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Investigating liquid-liquid phase separation of a monoclonal antibody using solution-state NMR spectroscopy: effect of Arg·Glu and Arg·HCl Priscilla Kheddo, Jack E. Bramham, Rebecca J. Dearman, Shahid Uddin, Christopher F. van der Walle, and Alexander P. Golovanov Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00418 • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 18, 2017

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Molecular Pharmaceutics

Investigating liquid-liquid phase separation of a monoclonal

antibody

using

solution-state

NMR

spectroscopy: effect of Arg·Glu and Arg·HCl Priscilla Kheddo,† Jack E. Bramham,† Rebecca J. Dearman,§ Shahid Uddin,‡ Christopher F. van der Walle,‡ Alexander P. Golovanov*,† †

Manchester Institute of Biotechnology and School of Chemistry, The University of Manchester,

Manchester, M1 7DN, UK; Manchester, M13 9PL, UK;

‡

§

School of Biological Sciences, The University of Manchester,

Formulation Sciences, MedImmune Ltd, Aaron Klug Building, Granta

Park, Cambridge, CB21 6GH, UK

*, corresponding author, Alexander P. Golovanov, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK Tel: +44161 3065813; Fax: +44161 3065201; Email: [email protected]

TOC Image

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ABSTRACT Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases, and should be avoided in the final drug product. Understanding the factors leading to LLPS, and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (‘mAb5’), its solubility and parameters extracted using 1H NMR spectroscopy, for various formulations. We show that the formulations demonstrating least LLPS lead to the largest mAb5 NMR signal intensities. In the formulations exhibiting the highest propensity to phase-separate the mAb NMR signal intensities are the lowest, even at higher temperatures without visible phase separation, suggesting a high degree of self-association prior to distinct phase separation. Addition of arginine glutamate prevented LLPS and lead to a significant increase in the observed mAb signal intensity, whereas the effect of arginine hydrochloride was only marginal. Solution NMR spectroscopy was further used to characterize the protein-lean and protein-rich phases separately and demonstrated that protein self-association in the protein-rich phase can be significantly reduced by arginine glutamate. Solution NMR spectroscopy may be useful as a tool to assess the propensity of mAb solutions to phase-separate.

KEYWORDS: arginine glutamate, arginine hydrochloride, translational diffusion coefficient, physical instability, protein self-association, formulation, NMR spectroscopy


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INTRODUCTION Monoclonal antibodies (mAbs) are an important class of therapeutic drugs 1, 2. Subcutaneous dosage is most practical but often requires high concentration mAb solutions (≥ 100 mg/mL) which may lead to high viscosity, aggregation, opalescence and phase separation

3-5

. The molecular mechanisms

underpinning liquid-liquid phase separation (LLPS) and linked solution opalescence associated with cold temperature storage are under investigation

5-8

. LLPS is a thermodynamically driven effect,

disrupting the homogeneity of protein solutions and ultimately resulting in two distinct phases which differ in concentration

5, 9

. Although mAb aggregation and reversible self-association has been

extensively described, there is less available literature concerning LLPS observed in relevant conditions 8, 10-13. LLPS is a metastable phenomenon not commonly observed or reproduced reliably in protein preparations in vitro

14, 15

; however, the rapid rise of protein therapeutics will increase its

occurrence and justifies studies using relevant models. During LLPS the initially uniform solution separates spontaneously into a lower, dense, proteinrich phase with high protein concentration and an upper, less dense, protein-lean (also often referred to as protein-poor) phase with a lower protein concentration. Before complete LLPS occurs, a sample may become highly opalescent due to suspended droplets of the protein-rich phase scattering visible light strongly, with a gradient of opalescence towards the bottom of a vial, as droplets partially settle. The lower protein-rich phase may become clear, or may retain some opalescence due to density inhomogeneity leading to light scatter; whilst high protein concentration in this phase may eventually lead to further physical instabilities such as irreversible protein aggregation or precipitation 16. LLPS is unacceptable in the final drug product since it presents an inhomogeneous formulation with different layer concentrations of protein and buffer components

17

. Although LLPS may occur spontaneously,

especially if triggered by lowering the solution temperature, its appearance may be somewhat variable due to the metastable nature of the process. Muschol and Rosenberger showed that LLPS may be spedup by temperature cycling through the liquid-liquid coexistence (binodal) curve 18. Therefore, repeated freeze-thaw can be a good test to assess the propensity of a mAb formulation to undergo LLPS during early developability studies where the same repeated freeze-thaw is also used to gauge the appearance of aggregates, e.g. as described by Yang et al.

19

. For a given mAb, LLPS and opalescence are

dependent on a number of formulation factors including ionic strength, pH, protein concentration, excipient(s), and temperature 9. In contrast to the atomic-level characterization of protein-protein packing in a rigid crystal revealed through X-ray diffraction, or the nucleating ‘equilibrium clusters’ of proteins in solution revealed at molecular level by small angle scattering 20, molecular arrangements in 3 ACS Paragon Plus Environment


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the condensed protein-rich phase of phase-separated samples remain to be fully characterized. Interestingly, recently a wider examples of LLPS in natural biological systems, including formation of sub-cellular membrane-less organelles in vivo, have been recognized, highlighting the general importance of molecular understanding of the detailed nature of LLPS 21, 22. We have previously demonstrated that 1H NMR spectroscopy is highly sensitive to the association state of mAbs in solution and can contribute to selection of the best formulation for monomeric content and protein stability

23

. We have also described the stabilizing effect of an

equimolar mixture of L-Arg and L-Glu (arginine glutamate, Arg·Glu) on proteins and mAbs Arg·Glu is also not toxic, with in vitro toxicity equivalent to that of NaCl

27

24-26

.

, and therefore may be

suitable as biopharmaceutical excipient. Although the effect of Arg·Glu on reducing protein aggregation and self-association, and increasing protein solubility is well-documented 23, 26, 28-32, to our knowledge, its effect on opalescence and LLPS of mAb or other protein samples has not been previously described. In this work, we use a IgG1 mAb (mAb5), which is extremely sensitive to small variation in solution conditions causing LLPS, as a model and demonstrate that observed mAb 1H NMR signal intensity can be used as an early indicator of protein propensity to exhibit LLPS in different formulations. We show that LLPS and opalescence caused by freeze-thaw cycling can be prevented or decreased by addition of Arg·Glu, and to a lesser extent, of Arg·HCl. Addition of these excipients increase the apparent solubility of mAb5 in the test formulations, as revealed by polyethylene glycol (PEG) protein solubility assay. We also use NMR to separately characterize protein-rich and -lean phases, and demonstrate that the addition of Arg·Glu dramatically reduces protein-protein interactions within the protein-rich phase, shifting equilibrium towards monomeric state, but without detectable effect on the state of mAb5 in the protein-lean phase.

Materials and Methods Sample preparation MAb5 (MW 149 kDa; pI 8.1-8.6) was supplied by MedImmune as a 56 mg/mL solution in 20 mM succinate, 95 mM Arg·HCl, 180 mM mannitol, 20 mM NaCl, pH 6.0 (‘succinate buffer’). To change the buffer, mAb5 was dialyzed exhaustively into 20 mM sodium acetate, pH 5.5 (‘acetate buffer’, prepared with sodium acetate trihydrate and glacial acetic acid, both USP grade, Avantor Performance Materials, Netherlands). For the initial visual screen, mAb5 was concentrated to 76.7 mg/mL using a 20 mL centrifugal concentrator (Amicon, 10 kDa MWCO) and 120 µL volumes distributed to ultra4 ACS Paragon Plus Environment

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micro UV cuvettes (Brand) with caps. The cuvettes were supplemented as required with stock solutions of buffer, NaCl, Arg·HCl or Arg·Glu, pH 5.5, to yield a final mAb5 concentration of 46 mg/ml in each vial. For selected conditions, 1 mL mAb5 samples were prepared in a similar way using glass vials. Samples for NMR were placed in 5 mm NMR tubes (~400 µL each), with an internal insert capillary filled with 2H2O for lock signal to avoid mAb dilution. Using a Pasteur pipette, separation of the protein-rich and -lean fractions for NMR was achieved by careful withdrawal of the upper phase, followed by a small volume immediately above and below the meniscus and finally the lower phase. In this manner, 180 µL of either protein-rich or -lean fractions were placed into 3 mm capillary insert NMR tubes placed inside 5 mM tubes filled with 2H2O, used as an external lock signal solution. Further addition of 100 mM Arg·Glu to these samples followed the previously described protocol 23, in which pre-measured amounts of Arg·Glu stock solution prepared at pH 5.5 (same as the sample) were placed in Eppendorf tubes and freeze-dried. The samples were then carefully removed from the NMR tubes, reconstituted with the dry Arg·Glu and placed back in the NMR tube, now with 100 mM Arg·Glu added but without any sample dilution or pH change.

Visual inspection of samples and image analysis To assess which formulations show instability and are prone to phase separations, mAb5 solutions and controls in cuvettes were subjected to three freeze-thaw cycles (-80 °C/4 °C) and inspected in a dark room at 4 °C with illumination from underneath by a fluorescent light box. After resting for 18 hours, color images were captured, blue hue enhanced for clarity before converting the images to greyscale, and pixel brightness along vertical sample slices analyzed with ImageJ 33. Plot profile grey values were blank corrected and expressed as a percentage of the maximum grey value. Processing was applied consistently between images. A set of selected mAb5 solutions in glass vials (used for Figure 2) were subjected to repeated (8x) freeze-thaw cycles (-80 °C/ambient temperature) in order to more clearly define the phase boundary and confirm the typical behavior of these formulations in larger volume sample vials with a different geometry and material. Images were captured at the room temperature and analyzed similarly; after which protein-lean and –rich fractions were extracted for the NMR analysis, as described above.

PEG solubility assay The apparent solubility of selected mAb5 solutions was examined by adding increasing amounts of polyethylene glycol (PEG, 8000 Da, Alfa Aesar), with sample turbidity measured using nephelometry. 5 ACS Paragon Plus Environment


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A 40 % w/v PEG solution in acetate buffer, a 10 mg/mL mAb solution, and acetate buffer containing either Arg·Glu, Arg·HCl or NaCl were aliquoted into 96-well UV-Star microplate (Greiner Bio-One, VWR, UK) using an automated liquid handling robot (Tecan, Freedom EVO®, Männedorf, Switzerland) such that each well contained 1 mg/mL mAb5 against a PEG gradient of 0-16 % w/v, in a variety of buffer conditions. Each individual condition was set in triplicate. Immediately after the final mixing step, the plate was transferred to the NEPHELOstar Plus microplate nephelometer (BMG Labtech, Ortenberg, Germany) to measure total light scatter at 90° to the incident beam at 635 nm (30 % laser intensity). These measurements reported the turbidity of each sample in Nephelometric Turbidity Units (NTU). Data were analyzed using the MARS-Omega analysis software, with a standard 4-parameter curve fitting applied for each data set to obtain the percentage PEG required to achieve mid-point maximum turbidity (EC50) which was used as a measure of apparent mAb5 solubility in that particularly formulation.

NMR spectroscopy All NMR experiments were acquired on Bruker 800 MHz Avance III spectrometer equipped with 5 mm TCI cryoprobe with variable temperature control unit, using standard pulse programs and parameters from Bruker library. The probe temperature was calibrated using methanol test sample, following the standard NMR procedure

34

, and additionally verified using an external thermocouple

placed in the test sample tube in the NMR probe. For variable temperature experiments, the starting sample temperature in each formulation condition tested was 20 ⁰C; the temperature was then decreased and spectra recorded with identical parameters at 15, 10, 8, 6, 4 and 2 ⁰C. For separate protein-rich and protein-lean samples the NMR spectra were recorded at 40 ⁰C. Spectrometer was shimmed at each temperature before the experiments. Proton 1D spectra were recorded using p3919gp pulse program using 16.0194 ppm spectral width and applying EM window function with typical 10 Hz broadening. Spectra were processed and analyzed using Topspin 3.1 (Bruker). The processed data was plotted in GraphPad Prism 6.0. To compensate for the change in water viscosity with decreasing temperatures, and for the difference in protein concentration in the succinate buffer to the other formulations, the corrected NMR []

signal intensities were calculated as: []

= []

(1)


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where is the signal intensity measured at a particular temperature T, [ ] is mAb concentration (either 56 mg/mL for the succinate buffer, or 46 mg/mL for all other formulations), and is the water viscosity at a particular temperature T. Although the measured signal intensities were represented in arbitrary units, they were directly comparable between different temperatures and formulations, as the acquisition parameters were identical throughout. Translational diffusion coefficients D were measured by diffusion-ordered spectroscopy (DOSY) using Bruker’s standard pulse program stebpgp1s19 with additional water signal presaturation during the relaxation delay. Standard DOSY processing was applied using Topspin 3.1. The diffusion time (∆) and the gradient length (δ) were set to 150 ms and 4.0 ms, respectively. The standard errors reported for each D were estimates based on the upper and lower limits for each DOSY peak. The measured diffusion coefficient D can be related to the apparent size of the molecule and apparent viscosity using the Stokes-Einstein equation: D=

(2)

where T is the absolute temperature, k is the Boltzmann constant; Rh is the hydrodynamic radius and η is the viscosity. The effective microscopic viscosity of different tested solutions at lower temperatures (compared to that at 20 ⁰C) was calculated as: =

(3)

where is water viscosity at 20 ⁰C (1.002 mPa s), and D20 and DT are measured diffusion coefficients for a chosen small-molecule probe (buffer component) at 20 ⁰C and temperature T, respectively, with the assumption that the effective molecular radius of this probe is insensitive to temperature changes.

RESULTS Initial LLPS screen Opalescence and LLPS can be observed visually; therefore an initial screen was set up for small volumes of mAb5 solutions in different formulations in transparent cuvettes, and subjected to freezethaw cycling. Although such cycling was initially suggested for detecting the appearance of protein aggregates as part of developability assessment studies

19

, in practice the same test can be extremely

useful to quickly identify potentially problematic formulations which may otherwise take a long time to phase-separate. Distinct phase separation of mAb5 was achieved by increasing ionic strength upon addition of 30, 50 or 100 mM NaCl to acetate buffer in the positive control group of samples (Figure 7 ACS Paragon Plus Environment


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1). Addition of both Arg·Glu and Arg·HCl reduced the appearance of opalescence and LLPS in a concentration dependent manner such that no distinct LLPS occurred in samples with these excipients. However, as can be seen on the images (Figure 1A), and also supported by consistently lower pixel brightness values and flatter curve profiles (Figure 1B), Arg·Glu reduced opalescence and the degree of LLPS significantly stronger than Arg·HCl at concentrations up to 150 mM. From this initial screen, four formulations showing mixed opalescence and LLPS behavior were chosen for further analysis and compared to the behavior of mAb5 in succinate buffer, each being subjected to eight freeze-thaw cycles (Figure 2). Here, we checked that the observed LLPS following freeze-thaw is reproducible for samples of larger volume in vials made of a different material (glass); larger volumes used made visual observations of LLPS more straightforward, and allowed acquirement of enough material for NMR analysis of protein-rich and protein-lean fractions separately (see below). As with the initial screen, addition of 100 mM Arg·Glu to acetate buffer and 30 mM NaCl significantly reduced the extent of opalescence and LLPS, whilst marked opalescence and partial LLPS was still present with 100 mM Arg·HCl. Mild opalescence was observed in the succinate buffer containing 20 mM NaCl, despite the presence of Arg·HCl and mannitol. The behavior of freshly-prepared mAb5 in five representative formulation conditions shown in Figure 2, upon just cooling, to observe the onset of LLPS, were later characterized by NMR spectroscopy (see below).

MAb5 solubility assessed by PEG-induced precipitation assay To assess the link between the relative apparent solubility of mAb5 in 15 different formulations and the appearance of opalescence and LLPS, we used a PEG-induced precipitation assay

35, 36

. The

relative solubility of mAb5 was represented as a PEG concentration at which 50 % of the maximum turbidity was reached (EC50), measured using nephelometry (see Supporting Information). Apparent mAb5 solubility showed a clear dependence on ionic strength: the EC50 values in 0, 15 and 30 mM NaCl/acetate buffer occurred at 16, 8 and 6 % w/v PEG, respectively (Figure 3). These results demonstrate that the addition of NaCl significantly reduced the apparent solubility of mAb5, agreeing with the increase in opalescence and LLPS observed in the positive control samples of the visual screens (Figure 1). Addition of Arg·Glu increased the apparent solubility of mAb5 more than Arg·HCl in all cases (Figure 3); specifically, the decrease in mAb5 solubility from addition of 30 mM NaCl was largely negated by 200 mM Arg·Glu but not Arg·HCl. Interestingly, the apparent solubility of mAb5 was the highest when in acetate buffer without salt (a condition where no plateau could be measured by nephelometry even at the highest PEG concentrations), any addition of Arg·Glu or Arg·HCl reduced 8 ACS Paragon Plus Environment

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this solubility value, most likely due to screening of electrostatic repulsion between mAb5 molecules. Although the solubility parameter EC50 determined in this assay refers to a liquid-solid phase separation (i.e., precipitation) in the presence of PEG, it correlated well with the visual observations for occurrence of opalescence and LLPS in the absence of PEG, suggesting that both effects are similarly modulated by the solution conditions, likely affecting the protein’s propensity to self-interact and hence form associates and/or aggregates.

NMR assessment of samples undergoing temperature-dependent LLPS Using freeze-thaw cycles provides a quick way to assess formulations for their propensity to undergo LLPS However, it is important to establish if there are any detectable differences in these formulations before phase separation occurs, and if these differences are enhanced at lower temperature, a usual trigger for LLPS onset. To further characterize the liquid formulations showing a characteristic LLPS behavior, as well as the control samples, we used 1H NMR spectroscopy. Fresh solutions corresponding to selected conditions described in Figure 2 were prepared and placed in the NMR tubes at room temperature before any phase separation occurred; a series of 1D NMR spectra were then recorded as sample temperature was lowered step-wise from 20 °C to 2 °C, where the transition leading to LLPS is expected to take place. Figure 4A shows an example of overlays of the characteristic methyl spectral region recorded for the different mAb5 formulations at 20 °C. Since the signal intensity represents the amount of soluble, freely tumbling protein present in the sample (e.g. the monomeric and lower-oligomeric population), we chose to follow the dependence of the intensity of the characteristic mAb5 signal at ~0.7 ppm on formulation composition and temperature. To compensate for increasing water viscosity with decreasing temperature, the signal intensities are []

presented as concentration-normalized and viscosity-corrected intensity parameter (Figure 4B). In []

the absence of temperature-induced protein self-association the temperature dependences of are []

expected to be largely flat; conversely, if the amount of non-associated protein decreases then also decreases. Here, all formulations showed a reduction in the signal intensities with decreasing temperature, indicating increased self-association at lower temperature. The initial intensity values varied significantly reflecting the difference in the initial degree of self-association, additionally modulated by the subtle differences in buffer viscosities in the samples with and without excipients (Figure 4B). The varying rates of the drops at lower temperatures revealed different propensities to further self-associate at lower temperature (Figure 4B). For example, mAb5 formulated in acetate buffer alone yielded the 9 ACS Paragon Plus Environment


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[]

greatest at 20 °C but subsequent steepest decline, most probably due to increased susceptibility to temperature induced self-association in the absence of excipients. The high initial intensity value (relative to other formulations containing various excipients) can be explained by the inherently low viscosity of this buffer in the absence of any excipients

23

. Conversely, mAb5 formulated in acetate

[]

buffer with 30 mM NaCl yielded the lowest across all formulations and temperatures implying the largest degree of protein self-association among the conditions tested here. Addition of 100 mM []

Arg·HCl increased only marginally, whereas addition of 100 mM Arg·Glu resulted in a significant []

[]

increase in , with values at lower temperatures surpassing even those in the absence of NaCl.

for mAb5 in the succinate buffer were very similar to those recorded in acetate buffer containing 30 mM NaCl and 100 mM Arg·HCl. This suggests that both the buffer species and mannitol did not have a significant influence on temperature-induced self-association of mAb5, consistent with the visual observations of opalescence and LLPS in the initial screen. To confirm that LLPS and protein self-association at lower temperatures did not alter microscopic solution viscosity and in turn distort mAb5 signal intensity, we employed diffusionordered spectroscopy (DOSY) to measure the temperature dependence of translational self-diffusion coefficients (D) of the small buffer molecules (acetate, or Arg, or Arg/Glu) used as probes present in each of the samples. As the formulations all had slightly different starting viscosities, and sizes of probe molecules differed as well, the initial values of D at 20 °C for these species did vary, however their temperature dependence showed similar trends (Figure 5A). Using the Stokes-Einstein equation and re-normalizing each relative solution viscosity to match the viscosity of water at 20 °C, the temperature dependencies of relative microscopic viscosities in the five selected formulations were compared with the viscosity of water, and found to overlap closely (Figure 5B). Since no significant deviations were identified, this indicated that the microscopic solution viscosities were almost entirely governed by the temperature-dependence of water viscosity, without contributions from protein selfassociation. []

Overall, the values correlated well with the tendency of mAb5 to phase-separate. Markedly, prior to cooling and displaying visible phase separation, mAb5 in the presence of 30 mM NaCl at 20 °C []

had very weak

and therefore the solution contained the largest protein population in a transient

NMR-invisible ‘dark state’ 37, a population forming large liquid molecular assemblies likely serving as an early precursor to protein-rich phase. Addition of Arg·Glu shifted the equilibrium towards monomeric state and decreased this dark state population significantly, unlike Arg·HCl; this was in 10 ACS Paragon Plus Environment

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agreement with the visual observations of LLPS. Therefore we suggest that quantification of this ‘dark state’ by NMR spectroscopy may enable early prediction and ranking of mAb formulations in terms of their likely propensities to show opalescence or LLPS at lower temperature, without the need to perform heat/cool cycles.

Solution NMR studies of protein-rich and protein-lean fractions NMR spectroscopy was further employed to separately characterize the state of mAb5 in the protein-rich and -lean fractions of the formulation which clearly underwent LLPS in the presence of 30 mM NaCl (cf. Figure 2). The concentration of mAb5 in the lean and rich phases was measured by absorbance at 280 nm as 8.6 and 114.0 mg/mL, respectively. Comparison of the NMR spectra recorded at 40 °C for the two fractions showed that despite the protein-rich phase being around 13-fold more concentrated than the protein-lean phase, signal intensity of the latter was 3-fold greater (Figure 6). Thus, the NMR signal for mAb5 in the protein-rich fraction was very weak, around 1/40th the intensity expected for this concentration, which implies that ca 97.5% of the protein population in this phase was present in a ‘dark state’. Although the protein-rich phase was not visibly aggregated or gelated, the molecules present in this ‘dark state’ must be self-associated with highly restricted mobility, e.g. existing as liquid aggregates. To check whether the degree of self-association, and hence signal intensities, in the protein-rich and -lean phases can be further affected, Arg·Glu was added directly to both samples, as described in the Methods. Briefly, the pre-measured amounts of concentrated Arg·Glu stock solution prepared at equivalent pH were freeze-dried and then reconstituted with the NMR samples, so that 100 mM was added but without sample dilution or change in pH. Addition of Arg·Glu caused a 3.5-fold increase in the intensity of the mAb5 signal in the protein-rich phase, but no change in protein-lean phase (Figure 6). This demonstrates that Arg·Glu disrupts the self-association between molecules in the protein-rich phase, decreasing the ‘dark state’ population. The observation that the relatively strong signal intensity of mAb5 in the protein-lean fraction was not affected by the addition of Arg·Glu indicates that the protein in this fraction was already largely monomeric. To assess further the differences in the molecular mobility under different conditions in the separated phases, the translational diffusion coefficients D were measured using DOSY NMR, before and after addition of 100 mM Arg·Glu (Figure 7). The diffusion of small probe molecules present in the samples is only 1.3-1.5 times slower in the protein-rich fraction, meaning that the effective microscopic viscosity in this fraction is only marginally increased compared with the protein-lean fraction. However, the diffusion rate of mAb5 itself is approximately ten times slower in the protein11 ACS Paragon Plus Environment


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rich fraction, both in the absence and presence of Arg·Glu (Figure 7). According to the Stokes-Einstein equation, this corresponds to around 10-fold increase in the effective radius of a diffusing particle, although this nominal figure should be taken with caution as the validity of this equation under extreme crowding conditions is somewhat limited. Moreover, the mAb diffusion in such conditions is heavily restricted by the caging effects, making this NMR-derived parameter D a poor reporter for the apparent size of protein clusters

23

. Addition of 100 mM Arg·Glu caused consistent, approximately 1.8-fold

decrease in all the diffusion coefficients which can be explained by a correspondent increase in the buffer viscosity

23

. Overall, addition of Arg·Glu to protein-rich fraction reduces self-association

allowing mAb5 molecules to tumble faster and more independently from each other, leading to higher observed signal intensities in 1H NMR spectra, however the translational diffusion rates of mAb5 molecules are not increased, probably due to extreme molecular crowding. Monitoring 1H signal intensity of mAb in different solution conditions therefore provides a valuable indicator of subtle differences in protein self-interactions and can be an early predictor for conditions leading to phase separation.

DISCUSSION The ability of mAb solutions to withstand stress (e.g. freeze/thaw cycling, storage at ambient and elevated temperatures, etc.) is considered as essential part of “developability assessment” which helps to identify early those mAbs and conditions which may lead to long term instabilities

19

. MAb5 at a

concentration of ~50 mg/mL undergoes reproducible LLPS caused by subtle changes in ionic strength and further triggered by variations in temperature, and so presents a useful model which is relevant to other mAb products. Although it is possible to systematically screen solution conditions for LLPS under repeated freeze-thaw cycles

19

, a quantitative and robust measurement made at a room

temperature would be beneficial to formulation practice. Such an analytical tool must be predictive of LLPS for a given mAb formulation and report a simple parameter as a measure of a preliminary critical quality attribute. In this study, a link between visually-observed LLPS of various mAb5 formulations []

triggered by freeze-thaw cycling and the quantitative NMR spectral parameter (signal intensity ) which showed characteristic changes upon cooling even before full LLPS occurred, has been established. Samples which subsequently underwent LLPS initially had a very large protein population invisible to NMR detection, as reported by weak signal intensities observed, and indicative of the presence of large transient liquid aggregates, which may nucleate LLPS upon cooling.


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An analogous interpretation of these NMR results is that the mAb5 molecules can be thought of as soft-sphere colloids which undergo ‘condensation’ leading to aggregation or LLPS 12. The likelihood of condensation is determined by mainly attractive short range interactions which are attributed to the metastable nature of LLPS, induced by the addition of NaCl

10, 14, 38-41

. Propensity of protein to self-

associate in different formulation conditions can be assessed using PEG-induced precipitation assay 35, 36

: the apparent solubility of mAb5 determined by this assay here correlated with the propensity for

LLPS. Despite some scatter in the data revealed by the repeats (Figure S1), the tendencies in relative shifts in the transition midpoints (EC50) were sufficiently clear. While LLPS and aggregation both arise from protein self-association under concentration dependent conditions, it is proposed that LLPS is predominant upon cooling and is reversible, with native protein structure maintained, whereas aggregation generally occurs at elevated temperatures and may lead to irreversible misfolding 13. The NMR spectra (Figure 6) also confirm that mAb5 maintained a folded, globular structure following LLPS, which is in agreement with previous data

3, 5, 10, 42

. Although LLPS and the fractionation of the

protein-rich phase may present a route to obtaining ultra-high concentration protein solutions for further bioprocessing or formulation steps 8, its highly self-associated nature will likely present significant problems for handling or long term stability. Nishi et al. (2010) proposed that protein selfassociation in the protein-lean phase is negligible, being dominated by monomeric species, but that in the protein-rich phase attractive intermolecular interactions dominate and are responsible for causing LLPS. The NMR results here support this proposition and further show that the freely tumbling behavior of mAb5 molecules in the protein-lean phase is not modulated by the addition of Arg·Glu. Importantly, the NMR data show that the tumbling of the mAb5 molecules in the protein-rich phase is severely restricted (while the microscopic viscosity reported by D of the buffer molecules is only marginally increased). We demonstrated that further addition of Arg·Glu to the protein-rich fraction significantly alleviates the self-association observed in this phase, which may provide a way forward in using LLPS as a method for protein concentration. This also fits recent observations that addition of Arg·Glu to high-concentration mAb solutions removes the shear-thinning effect and hence the presence of protein networks, while significantly reducing solution viscosity 32. When analyzing the dependence of the NMR signal intensities in the presence of salts, it is important to consider other experimental factors which can perturb the results. It is well known that NMR sample tube geometry may affect the sensitivity of experiments conducted with a cryogenicallycooled probehead (cryoprobe), especially when using highly-conductive solutions with high ionic strength

25, 43

. This is because high-conductivity buffers can decrease signal intensity due to radio13 ACS Paragon Plus Environment


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frequency losses, with this effect being more prominent for larger (i.e. 5 mm) diameter sample tubes. However, here the 30 mM NaCl concentration in 20 mM acetate buffer was too low to cause a significant fall in sensitivity. Indeed, the observed increase in signal intensities upon Arg·Glu addition ran counter to this experimental artefact, as such, if anything, leading to an underestimation of the true increase in the population of freely tumbling mAb5 molecules. For similar cryoprobe experiments with high concentrations of conductive salts (e.g., NaCl near isotonicity) thin capillary (≤3mm) NMR tubes should be preferably employed 25, 43. In summary, 100 mM Arg·Glu could be used as an effective excipient to reduce LLPS of mAb5 formulations under typical ionic strength and pH conditions, whereas Arg·HCl at the same concentration had much weaker effect. Solutions undergoing little or no LLPS if subjected to freezethaw cycling, in freshly-prepared samples generally gave rise to a greater NMR signal intensities at low temperature, indicating a larger fraction of freely tumbling molecules corresponding to a monomeric state. Conversely, solutions undergoing LLPS after freeze-thaw cycles had an increased population of NMR-invisible protein species even before such sample stress. One important advantage of NMR spectroscopy is that it does not require sample dilution and can assess mAb formulations in situ in a very wide range of concentrations []

signal intensities

23

. The concentration-normalized and viscosity-corrected NMR

therefore may prove to be a useful, predictive parameter of LLPS of high

concentration mAb solutions.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources PK was supported by a BRIC PhD studentship BB/K004379/1, and JEB by CASE DTP PhD studentship BB/M011208/1 from the UK Biotechnology and Biological Sciences Research Council (BBSRC) in partnership with MedImmune Ltd.

Acknowledgement PK was supported by a BRIC PhD studentship BB/K004379/1, and JEB by CASE DTP PhD studentship BB/M011208/1 from the UK Biotechnology and Biological Sciences Research Council


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(BBSRC) in partnership with MedImmune Ltd. We are grateful to Matthew Cliff for maintaining the NMR Facility.

Supporting Information Figure S1, Nephelometric turbidity graphs as a function of PEG concentration in mAb5 formulations, for solubility assay.

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FIGURES

Figure 1. LLPS and opalescence in mAb5 formulations. (A) Visual screen of effects of NaCl, Arg·Glu and Arg·HCl on mAb5 solution opalescence and LLPS in acetate buffer. All images were processed consistently by 100% blue color saturation using GIMP to make opalescence clearer. No opalescence was observed in the negative controls, solutions without mAb. (B) Pixel brightness analyses of sample opalescence and LLPS in the visual screen sample images. First, curve slope and shape reflect the degree of LLPS, with deviation from the flat vertical indicating greater density non-uniformity and a presence of a distinct phase boundary (for example, this is most distinctly observed for the Positive Control profiles shown in red). Secondly, the pixel brightness value indicates the degree of opalescence at a given position, with a higher value indicating greater opalescence.


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Figure 2. Selected representative mAb5 formulations after 8 freeze-thaw cycles. (A) Samples showing visually strong or weak LLPS are marked with solid- and dotted-line arrows, respectively. (B) Pixel brightness analysis of opalescence and LLPS. Profiles shifted to the right, to more pixel brightness, correspond to more opalescent samples. Higher pixel density towards the bottom of the sample is a marker for LLPS (cf. Figure 1 legend).



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Figure 3. Assessment of mAb5 solubility in selected formulations, as labelled. Solubility is represented as concentration of PEG required to induce 50% of maximal turbidity in mAb5 formulation (EC50). Asterisk denotes no curve plateau reached in this condition.


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Figure 4. NMR analysis of the selected mAb5 formulations. (A) Overlay of 1H NMR spectra acquired at []

20 °C showing raw signal intensities between 0.9 and -1.0 ppm. (B) Values of the intensity parameter (arbitrary units) plotted as a function of temperature.



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Figure 5. Temperature-dependence of the relative microscopic viscosity ɳ for the selected mAb5 formulations. (A) Translational self-diffusion coefficients D of the small molecules (acetate, Arg, or Arg and Glu) measured for the selected formulations. (B) Microscopic viscosities ɳ of the selected formulations calculated from the values of D using the Stokes-Einstein equation, overlaid with the theoretical values for water. The error bars represent the estimates of the upper and lower limit of ɳ based on the error limits from D.


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Figure 6. Overlay of 1H NMR spectra of the protein-rich (A) and -lean (B) fractions of phase-separated sample of mAb5 prepared at 46 mg/mL in 20 mM acetate, 30 mM NaCl, pH 5.5. Arg·Glu was further added to the samples to a final concentration of 100 mM.



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Figure 7. Translational diffusion coefficients (D) of mAb5 and small-molecule probes in phase-separated fractions. The values of D were measured by DOSY NMR for protein-rich and -lean fractions, before (without) and after (with) the addition of 100 mM Arg·Glu. Right y-axis shows logD scale, with the corresponding value of D for the signal marked with dotted line displayed in the inserts. The uncertainty of D is defined by the width of the DOSY peak projection shown on the left of each panel. In the absence of Arg·Glu, acetate ion was used as a small-molecule probe. The peaks from acetate (Ac), Arg and Glu are labelled for clarity, and additional artefact peaks originating from DOSY processing are marked with dotted circles; these peaks were ignored while determining the values of D.


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