at ~150 kDa) and large contaminant bands at ~20 and ~250 kDa. This section therefore demonstrates the ability to conveniently terminate nano-buffering using a low concentration of strong ion-pairing salts, which enabled the isolation of two mAbs from complex media at a stoichiometry identical to the solution binding stoichiometry of the native Protein-A.


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Figure 3. Quantitative release of IgG and mAbs from the scavenger by termination of nanobuffering using strong ion-pairing salts. (a) The buffering capacity of the scavenger library (1.58 µM) decreased by a factor of ~10 in the presence of 60 mM NaClO4. (b) Opacity of solutions of Scavenger 6 (1.58 µM, 1 mM phosphate buffer, pH 4) in the presence of 60 mM of different salts. (c) Quantitative recovery of IgG, and two mAbs using two different amounts of Scavenger 6. (d) SDS-PAGE analysis of MEDI-573 purified by affinity precipitation using Scavenger 6. Protein ladder (lane 1), Cell culture supernatant containing MEDI-573 (lanes 2 and 3; non-reducing and


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reducing, respectively) and purified mAb (lanes 4 and 5; non-reducing and reducing, respectively). Data in panes (a–c) are presented as Mean + SD, n = 3.

Recyclability of the scavenger Cost is a major driving force in the development of new mAb purification strategies. Therefore, considering the cost of recombinant Protein-A, it would be desirable to recycle the scavenger for re-use. As such, the protective character of the co-polymer on the Protein-A component of the scavenger was evaluated. At first, Scavenger 6 was challenged to repetitive precipitation/redissolution cycles (basification and acidification, respectively), and then IgG binding stoichiometry evaluated. No appreciable change in binding stoichiometry was observed, even after 10 cycles of acid/base treatment (Supplementary Fig. S6). Given these promising results, the scavenger was challenged to four consecutive rounds of MEDI-573 purification from cell culture supernatant. For these tests, 0.5 or 1 molar eq. of Scavenger 6 was used relative to mAb owing to its ~2:1 binding stoichiometry (above). As illustrated in Figure 4a, a cumulative ~5–13% loss of the ability of the scavenger to retrieve mAb from cell culture supernatant was observed in rounds 2–4. Comparable results were obtained with IgG and suggests partial fouling of the scavenger. Based on this result, it is expected that the scavenger can be used for multiple rounds of mAb recovery before replacement is required. Gel electrophoresis of the purified mAbs are very similar for all four rounds of purification, indicating that the scavenger is not undergoing non-specific interaction with other impurities from the crude mAb solutions (Fig. 4b). Also, a recyclability experiment performed at pH 6.0 (Supplementary Fig. S8) yielded similar results, indicating that antibody release under less acidic conditions is possible. Overall, this section demonstrates


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recyclability of the scavenger over multiple rounds of purification, while still maintaining high binding stoichiometry to mAbs. More importantly, these results hold substantial promise for future improvements to Scavenger 6, possibly by using other engineered forms Protein-A that have been designed for lesser fouling.33, 34

Figure 4. Recyclability of the scavenger. (a) Recovery of MEDI-573 or IgG from cell culture supernatant after each cycle of purification using the same scavenger (1 eq. of Scavenger 6) (Mean + SD, n = 3). Values provided as a percentage of antibody titer of the crude solution. (b) SDSPAGE of recovered MEDI-573 after each cycle of purification. For each cycle, non-reducing (left) and reducing conditions (right) were employed.


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Discussion pH-responsive polymers differ from traditional small-molecule buffering agents in that their buffering side-chains are not homogenously distributed in solution. Indeed, on the nanoscale, the buffering side-chains of the polymer are confined to a small volume (via the polymer backbone), which dramatically increases their local concentration at this location. When adapted to a single polymer chain at the nanoscale, the van Slyke equation35 predicts infinite buffering capacity at the immediate surface of the polymer, but a rapid decreases thereafter (proportionate to the cube of distance). This suggests that the environment affected by nano-buffering is very small, and highly confined to the immediate vicinity of the polymer. In fact, at a concentration of 8 µM IgG (typical value used in this study), the average volume per scavenger is 2 × 105 nm3, which corresponds to a cube with an edge of ~60 nm. Given the anomalously high diffusion coefficient of protons in water achieved via the Grotthuss mechanism36 (also known as ‘proton jumping’), the time required for a proton to diffuse between complexes is on the order of 10–7 s. This is significantly faster than typical timescales for protonation/deprotonation37 and thus it is unlikely that the system can be divided into regions of ‘local’ and ‘bulk’ pH. Indeed, the fact that IgG (hydrodynamic radius ~6 nm) complexed to the scavenger remained sensitive to bulk pH equally suggests that nanobuffering is restricted to well below this distance from the polymer. Thus, to reconcile the microenvironmental effect of pH responsive polymers observed in this study (and others)19, a more detailed look into the nanoscale range of nano-buffering became necessary. It is well-known from the protein literature that the manner in which functional groups are positioned in 3D at the nanoscale influences their local acidity constants. Indeed, the pKa of amino acid residues on the surface of a protein can sometimes be very different from those of free amino acids in solution, because of close-range interactions. As such, this reasoning suggests that nano-


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buffering could exist at the interfacial contact zone between Protein-A and the (co)polymer, where intimate contact might overcome high proton diffusion. Indeed, this contact is possible not only because Protein-A and the polymer are covalently bound to one another, but also due to other possible mechanisms including electrostatic interactions, hydrogen bonding, etc. Indeed, the surface of Protein-A pointing away from IgG (when complexed) is negatively charged (Fig. 5a) and could interact with the positively-charged (co)polymer (on the same or a different scavenger). To investigate this aspect using a complementary technique, Scavenger 6 was incubated with 6,8dihydroxy-1,3-pyrenedisulfonic acid disodium salt (DHPDS), which is a small molecule fluorophore with two negatively-charged sulfonate groups that can electrostatically interact with the positively-charged (co)polymer. The fluorescence properties of DHPDS are sensitive to pH, and Fig. 5b depicts a linear dependence of fluorescence intensity with respect to bulk solution pH. A similar trend was observed for DHPDS in the presence of Protein-A, with slight differences suggesting interaction with the protein. However, in the presence of either Scavenger 6 or the equivalent pH-responsive (co)polymer (i.e., without Protein-A), the fluorescence intensity was more or less stable between pH 6–7.5, though it decreased thereafter (due to scavenger/polymer precipitation). Remarkably, the local pH experienced by DHPDS estimated from this figure was in the vicinity of ca. ~8, which was close to the pKa of the polymers used. As such, this experiment supports that intimate contact with the pH-responsive polymer (e.g., covalent bond, electrostatic interactions, hydrogen bonding, or other) is required to observe nano-buffering. This reasoning therefore explains why Protein-A, which is covalently bound to the (co)polymers, experiences a buffering phenomenon, but not IgG.


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Figure 5. Intimate contact is required for nano-buffering. (a) Protein-A domain C (green) bound to the Fc fragment of IgG (purple) (pdb 4wwi). Nine negatively-charged residues (blue) on the Protein-A domain C are pointing away from the antibody, in comparison to only four positivelycharged ones (red), suggesting possible interaction with the positively-charged (co)polymer on the scavengers. (b) The fluorophore DHPDS responded in a linear manner with bulk pH when either alone or in the presence of Protein-A. However, in the presence of Scavenger 6 or polymer alone (i.e., Scavenger 6 without the Protein-A component), the fluorescence of the dye remained unchanged. Data presented as Mean ± SD, n = 3.


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The second interesting observation from this study is that low concentrations of strong ioncomplexing agents terminate the buffering effect of the polymers. This effect occurred independently of pH by preventing proton exchange between the polymer and the solvent, and was necessary in this study to release the antibody from the scavenger. While perchlorate had the most pronounced effect of all salts examined herein Fig. 3b, it is very likely that negatively-charged polyanions may have a similar effect as perchlorate because of cooperativity resulting from multiple ion-pairing interactions between the two species. Thus, for many nanoscale systems formed by complex coacervation (micelles, DNA delivery vehicles),38 nano-buffering may intrinsically be terminated by the presence of polyanions, which suggests that the effect might be restricted to certain types of nanoscale systems, and depend on their composition.

Conclusions In summary, this study demonstrates that pH-responsive polymers have a very high buffering capacity in their immediate vicinity, which can be exploited to dissociate local nanoscale pH from bulk solution pH. This effect was exploited to control the binding affinity between two proteins. The effect requires intimate contact between the protein and the pH-responsive polymer. Based on this principle, a homogenous scavenger was prepared for antibody purification. Native Protein-Alike binding stoichiometry was achieved, the scavenger could be recycled, and used in a manner that did not require lengthy/tedious physical purification processes (e.g., tangential flow filtration, affinity columns, etc.). Moreover, the nano-buffering effect could be terminated using low concentrations of strong ion-pairing salts, to achieve quantitative release of the antibodies from the bio-conjugate. As therapeutic strategies involving mAbs are becoming increasingly


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prevalent,39,

40

market demands are exerting considerable pressure to make the manufacturing

process more efficient to reduce their cost. In fact, while substantial engineering has gone into optimizing the performance of solid-supported mAb scavengers based on Protein-A, Protein-G, or aptamers (etc.),32,39-43 little work has been reported on homogenous Protein-A-based scavengers. As such, this system is relevant because homogenous scavengers can be more rapidly processed (centrifugation, filtration) and recycled (for reuse). Finally, considering the abundance of pHresponsive polymeric (or other) systems described in the literature, this study provides perspectives on their very local pH properties, which may differ significantly to those of bulk solution pH. Moreover, this study establishes lines of thought for predictably manipulating these local pH properties, and even terminating the nano-buffering effect, to achieve new or tailored effects.

Methods Preparation of scavengers. The scavengers were synthesized by ATRP of alkylated aminoethyl methacrylates from the Protein-A macro-initiator. The characteristics of the polymer were controlled by the composition of monomers and their feed ratio vs. the macro-initiator (Table S1). As a representative example (to prepare 1), CuBr (13.2 mg, 0.092 mmol) and 2,2’-bipyridine (14.35 mg, 0.092 mmol) were added to a cylindrical flask containing 4 mL of phosphate buffer (100 mM, pH 7). The flask was sealed with a rubber septum and the solution purged by bubbling nitrogen for 15 min. In a separate flask, Protein-A macro-initiator (10 mg, 0.2914 µmol) and DIPAEMA (137.97 µL, 0.5828 mmol) were added to 2 mL PBS (100 mM, pH 7) and purged as above. To the macro-initiator solution was added 150 µL of the copper solution using a gas-tight syringe, and the solution gently stirred for 24 h at 4 °C. The polymerization reaction was quenched


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by exposure to air. Thereafter, monomer conversion was evaluated by diluting a drop of the crude mixture in 500 µL D2O containing 5 µL D3PO4, and comparing the intensity of the residual monomer peaks at 5.86 and 5.47 ppm relative to an internal standard, dimethyl sulfoxide-d6 (DMSO-d6) at 2.50 ppm. To purify the scavenger, the reaction mixture was acidified to pH by incremental addition of 1 M HCl (not all scavengers were soluble at pH 7, used for polymerization). Subsequently, the solution was basified to pH 8–9 by incremental addition of 1 M NaOH, and the precipitated scavenger recovered by centrifugation (4,000g for 20 min at 4 ºC). The precipitate was then re-dissolved in 5 mL of PBS (1 mM, pH 4), and the process repeated three more times. Note that for some scavengers, dissolution was facilitated by bubbling with CO2. The final precipitate was lyophilized and stored at –20 °C until used. 1HNMR of the freeze dried samples did not give any characteristic peak of monomer molecule (5.86 and 5.47 ppm) implying the high purity of scavengers. Preparation of polymer (i.e., scavenger without Protein-A; Scheme S3). A control pHresponsive polymer was prepared by ATRP using an mPEG macro-initiator (1 mg, 1.66 mmol) and TBAEMA (336.96 µL, 1.660 mol) using the same procedure as for the scavengers. Analysis of molecular weight. Owing to the difficulty in analyzing the molecular weight of protein–polymer conjugates, this value was determined by three complementary techniques: 1H NMR spectroscopy, size-exclusion chromatography, and by UV-Vis spectrometry (Tables S1). 1H NMR spectroscopy: The molecular weight was determined from the initial feed ratio of macroinitiator to monomer, and monomer conversion; Size-exclusion chromatography: An Agilent technologies 1260 high pressure liquid chromatography system equipped with pump (Agilent 1260 Infinity Bio-inert Quaternary Pump (G5611A)), auto-sampler (Agilent 1100 HPLC G1367A Auto-sampler), thermostat (Agilent 1100 G1330B ALS Therm), two Agilent Bio SEC-5 columns


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(7.8 × 300 mm) with 5 µm particle size and 1000 and 2000 Å nominal pore sizes place in series, and an evaporative light scattering detector (1290 Infinity II ELSD) were used to determine molecular weight and dispersity. 50 µL of 3 g/L sample solutions were injected and eluted with 1% formic acid at a flow rate of 1 mL/min. Apparent number-average molecular weight (Mn) and dispersity (Đ) were established relative to methoxy polyethylene glycol standards. UV-Vis spectroscopy: A known mass of lyophilized scavenger was dissolved in a known volume PBS (1 mM, pH 4). The number of moles of scavenger in the solution was determined using the extinction coefficient of Protein-A at 280 nm. Molecular weight corresponds to the dry mass of conjugate divided by the number of moles of conjugate established spectroscopically. (Note that the polymer does not absorb at 280 nm) Buffer capacity experiment. 5 mg of scavenger was dissolved in 1 mL of phosphate buffer (PBS; 1 mM, pH 4) and diluted as necessary with this same buffer to achieve a concentration of 3.16 × 10–5 M, determined by optical absorbance at 280 nm (ɛ280, Protein-A = 7582 cm–1 M–1). 100 µL of scavenger stock solution was diluted with 2 mL water containing 1.5 mM NaCl, pH 5. pH titration was carried out by repeatedly adding 2 µL of 0.1 M NaOH solution under stirring. The pH increase in the range of 5.5–9 was monitored as a function of added volume of NaOH. Buffer capacity (β = –dnH+/dpH, where dnH+ is the moles of added H+ and dpH is the associated pH change) for each scavenger as a function of pH is plotted in Fig. 1a. For each sample, the pKa corresponded to the pH at which maximum buffer capacity was observed. Cloud-point assays. 50 µL of stock scavenger solution (above) and 50 µL of IgG (1.6 × 10–4 M, 1 mM PBS pH 4) were added to 1 mL of PBS (1 mM, pH 4) in a glass cuvette with a 1-cm optical path-length. The pH of the solution was incrementally raised by repetitive addition of 1–2 µL of


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0.05 M NaOH. The cloud-point of the IgG–scavenger complex was monitored via opacity at 600 nm. Binding stoichiometry. As a representative example to determine binding stoichiometry, 50 µL of Scavenger 6 (3.16 × 10–5 M) was added 1 mL PBS (1 mM, pH 4) at room temperature in a glass cuvette with an optical path length of 1 cm. The absorbance of this solution at 280 nm (A280) was measured. To this solution, 50 µL of IgG (1.6 × 10–4 M in 1 mM PBS; i.e., 5 molar eq. (excess)) were added and A280 measured again. This solution was transferred to a 1.5 mL Eppendorf tube and the pH was increased incrementally by repetitive addition of 0.05 M NaOH. Once the desired pH was reached, the solution was centrifuged at 14,000 g for 30 min at 4 ºC to remove the precipitated IgG–scavenger complex. The supernatant was then carefully collected and A280 measured to establish the amount of IgG remaining in solution. Binding stoichiometry was calculated according to the following equation:

[

𝐵𝑖𝑛𝑑𝑖𝑛𝑔 𝑠𝑡𝑜𝑖𝑐ℎ𝑖𝑜𝑚𝑒𝑡𝑟𝑦, 𝑁 = 1 ―

]

𝑐 [𝐼𝑔𝐺] × 𝑏―𝑎 [𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑒𝑟]

where “a” corresponds to A280 of scavenger solution at pH 4, “b” corresponds to A280 of the scavenger + IgG solution at pH 4, and “c” is the A280 of the supernatant following removal of the precipitated IgG–scavenger complex. Isothermal titration calorimetry. Isothermal titration calorimetry was performed with a Nano ITC (TA instruments Inc.) at 25 °C and a stirring speed of 250 rpm. Protein concentration was determined using its absorbance at 280 nm using the following molar extinction coefficients: 7582 cm–1 M–1 for Protein-A or for Protein-A macro-initiator, and 210,000 cm–1 M–1 for IgG. The solutions of Protein-A, Protein-A macro-initiator, and IgG dissolved in 1 mM PBS, pH 4 were


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diluted in potassium phosphate buffer (100 mM, in desired pH) to a final concentration of 15 µM (or 300 µM for IgG) and the buffer exchanged with the same buffer using a centrifugal dialysis unit (3 kDa MWCO) to reduce background signal from buffer mismatches. All solutions were degassed by stirring under vacuum before use. Fifty-µL of IgG was aspirated into the syringe and injected into 300 µL of Protein-A or Protein-A macro-initiator solution, in the cell. Titrations were carried out with 25 injections (each 1.96 µL) in 130 s intervals. Results were analyzed with ITCRun and NanoAnalyze and fitted to an independent model. Salt-induced precipitation. Fifty-µL of Scavenger 6 (3.16 × 10–5 M in 1 mM PBS, pH 4) was dissolved in 1 mL of PBS (1 mM, pH 4) at room temperature in a glass cuvette with a path length of 1 cm. To this solution, 0–30 µL of salt (NaClO4, NH4ClO4; etc.) solution (2 M in 1 mM PBS, pH 4) was added and the opacity at 600 nm measured. Stability of scavengers during precipitation. The stability of the scavenger during repeated precipitation and dissolution was evaluated by the alternating addition of 0.5 M HCl and NaOH to 5 mL of Scavenger 6 (3.16 × 10–5 M) in PBS (1 mM, pH 4). This procedure was repeated 10 times and an aliquot of 50 µL was withdrawn for determination of pulldown stoichiometry each time the scavenger was made soluble. The binding stoichiometry of the acid/base treated sample with IgG was determined as described above. Reusability studies. Fifty-µL of Scavenger 6 (3.16 × 10–5 M in 1 mM PBS, pH 4) was dissolved in 1 mL of PBS (1 mM, pH 4) containing 8 µM IgG. The pH of the solution was increased to 7.8 by the incremental addition of 0.1 M NaOH (approx. 6 × 2 µL). The turbid solution was centrifuged (14,000 g, 30 min, 4 ºC) and the supernatant, containing unbound IgG, was carefully removed. The precipitated affinity complex left in the Eppendorf tube was dissolved by gentle agitation in 1


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mL PBS (1 mM, pH 4). The complete dissolution was ascertained by measuring opacity at 600 nm. To this clear solution, 30 µL of 2M NaClO4 in PBS (1 mM, pH 4) was added, and the solution thoroughly mixed. The resulting turbid solution was centrifuged (14,000 g, 30 min, 4 ºC) and the supernatant (containing the released IgG) was carefully removed. The absorbance of the supernatant at 280 nm was recorded to quantify the amount of released IgG (ɛ = 210,000 cm–1 M–1). Separately, the precipitated scavenger was dissolved in 1 mL of PBS (1 mM, pH 4) containing 8.0 µM IgG, and the entire procedure was repeated four times. Purification of mAb from cell culture supernatant. Three crude monoclonal antibodies solutions (Table S2) in production media were graciously provided Dr. Yves Durocher (NRC, Royalmount, Canada). These crude solutions were transferred to a 2 mL Eppendorf tube. For the purification of antibody sample 1 using 1 eq. of scavenger, 50 µL of scavenger 6 solution (1.35 × 10–4 M in 1 mM PBS, pH 4) was then added and the pH of the mixture was gradually increased to pH 7.8 by addition of 1 M NaOH. The antibody–scavenger complex was recovered by centrifugation (14,000 g, 30 min, 4 ºC) as a white precipitate. The precipitate was dissolved in 1 mL PBS (1 mM, pH 4) and the pH of the solution adjusted to 4.0 by addition of 1 M HCl. Full dissolution of the precipitate was verified by measuring opacity at 600 nm. Thereafter, the scavenger was selectively precipitated with 30 µL of 2 M NaClO4 (1 mM PBS, pH 4) and removed by centrifugation (14,000 g, 30 min, 4 ºC). The supernatant containing the purified mAb was carefully collected and the recovery yield (vs. titer of the crude mixture) was determined via absorbance at 280 nm (corresponding to mAb, ɛ = 210,000 cm–1 M–1). The antibody titer was determined by Protein-A HPLC, which provides a titer value that is insensitive to contaminants. The purity of the purified antibody was analyzed by SDS-PAGE under reducing and non-reducing conditions using Mini-PROTEIN TGX Stain-Free Precast Gels (4–15% acrylamide). A kit of


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marker proteins (Bio-Rad Precision Plus Protein Unstained Standards) was used as standard proteins (lane 1, Fig. 3d). 10 µL of cell lysate (lane 2 and 3) and purified antibody (lane 4 and 5) were mixed with 3 µL of loading buffer (65 mM Tris-HC1, pH 6.8, 2% SDS, 10% glycerol, and 0.1% bromophenol blue) in the presence and absence of 5% 2-mercaptoethanol (reducing agent). Prior to loading, samples with 2-mercaptoethanol were heated to 110 °C for 10 min and cooled. Gels were run in Tris/glycine/SDS (3.0/14.4/1.0 g/L) buffer, pH 8.3 under constant voltage (100 V) for about 1 h 25 min. The gels were imaged using UVP Biodoc-it imaging system. The differences in intensity (which are typically qualitative in SDS-PAGE) are the result of the staining method employed (not Coomassie staining). Recycling of scavenger after use in production media. After having been used as described above for purifying a monoclonal antibody from production media, the precipitated scavenger was dissolved in 100 µL PBS (1 mM, pH 4). This scavenger solution was used for another cycle of purification using the same procedure as above. Purification was repeated for 4 cycles. The amount of released mAb and their purity were analyzed after each cycle of purification as described above.

ASSOCIATED CONTENT Materials, instruments, synthesis and characterization of scavengers or polymer, experimental procedures, and supplementary figures, schemes and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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*Marc A. Gauthier Institut National de la Recherche Scientifique (INRS), EMT Research Center, 1650 boul. LionelBoulet, Varennes, J3X 1S2, Canada E-mail: [email protected] Telephone: +1 514 228 69 32 Fax: +1 450 929 81 02 Author Contributions PA, YZ, NC, and MAG designed and conceived the study. PA and YZ performed all experiments, with the help of AAG and TRC. HdH assisted in modeling the existence of local pH effects. PA and MAG wrote the manuscript. All authors discussed the results and their implications, and commented on the manuscript at all stages. Funding Sources PA and TRC acknowledge postdoctoral scholarships from the Fonds de Recherche du Québec Santé (FRQS). AAG acknowledges postdoctoral scholarships from the Fonds de Recherche du Québec Nature et Technologies (FRQNT) and the Canadian Institutes for Health Research (CIHR). Further, she acknowledges the Chu Family Scholarship for a career award. This work was further supported by the Natural Science and Engineering Council of Canada (CRDPJ 493244 – 15; RGPIN-2015-04254). MAG is a Research Scholar of the FRQS. ACKNOWLEDGMENT Yves Durocher from the National Research Council of Canada is thanked for donation of monoclonal antibodies and for scientific discussions. Nicolas Doucet and Jacinthe Gagnon from INRS Centre Armand-Frappier are acknowledged for assistance with ITC experiments.


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TOC entry

pH-responsive polymer nano-buffer pH in their immediate vicinity and can be used to control protein–protein interactions.


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Evidence, Manipulation, and Termination of pH 'Nanobuffering' for

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