Facile Preparation of Stable AntibodyâGold Conjugates and

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A facile method for preparing stable antibody-gold conjugates and application to affinity-capture self-interaction nanoparticle spectroscopy Steven B. Geng, Jiemin Wu, Magfur E. Alam, Jason S. Schultz, Craig D. Dickinson, Carly R. Seminer, and Peter M. Tessier Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Bioconjugate Chemistry

A facile method for preparing stable antibody-gold conjugates and application to affinity-capture self-interaction nanoparticle spectroscopy Steven B. Geng1,*, Jiemin Wu1,*, Magfur E. Alam1, Jason S. Schultz2, Craig D. Dickinson2, Carly R. Seminer1, and Peter M. Tessier1, † 1

Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 2 Eli Lilly Biotechnology Center, San Diego, CA 92121, USA

Protein-nanoparticle conjugates are widely used for conventional applications such as immunohistochemistry and biomolecular detection as well as emerging applications such as therapeutics and advanced materials. Nevertheless, it remains challenging to reproducibly prepare stable protein-nanoparticle conjugates with highly similar optical properties. Here we report an improved physisorption method for reproducibly preparing stable antibody-gold conjugates at acidic pH using polyclonal antibodies from a range of species (human, goat, rabbit, mouse and rat). We find that gold particles synthesized using citrate alone or in combination with tannic acid are similar in size but display variable colloidal stability when conjugated to polyclonal antibodies. The variability in conjugate stability is due to differences in the pH and composition of the original gold colloid, which prevents reproducible preparation of stable antibody conjugates without additional purification of the particles prior to conjugation. Sedimentation-based purification of gold particles synthesized using different methods enabled reproducible generation of antibody-gold conjugates with high stability and similar plasmon wavelengths. We also find that antibody conjugates prepared using our improved procedure display excellent performance when applied to a high-throughput immunogold assay (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) for identifying monoclonal antibodies with low self-association, high solubility and low viscosity. The stable antibody conjugates prepared with various types of gold colloid result in robust and reproducible AC-SINS measurements of antibody selfassociation using extremely dilute (microgram per mL) and unpurified antibody solutions. We expect that this improved methodology will be useful for reproducibly preparing stable antibody-gold conjugates for diverse applications. Keywords: mAb, monoclonal, conjugation, nanoparticle, aggregation, solubility, viscosity Running title: Preparation of stable antibody-gold conjugates *equal contribution †

corresponding author

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INTRODUCTION The importance of protein-gold nanoparticle conjugates has motivated the development of many procedures for immobilizing proteins on gold nanoparticles via non-covalent and covalent methods.1-6 Noncovalent protein immobilization is most common, and many such physisorption methods share several features: i) dialysis (or buffer exchange) of the target proteins into water or low ionic strength solutions to reduce the concentration of ionic species that are destabilizing to gold colloid prior to conjugation; ii) adsorption of the target proteins at a pH that promotes both protein adsorption and stability of the resulting conjugates; iii) stabilization of the resulting protein-gold conjugates with molecules such as polyethylene glycol (PEG) or bovine serum albumin; and iv) removal of unadsorbed proteins using sedimentation or filtration. These procedures have been widely used to prepare a large number of different types of proteingold conjugates.1, 3, 4 Nevertheless, it remains challenging to reproducibly conjugate various types of proteins to gold particles and achieve conjugates with extremely similar optical properties (e.g., initial plasmon wavelengths and absorbances, maximum plasmon shifts and absorbance changes). This is particularly important for proteingold conjugates that are used in detection applications because the initial plasmon wavelengths of the conjugates as well as their maximum plasmon shifts must be reproducible for different batches of gold colloid and target proteins. We have encountered this general problem during the development of an immunogold assay (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) for measuring monoclonal antibody (mAb) self-association.7, 8 The premise of AC-SINS is that polyclonal antibodies specific for human or non-human mAbs are conjugated to gold nanoparticles (radii of 5–10 nm), and the resulting conjugates are used to capture the mAbs of interest. The high local concentration of adsorbed mAbs magnifies their self-interactions and enables detection of weak self-interactions. Attractive mAb selfinteractions (which generally reduce mAb solubility) lead to reduced interparticle distances and increased plasmon wavelengths, and vice versa for repulsive interactions (which generally increase mAb solubility).7-14 We find that AC-SINS measurements for extremely dilute (0.001–0.01 mg/mL) and unpurified mAb solutions are correlated with solubility measurements for purified mAbs at 3–5 orders of magnitude higher antibody concentrations.7, 8 Importantly, we find that the reproducibility of our assay is strongly influenced by the properties of the original, unmodified gold particles. Interestingly, some batches of gold colloid obtained from the manufacturer are incompatible with our AC-SINS assay because they rapidly aggregate when conjugated to polyclonal antibodies.7 In contrast, other batches of gold colloid with the same size and obtained from the same manufacturer result in stable antibody conjugates that are readily compatible with AC-SINS. Others have also reported similar problems related to variable stability of protein-gold conjugates.5, 6 Here we have investigated methods for purifying unmodified gold particles that have been generated using different 2 ACS Paragon Plus Environment

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synthesis procedures to make them compatible with stable and reproducible conjugation to diverse types of antibodies at acidic pH. Moreover, we have applied these improved methods for evaluating the selfassociation behavior of extremely dilute and unpurified mAbs using AC-SINS.

RESULTS Stability of antibody-gold conjugates is improved by first purifying gold particles We routinely use commercially available gold nanoparticles (nominal radii of 10 nm) that are produced by reducing gold salt with tannic acid and citrate (the details of the synthesis are proprietary and not available from the manufacturer). The citrate-stabilized particles are reported to contain trace amounts of tannic acid and potassium carbonate in addition to citrate. We have previously reported that we occasionally obtain batches of gold particles from the manufacturer that lead to rapid particle aggregation when mixed with polyclonal antibodies at acidic pH (pH 4–5, 2 mM acetate), while most batches of the same gold particles result in stable antibody-gold conjugates.7 Our previous studies demonstrated that the problem is due to differences in the unmodified gold colloid. Interestingly, this problem is not due to obvious differences in particle size or particle aggregation (as judged by dynamic light scattering). Moreover, we find that a simple filtration assay can robustly distinguish between the different batches of unmodified gold

Figure 1. Evaluation of the stability of gold nanoparticles and antibody-gold conjugates for multiple batches of gold colloid. Gold nanoparticles (AuNP) S (Stable) and U1 (Unstable, batch 1) are two batches of gold particles (nominal radii of 10 nm) obtained from the manufacturer. (A) Dynamic light scattering measurements of unwashed and washed gold colloid. The particle radii are reported on the primary y-axis and the sum of squares (a measure of the goodness of fit of the light scattering data) are reported on the secondary y-axis. (B) pH, (C) plasmon wavelength and (D) absorbance measurements of unwashed and washed gold colloid before and after conjugation to goat anti-human Fc polyclonal antibodies. The anti-Fc antibodies were buffer exchanged to pH 4.3 (20 mM acetate), and the final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 2 mM, respectively. The washed gold particles were washed once with water. The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments.

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particles.7 Unstable batches of gold particles show reduced particle recovery when filtered (95%). Importantly, filtration of the unstable gold particles does not make them compatible with stable conjugation to polyclonal antibodies. Another attribute of unstable batches of gold particles is that they typically have a slightly higher pH (pH ~5.5–5.8) than stable batches of gold (pH ~5–5.3).7 However, simply decreasing the pH of the unstable gold particles does not make them compatible with stable conjugation to polyclonal antibodies. Thus, we evaluated whether washing the unmodified gold particles via sedimentation and resuspension in water would reduce the differences between stable and unstable batches as well as improve the compatibility of unstable gold particles with conjugation to anti-Fc antibodies (Fig. 1). Importantly, we find that the sizes of stable (S) and unstable (U1) batches of gold are similar prior to washing (radius of ~9–10 nm) and consistent with the nominal particle size (radius of 10 nm), as judged by dynamic light scattering (Fig. 1A). However, a measure of the quality of the light scattering analysis (sum of squares, SOS) shows that the stable particles have better (lower) SOS values (7±3) before washing than the unstable particles (33±21), suggesting that the stable particles are more homogeneous. Next, we washed both batches of gold particles by i) sedimenting them at 21130 rcf for 5–6 min, ii) removing 95% of the supernatant volume, and iii) resuspending the particles in Milli-Q water to achieve the initial volume. This sedimentation process typically resulted in a small, dense pellet that does not resuspend and a much larger (loose) pellet that readily resuspends in water. The sedimentation time (5–60 min), sedimentation temperature (4–25 °C) and source of water (distilled water or Milli-Q purified distilled water) had little impact on the pelleting and resuspension process. Moreover, untreated polypropylene microcentrifuge tubes from multiple vendors displayed excellent sedimentation and resuspension properties (Fig. S1). However, low-binding tubes from multiple vendors caused particle aggregation during sedimentation and such particle aggregates could not be resuspended in water (Fig.

Figure 2. Effect of pH on the stability of gold particles conjugated to anti-Fc antibodies. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by adsorbing goat anti-human Fc antibodies on gold particles at various pH values. Stable gold particles (AuNP: S) were washed once with water and mixed with anti-Fc antibodies, the latter of which were buffer exchanged to pH 4– 5.5 (200 mM acetate). The pH values for the antibody-gold conjugates were estimated using pH paper, and the final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 20 mM, respectively. The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments.


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S1). This destabilizing effect was specific to the type of low-binding tubes, as we identified one type of lowbinding tube (Sorenson BioScience, 39640T) that is treated with a Teflon substance and is compatible with gold sedimentation and resuspension (data not shown). Dynamic light scattering analysis revealed that washing the unmodified particles led to little change in size or SOS values for both types of particles (Fig. 1A). Importantly, we find that washing the two batches of unmodified particles results in detectable changes in pH (Fig. 1B). The initial pH values of the stable and unstable particles were pH ~5–5.3 and ~5.5–5.8, respectively. However, the pH of both batches of particles was pH ~4.4–4.7 after washing. Moreover, the higher initial pH of the unstable (unwashed) particles resulted in a higher pH during conjugation of gold particles to goat anti-human Fc antibody (which was originally buffer exchanged to pH 4.3, 20 mM acetate). The pH of the antibody-gold mixture was pH ~4.4–4.7 for the unstable (unwashed) particles and pH ~4–4.4 for the stable (unwashed) particles. In contrast, the pH of the antibody-gold mixtures was similar (pH ~4– 4.4) after washing for stable and unstable gold particles. We next evaluated the effect of washing the gold colloid on the plasmon wavelengths (Fig. 1C) and absorbances (Fig. 1D) for the particles before and after conjugation to goat anti-human Fc antibodies. The plasmon wavelengths (~523–524 nm) and absorbances (0.25–0.29 AU) were similar for both stable and unstable gold particles before and after washing, which is consistent with their similar sizes (Fig. 1A). However, unstable (unwashed) particles readily aggregated when mixed with antibody, as detected by large plasmon wavelengths (576 nm; Fig. 1C) and low absorbances (0.003 AU; Fig. 1D). Interestingly, washing the unstable particles and then conjugating them to antibodies resulted in stable antibody-gold conjugates with plasmon wavelengths and absorbances that were generally similar (536 nm, 0.17 AU) to those obtained using stable gold particles either before or after washing (532–534 nm, 0.21–0.22 AU). As noted earlier, we were unable to prevent particle aggregation simply by reducing the pH of the unstable particles or the anti-Fc antibody prior to conjugation (data not shown). Identification of factors that influence the stability of antibody-gold conjugates We next sought to evaluate in more detail the impact of solution pH during antibody conjugation on the stability of the resulting conjugates (Fig. 2). To accomplish this, we washed stable gold particles and mixed them with goat anti-human Fc antibody (initially at pH ~4–5.5) to achieve final pH values of pH ~4–5.1 for the antibody-gold conjugates. The highest pH values (pH ~5.0–5.1) resulted in significant increases in the plasmon wavelengths (Fig. 2A) and reductions in the absorbances (Fig. 2B), which is consistent with conjugate aggregation. In contrast, lower pH values resulted in little change (pH ~4–4.3) or intermediate changes (pH ~4.4–4.9) in plasmon wavelengths and absorbances. The plasmon wavelengths for the conjugates prepared at pH ~4–4.3 (530–531 nm) are larger than the original gold particles (523–524 nm),



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which is consistent with antibody immobilization.7, 8 Antibody conjugation at pH ~4–4.3 also resulted in stable conjugates for polyclonal antibodies from several different species (human, rat, mouse, rabbit) in addition to those from goat (Fig. S2). Moreover, we evaluated a larger range of pH values for conjugating goat polyclonal antibodies to gold particles that had been washed (pH ~4–10), and found that antibody conjugates with high stability could only be prepared at pH values ~7.4 (Fig. 3). We quantified the number of antibodies bound per particle at low pH (~4.3) and high pH (~9), and found that both conditions led to a similar extent of immobilization (19.9 ± 3.7 antibodies per particle at pH ~4.3 and 20.4 ± 0.5 antibodies at pH ~9). Collectively, these results demonstrate that solution pH impacts the stability of antibody-gold conjugates even for washed stable particles. Moreover, our findings suggest that pH ~4–4.3 is a useful conjugation pH that is similar to the pH of the washed gold colloid (pH ~4.7) and which promotes high stability of the resulting antibody-gold conjugates for a wide range of antibodies. We also sought to identify other factors that may influence the stability of the antibody-gold conjugates. The fact that the gold particles are stabilized by citrate suggests that differences in citrate concentration may also influence the stability of the conjugates. Thus, we evaluated the impact of the citrate concentration on the stability of conjugates prepared at pH 4.3 using stable (washed) particles (Fig. 4). While the amount of residual citrate in the gold colloid is unknown (this is not specified by the manufacturer), we evaluated citrate concentrations (0–10 mM) that span the concentrations of citrate (~1–10 mM) reported for synthesizing similar gold particles.15 Moderate increases in plasmon wavelength (~2 nm) and decreases in absorbance (~0.06 AU) are observed for

Figure 3. Effect of pH on the stability of gold particles conjugated to anti-Fc antibodies. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by absorbing goat anti-human Fc antibodies on gold particles at various pH values. Stable gold particles (AuNP: S) were washed once with water and mixed with anti-Fc antibodies (final concentration of 40 µg/mL), the latter of which were buffer exchanged to pH ~4.3–10 (40 mM buffer). The buffers used were acetate (pH 4.3 and 5.5), phosphate (pH 6.5, 7.4 and 8), and borate (pH 9 and 10). The pH values for the antibody-gold conjugates were estimated using pH paper, and the final buffer concentration (after mixing the gold and antibody) was 20 mM. The antibody-gold conjugates were resuspended in the corresponding buffer used for conjugation (final buffer concentration of 20 mM). The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from two independent experiments.

citrate concentrations up to 10 mM. On one hand, this finding suggests that differences in the residual amount of citrate are unlikely to be responsible for the variable stability of the conjugates prepared using unwashed particles. However, this finding also suggests that differences in citrate concentration may be responsible for small variations in baseline plasmon wavelengths


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for antibody-gold conjugates prepared using different batches of gold. Thus, washing gold particles (even those that are stable without washing) prior to antibody conjugation may be helpful for eliminating variations in baseline plasmon wavelengths, which is important for improving the reproducibility of assays such as AC-SINS. The lack of precise information about the composition of the gold particles obtained from the manufacturer led us to test the generality of our findings for gold particles that we synthesized using a published procedure.16 We prepared gold particles via citrate reduction of chloroauric acid in the absence of tannic acid or other additives. The resulting particles (U2) possessed the expected size (radius of 12.1±0.5 nm) and similar SOS values (7±2) as gold obtained from the manufacturer (SOS values of 7–33). Importantly, the pH of our synthesized particles (pH ~5.5–5.8 for U2; Fig. 5A) was similar to that for unstable particles from the manufacturer (pH ~5.5–5.8 for U1; Fig. 1B). However, washing the U2 particles resulted in pH values (pH ~4.7; Fig. 5A) similar to those for washed particles from the manufacturer (pH ~4.4–4.7; Fig. 1B). The relatively high pH of unwashed U2 particles (pH ~5.5–5.8) resulted in higher-than-expected pH values during conjugation to anti-Fc antibody (pH ~5.3–5.5; Fig. 5A). However, washing the U2 particles resulted in pH values during conjugation (pH ~4–4.3) that were similar to those observed for stable (unwashed or washed) or unstable (washed) particles from the manufacturer (pH ~4–4.3). The unwashed U2 particles also aggregated when mixed with anti-Fc antibody, as evidenced by increased plasmon wavelengths (Fig. 5B) and reduced absorbance values (Fig. 5C). However, mixing the antiFc antibody with the washed U2 particles led to stable antibody-gold conjugates with similar plasmon wavelengths (530 nm) as observed

Figure 4. Effect of citrate concentration on the stability of antibody-gold conjugates. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by adsorbing goat anti-human Fc antibodies on gold particles at different citrate concentrations (0–10 mM final concentration; pH 4.3). Stable gold particles (AuNP: S) were washed once with water and mixed with anti-Fc antibodies, the latter of which were buffer exchanged to 0– 100 mM citrate (10 fold higher than the final citrate concentrations) at a constant concentration of 20 mM acetate (pH 4.3). The final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 2 mM, respectively. The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments.

for conjugates prepared with washed particles from the manufacturer (530–531 nm). These results suggest that our methods for preparing stable antibody-gold conjugates are applicable to gold colloid synthesized using different procedures and which contain different initial solution compositions and pH values. We have previously reported that the anti-Fc antibody concentration also significantly impacts the stability of antibody-gold conjugates because subsaturating antibody concentrations cause particle bridging 7 ACS Paragon Plus Environment


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and aggregation.7, 10 Therefore, we investigated the impact of the anti-Fc concentration during conjugation on the stability of the resulting conjugates (Fig. 6). Relative to the anti-Fc concentration that we used for our initial studies (40 µg/mL; Figs. 1–5), we evaluated lower and higher concentrations as well (2.5–160 µg/mL). Stable (unwashed) particles from the manufacturer aggregate at 2.5 µg/mL of anti-Fc antibody, while they are stabilized at higher antibody concentrations (Fig. 6A). Interestingly, washed stable (Fig. 6B) and washed unstable (U2, Fig. 6C) particles are more stable at low anti-Fc concentration (2.5 µg/mL) than unwashed stable particles. Although conjugates prepared using 40 and 160 µg/mL anti-Fc antibody display similar plasmon wavelengths and absorbances, the recovery after resuspension was modestly higher for the highest anti-Fc antibody concentration (>95% for 160 µg/mL, >90% for 40 µg/mL). This is also consistent with our previous findings for unwashed particles7 and suggests that the highest anti-Fc antibody concentration (160 µg/mL) is best for reproducibly preparing stable antibody-gold conjugates. We also compared the impact of anti-Fc antibody concentration on the stability of conjugates prepared at low pH (~4–4.3) versus high pH (~9; Fig. S3). Interestingly, subsaturating concentrations of anti-Fc antibody (e.g., 1–5 µg/mL) at high pH (~9, 2 mM borate) yield small plasmon shifts (95%) of the conjugates after sedimentation and resuspension, and lower recoveries are indicative of poor conjugate stability and should be avoided. Moreover, we recommend


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removing ~96% of the supernatant volume, measuring the residual volume with a pipette, and modestly diluting the concentrated conjugates with the conjugation buffer to 5% of the original volume. This is important because changes in the concentration of the conjugates due to different residual volumes after sedimentation can lead to variability in the resulting plasmon wavelengths. Previous studies have required the use of glycerol gradients to remove unbound antibody and/or aggregated conjugates as well as dialysis to remove glycerol after conjugate sedimentation,5, 6 which makes the conjugation process much more complex and harder to reproduce. Nevertheless, a drawback of our approach that uses relatively high antibody concentrations (such as 160 µg/mL anti-Fc antibody during conjugation) is that it requires at least one sedimentation step to remove free (unbound) antibody for applications in which free antibody can interfere with the intended use of the conjugates. However, we find that conjugate sedimentation and resuspension is simple and reproducible, and can be used to efficiently deplete unbound antibody. Finally, we find that the plasmon wavelengths of the resulting antibody-gold conjugates (pH 4.3, 2 mM acetate) are typically ~529–531 nm for conjugates prepared with thiolated PEG and ~533–534 nm for conjugates prepared without thiolated PEG, and higher plasmon wavelengths are indicative of suboptimal antibody conjugation and/or purification. Our findings also provide some insights for improving the sensitivity of AC-SINS measurements of mAb self-association. We suggest only using one sedimentation step to remove the unbound anti-Fc antibody (prior to mixing the conjugates with mAbs) because this leads to maximum assay sensitivity. The plasmon shifts observed for highly associative mAbs (human mAb CNTO607 and rabbit mAb 1H9) are ~10 nm (~40%) larger after a single sedimentation step than after two sedimentations steps to remove unbound antiFc antibody. The origin of this signal enhancement is unknown and will need to be investigated in the future. Nevertheless, a disadvantage of using only one sedimentation step to remove unbound anti-Fc antibody is that higher mAb concentrations (~5–10 µg/mL) are needed to saturate the particles than if two sedimentation steps are used. However, we find that mAb concentrations of ~5–10 µg/mL are still low enough to be compatible with unpurified antibody samples produced at small scale and which have original mAb concentrations of at least ~50 µg/mL. We also suggest that the mAbs of interest be prepared with excess non-specific polyclonal antibody (such as goat non-specific polyclonal antibody) prior to mixing with anti-Fc antibody conjugates. We find that this promotes high assay reproducibility, which may be due to the prevention of non-specific mAb adsorption on the microwell plates and/or gold conjugates. Another important consideration is the control samples that are used to calculate the plasmon shifts for AC-SINS. While most of our experiments were performed using anti-human Fc controls without human polyclonal antibody, we found at the end of our studies that anti-human Fc controls with human polyclonal antibody yielded similar plasmon shifts (Fig. S7). We recommend evaluating the use of anti-human Fc conjugates with and without human polyclonal antibody 17 ACS Paragon Plus Environment


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as the controls to determine which is more suitable for the intended application. It may be that the use of human antibody controls leads to higher reproducibility because of the increased stability of the controls with bound human antibody as well as measurements that are more reflective of the properties of the human antibodies and less dependent on the properties of the non-human capture antibodies. We expect that these modifications to our previously described AC-SINS assay7, 8 as well as our improved procedure for preparing stable anti-Fc conjugates will enable robust identification of mAb variants that possess low self-association and favorable biophysical properties at high concentration (high solubility and low viscosity).29, 30

CONCLUSIONS Our findings demonstrate that reproducible preparation of antibody-gold conjugates requires purification of gold colloid prior to antibody conjugation. While there are many approaches to purifying gold colloid, we find that a single sedimentation and resuspension step in water is superior for both removing gold particle aggregates and reducing the concentration of non-gold solution components. This simple procedure enables stable and reproducible conjugation at acidic pH (pH 4-4.3), which is close to the pH of the washed gold colloid and well below the expected antibody isoelectric point. Our findings are dissimilar to previous recommendations that protein-gold conjugates be prepared at pH values above the protein isoelectric point. Additional work is needed to more thoroughly evaluate the stability of antibody-gold conjugates prepared at acidic and basic pH to better understand advantages and disadvantages of each approach. Moreover, it will be interesting to evaluate if acidic pH is useful for improving the preparation of nanoparticle conjugates using non-antibody proteins, especially those with neutral-to-basic isoelectric points that are known to be highly soluble at acidic pH.

EXPERIMENTAL METHODS Materials Unconjugated, citrate-stabilized gold particles (nominal radii of 10 nm, 15705) were obtained from Ted Pella, Inc. (Redding, CA) and stored at 4 °C (freezing the particles is strongly destabilizing and should be avoided). Polyclonal goat IgG (005-000-003), polyclonal goat anti-human antibodies (Fc specific, 109-005098), and polyclonal goat anti-rabbit antibodies (Fc specific, 111-005-008) were obtained from Jackson ImmunoResearch (West Grove, PA). Purified and unpurified mAbs were obtained from Eli Lilly, as described previously.7 Polyclonal IgG from human serum (Reagent grade, I4506), chloroauric acid (254169), sodium citrate (USP grade, S1804), citric acid (ACS grade, 251275), potassium phosphate monobasic (ACS grade, P0662), sodium phosphate dibasic dihydrate (reagent grade, 30435), and potassium chloride (molecular biology grade, P9541) were obtained from Sigma Aldrich (St. Louis, MO). Glacial acetic acid (ACS grade, AC12404-0010), potassium acetate (ACS grade, P171) and sodium chloride (ACS grade, S271) were obtained from Thermo Fisher Scientific (Waltham, MA). Methoxy polyethylene glycol thiol (MW 18 ACS Paragon Plus Environment

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2000, PG1-TH-2k) was acquired from Nanocs Inc. (New York, NY). Microcentrifuge tubes (1.5 mL, 16155500) were purchased from USA Scientific (Ocala, FL). Non-bleeding pH indicator strips were obtained from EMD Millipore (mColorpHast, 1.09542.0001 and 1.09543.0001, Billerica, MA). The micro BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (23225).

Methods Gold nanoparticle synthesis A solution (22.5 mL) containing 3 mg of chloroauric acid was prepared in water and brought to boil in a 500 mL Pyrex bottle (1395500, Corning) with vigorous stirring (Model H4000 HS hotplate, Biomega) using a Teflon-coated magnetic stir bar (1.5 in). A second solution (2.5 mL) with 2% wt/vol sodium citrate (pH ~8–9) was then added to the chloroauric acid and allowed to react for ~10 min until the solution color turned dark red. The solution was then cooled to room temperature before evaluation. Purification and evaluation of gold nanoparticles Gold nanoparticles were washed by centrifuging the gold solution (~1 mL) at 21130 rcf for 5–6 min in untreated 1.5 mL microcentrifuge tubes (1615-5500, USA Scientific) using an Eppendorf 5424 microcentrifuge (without temperature control). Afterward, 95% of the supernatant was removed (without disturbing the sedimented gold), and the sedimented gold was then resuspended with purified (Milli-Q) water (unpurified tap water is destabilizing and should be avoided). A small (compact) black pellet that was distinct from the majority of the sedimented (loosely associated) gold was sometimes observed and was not resuspended (it does not disperse when adding water and should not be perturbed physically). For limited experiments that investigated the impact of anti-Fc conjugation at low versus high pH on the AC-SINS measurements (Figs. S5 and S6), the gold was resuspended in water for conjugation at pH ~4-4.3 or 2 mM borate for conjugation at pH ~9. Additional studies were also performed using low-binding tubes from USA Scientific (1415-2600) as well as 1.7 mL untreated (16070) and low-binding (11700) tubes from Sorenson Bioscience (Fig. S1). The pH of these solutions was evaluated by depositing 15–20 µL on pH paper and comparing the color of the pH paper to that of buffered solutions (without antibody or gold particles) containing the corresponding buffer and whose pH was measured via a pH meter. The color of the pH paper – if not directly compared to known pH solutions – can lead to errors in pH measurements. For the experiments performed in this work, the potential for misleading measurements using pH paper (if not compared to the corresponding buffer standards) was greatest for weakly buffered solutions at high pH (e.g., 2 mM borate, pH ~9–10) and least for strongly buffered solutions (e.g., 20 mM buffer) or weakly buffered solutions at low pH (e.g., 2 mM acetate, pH ~4–5).



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Dynamic light scattering (DLS) measurements of the gold particles without antibody were performed using a DynaPro Titan light scattering system (Wyatt, Santa Barbara, CA). Each sample (80 µL) was added without dilution to a disposable cuvette (Eppendorf, 952010069) and evaluated. The laser power was adjusted to maintain approximately one million counts per second. The scattering data were fit assuming the particles to be Rayleigh spheres (Dynamics software, Wyatt). The absorbance spectra (450–650 nm) of the gold nanoparticles were measured using a Tecan Safire2 plate reader. Each plasmon wavelength was calculated by fitting forty data points (1 nm increments) around the wavelength of maximum absorbance to a second order polynomial function and setting the first derivative to zero. The corresponding absorbance value was calculated as the difference between the maximum absorbance value at the plasmon wavelength and the minimum absorbance value between 510 and 610 nm. Preparation of antibody-gold conjugates In preparation for mAb immobilization, goat anti-human Fc or goat anti-rabbit Fc antibody was buffer exchanged twice into 20 mM potassium acetate (pH 4.3) using Zeba desalting columns (PI-89882, Thermo Fisher Scientific) and subsequently filtered using 0.22 µm PVDF syringe filters (4 mm, Merck Millipore, SLGV004SL). For evaluation of gold conjugate stability, the capture antibody was buffer exchanged twice into solution conditions of varying pH (pH 4.0–5.5, 200 mM acetate) or citrate concentration (0–100 mM citrate, 20 mM acetate, pH 4.3). For experiments comparing the impact of conjugating anti-Fc antibodies at low versus high pH on the AC-SINS measurements (Figs. S5 and S6), the capture antibody was buffer exchanged twice into either pH 4.3 (20 mM acetate) or pH 9 (2 mM borate). The polyclonal antibody concentrations after buffer exchanging and filtering were calculated using UV absorbance measurements at 280 nm and extinction coefficients of 1.26 mL/(mg⋅cm) for goat anti-human Fc antibody or 1.35 mL/(mg⋅cm) for goat anti-rabbit Fc antibody. The capture antibodies were then typically diluted to 0.4 or 1.6 mg/mL with the appropriate buffer (typically 20 mM acetate, pH 4.3). Next, one part (100 µL) of the capture antibody was first added to a new 1.5 mL microcentrifuge tube (1615-5500, USA Scientific), and then nine parts (900 µL) of either washed or unwashed gold nanoparticles (nominal concentration of 4.67×1011 particles/mL, diluted from the original gold stock solution of 7×1011 particles/mL using Milli-Q water) were added. The solution (1000 µL) was rapidly mixed by pipetting 900 µL (of the 1000 µL) up and down ten times immediately after addition of the gold, and the conjugates were then incubated overnight at room temperature. The next day, one part (typically 111 µL) of 1 µM thiolated PEG (MW 2000) in 2 mM acetate (pH 4.3) was typically added to nine parts (typically 1000 µL) of the conjugate solution and incubated for 1 h. To evaluate the role of thiolated PEG on the AC-SINS measurements, control experiments were also performed 20 ACS Paragon Plus Environment

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without thiolated PEG (111 µL of 2 mM acetate buffer was added instead for the control without thiolated PEG, Fig. S8). The conjugates were then centrifuged for 6 min at 21130 rcf, after which ~96% of the supernatant (typically 1067 µL) was removed. The pelleted conjugates (~44 µL) were resuspended in the residual buffer and the volume of the residual conjugates was measured via a pipette. The conjugates were then modestly diluted using the conjugation buffer to 5% of the original volume (~56 µL), which is important to ensure reproducible concentrations of the sedimented conjugates. The resuspended conjugates were then diluted 10-fold in 2 mM acetate (pH 4.3). For analysis of the impact of anti-Fc conjugation at pH ~9 (2 mM borate final concentration) on the AC-SINS measurements (Figs. S5 and S6), the thiolated PEG solution was prepared in 2 mM borate (pH 9) and used to block the conjugates as described for the low pH experiment. Afterward, the conjugates prepared at high pH were resuspended in 2 mM borate instead of 2 mM acetate. For limited experiments, the pelleting process was conducted twice to further reduce the concentration of unbound anti-Fc antibody (Fig. S4). To quantify the number of polyclonal antibodies immobilized per gold particle, goat anti-human Fc antibody was buffer exchanged twice against 20 mM potassium acetate (pH 4.3) or 20 mM borate (pH 9). The concentration of polyclonal antibody was then measured via UV absorbance, and the antibody solutions were diluted to 150 and 250 µg/mL with the corresponding buffers. Next, the anti-Fc antibodies were conjugated to the gold nanoparticles overnight at 15 and 25 µg/mL. Afterward, the conjugates were blocked with thiolated PEG and then centrifuged for 1 h at 21130 rcf (4 °C). The unbound concentration of antibody in the supernatant was quantified using the micro-BCA protein assay (Thermo Fisher Scientific). The amount of immobilized antibody was determined via the difference in the amount of the antibody in the supernatant after sedimentation for control samples without nanoparticles relative to those with nanoparticles. mAb immobilization and plasmon analysis of nanoparticle conjugates For AC-SINS experiments using human and rabbit mAbs, polyclonal goat non-specific antibody (2–5 mg/mL) was buffer exchanged twice into 11.1 mM sodium citrate (pH 6) with 0 mM NaCl (for rabbit mAbs) or 55.6 mM NaCl (for human mAbs). The goat non-specific antibody concentration was determined using UV absorbance measurements at 280 nm and an extinction coefficient of 1.28 mL/(mg⋅cm). The non-specific antibody was subsequently diluted to 117 µg/mL in the corresponding solution used for buffer exchange. For purified mAbs (rabbit or human), the stock solution was typically diluted in the appropriate citrate buffer (0 or 55.6 mM NaCl, pH 6) to 111 µg/mL of mAb. Afterward, one part of the diluted mAb (typically 12 µL) was combined with nineteen parts (typically 228 µL) of the non-specific goat antibody solution (117 µg/mL) to make a solution with 5.6 µg/mL mAb, 111 µg/mL non-specific goat polyclonal antibody, 11.1 mM citrate (pH 6), and 0 or 55.6 mM NaCl. Finally, one part (8 µL) of the resuspended and diluted capture conjugates was first added to transparent and flat21 ACS Paragon Plus Environment


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bottom 384-well polystyrene plates (Thermo Fisher Scientific, 12565506), and then nine parts (72 µL) of the mAb solution (with non-specific goat antibody) was added to achieve a final volume of 80 µL per well (the solutions were mixed by pipetting up and down ten times). Final conditions in the well plate were 5 µg/mL mAb, 100 µg/mL non-specific goat antibody, and 10 mM citrate (pH 6) with 0 or 50 mM NaCl. After incubation for 2 h, the absorbance spectra for the samples were typically measured using a Tecan Safire2 plate reader (limited experiments were also conducted using Molecular Devices SpectraMax M5 and Biotek Synergy 4 plate readers). Each well was read twenty times in 1 nm intervals from 450-650 nm. For unpurified human mAbs, the stock solutions were diluted to 55.6 µg/mL in clarified cell culture media instead of the citrate buffer. Additionally, the non-specific goat antibody (2–5 mg/mL) was buffer exchanged twice into 13.9 mM citrate (pH 6) with 69.4 mM NaCl, and then diluted to 139 µg/mL in the buffer exchange solution. Afterward, one part of the diluted mAb was mixed with four parts of the nonspecific goat antibody to make a solution with 11.1 µg/mL mAb, 111 µg/mL non-specific goat antibody, 11.1 mM citrate (pH 6), 55.6 mM NaCl and 20% media. Finally, one part (5 µL) of anti-Fc conjugates (prepared as described above) was first added to the 384-well plates, and then nine parts (45 µL) of the mAb solution (with non-specific goat antibody) was added (the solutions were pipetted up and down ten times). Final conditions in the well plate were 10 µg/mL mAb, 100 µg/mL non-specific goat antibody, 10 mM citrate (pH 6), 50 mM NaCl and 18% media (50 µL total per well). After incubation for 2 h, the absorbance spectra of the samples were measured using a Tecan Safire2 plate reader. To evaluate the stability of the capture conjugates as a function of storage time (Fig. S10), the goat antihuman Fc conjugates were prepared as described above. After the overnight incubation with anti-Fc antibody (160 µg/mL) but before the addition of thiolated PEG and sedimentation to remove free anti-Fc antibody, the conjugates were stored for 1-7 days at 4 °C. On each day of evaluation, the conjugates were shifted from 4 °C to room temperature, mixed with thiolated PEG and processed as described above. A single sedimentation step (21130 rcf for 6 min) was used to remove unbound anti-Fc antibody. The final concentration of the purified mAbs for these experiments was 5 µg/mL, and the final solution conditions were 100 µg/mL nonspecific goat antibody, 10 mM citrate (pH 6) and 50 mM NaCl (80 µL total per well).

SUPPORTING INFORMATION Analysis of gold nanoparticle sedimentation and resuspension in different microcentrifuge tubes, stability of gold conjugates prepared using polyclonal antibodies from different species, stability of antibody conjugates at subsaturating and saturating antibody concentrations prepared at acidic and basic pH, measurements of mAb self-association using antibody-gold conjugates prepared at acidic and basic pH, and impact of using different control samples, blocking procedures and plate readers on measurements of mAb self-association. This material is available free of charge via the Internet at http://pubs.acs.org. 22 ACS Paragon Plus Environment

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ACKNOWLEDGEMENTS We thank members of the Tessier lab for their assistance editing the manuscript. This work was supported by Eli Lilly.

CONFLICTS OF INTEREST P.M.T. has received consulting fees and/or honorariums for presentations of this and/or related research findings at Eli Lilly, MedImmune, Bristol-Myers Squibb, Janssen, Merck, Genentech, Amgen, Pfizer, Adimab, Abbott, DuPont, Danisco, Bayer, Abbvie, Roche, Boehringer Ingelheim and Regeneron.



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Rana, S., Yeh, Y. C., and Rotello, V. M. (2010) Engineering the nanoparticle-protein interface: applications and possibilities. Curr Opin Chem Biol 14, 828-34. Aubin-Tam, M. E. (2013) Conjugation of nanoparticles to proteins. Methods Mol Biol 1025, 19-27. Albanese, A., Tang, P. S., and Chan, W. C. (2012) The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu Rev Biomed Eng 14, 1-16. Mout, R., Moyano, D. F., Rana, S., and Rotello, V. M. (2012) Surface functionalization of nanoparticles for nanomedicine. Chem Soc Rev 41, 2539-44. Yokota, S. (2010) Preparation of colloidal gold particles and conjugation to protein A, IgG, F(ab')(2), and streptavidin. Methods Mol Biol 657, 109-19. Oliver, C. (2010) Conjugation of colloidal gold to proteins. Methods Mol Biol 588, 369-73. Wu, J., Schultz, J. S., Weldon, C. L., Sule, S. V., Chai, Q., Geng, S. B., Dickinson, C. D., and Tessier, P. M. (2015) Discovery of highly soluble antibodies prior to purification using affinity-capture selfinteraction nanoparticle spectroscopy. Protein Eng Des Sel 28, 403-14. Sule, S. V., Dickinson, C. D., Lu, J., Chow, C. K., and Tessier, P. M. (2013) Rapid analysis of antibody self-association in complex mixtures using immunogold conjugates. Mol Pharm 10, 1322-31. Geng, S. B., Wittekind, M., Vigil, A., and Tessier, P. M. (2016) Measurements of monoclonal antibody self-association are correlated with complex biophysical properties. Mol Pharm 13, 1636-45. Li, X., Geng, S. B., Chiu, M. L., Saro, D., and Tessier, P. M. (2015) High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum. Bioconjug Chem 26, 520-8. Liu, Y., Caffry, I., Wu, J., Geng, S. B., Jain, T., Sun, T., Reid, F., Cao, Y., Estep, P., Yu, Y., et al. (2014) High-throughput screening for developability during early-stage antibody discovery using selfinteraction nanoparticle spectroscopy. mAbs 6, 483-92. Jayaraman, J., Wu, J., Brunelle, M. C., Cruz, A. M., Goldberg, D. S., Lobo, B., Shah, A., and Tessier, P. M. (2014) Plasmonic measurements of monoclonal antibody self-association using self-interaction nanoparticle spectroscopy. Biotechnol Bioeng 111, 1513-20. Kelly, R. L., Sun, T., Jain, T., Caffry, I., Yu, Y., Cao, Y., Lynaugh, H., Brown, M., Vasquez, M., Wittrup, K. D., et al. (2015) High throughput cross-interaction measures for human IgG1 antibodies correlate with clearance rates in mice. mAbs 7, 770-7. Estep, P., Caffry, I., Yu, Y., Sun, T., Cao, Y., Lynaugh, H., Jain, T., Vasquez, M., Tessier, P. M., and Xu, Y. (2015) An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies. mAbs 7, 553-61. Slot, J. W., and Geuze, H. J. (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38, 87-93. Turkevich, J., Stevenson, P. C., and Hillier, J. (1951) A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss Faraday Soc 11, 55-75. Bethea, D., Wu, S. J., Luo, J., Hyun, L., Lacy, E. R., Teplyakov, A., Jacobs, S. A., O'Neil, K. T., Gilliland, G. L., and Feng, Y. (2012) Mechanisms of self-association of a human monoclonal antibody CNTO607. Protein Eng Des Sel 25, 531-7. Wu, S. J., Luo, J., O'Neil, K. T., Kang, J., Lacy, E. R., Canziani, G., Baker, A., Huang, M., Tang, Q. M., Raju, T. S., et al. (2010) Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng Des Sel 23, 643-51. Sule, S. V., Sukumar, M., Weiss, W. F., Marcelino-Cruz, A. M., Sample, T., and Tessier, P. M. (2011) High-throughput analysis of concentration-dependent antibody self-association. Biophys J 101, 174957. Burckbuchler, V., Mekhloufi, G., Giteau, A. P., Grossiord, J. L., Huille, S., and Agnely, F. (2010) Rheological and syringeability properties of highly concentrated human polyclonal immunoglobulin solutions. Eur J Pharm Biopharm 76, 351-6. Szenczi, A., Kardos, J., Medgyesi, G. A., and Zavodszky, P. (2006) The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution. Biologicals 34, 5-14.


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23. 24. 25. 26. 27. 28. 29. 30.


Geoghegan, W. D., and Ackerman, G. A. (1977) Adsorption of horseradish-peroxidase, ovomucoid and antiimmunoglobulin to colloidal gold for indirect detection of concanavalin-A, wheat-germ agglutinin and goat antihuman immunoglobulin-G on cell-surfaces at electron-microscopic level: New method, theory and application. J Histochem Cytochem 25, 1187-1200. Birrell, G. B., Hedberg, K. K., and Griffith, O. H. (1987) Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy. J Histochem Cytochem 35, 843-53. Yeo, E. L., Chua, A. J., Parthasarathy, K., Yeo, H. Y., Ng, M. L., and Kah, J. C. (2015) Understanding aggregation-based assays: nature of protein corona and number of epitopes on antigen matters. RSC Adv 5, 14982-14993. Sule, S. V., Cheung, J. K., Antochshuk, V., Bhalla, A. S., Narasimhan, C., Blaisdell, S., Shameem, M., and Tessier, P. M. (2012) Solution pH that minimizes self-association of three monoclonal antibodies is strongly dependent on ionic strength. Mol Pharm 9, 744-51. Sahin, E., Weiss, W. F., Kroetsch, A. M., King, K. R., Kessler, R. K., Das, T. K., and Roberts, C. J. (2012) Aggregation and pH-temperature phase behavior for aggregates of an IgG2 antibody. J Pharm Sci 101, 1678-87. Brummitt, R. K., Nesta, D. P., Chang, L., Chase, S. F., Laue, T. M., and Roberts, C. J. (2011) Nonnative aggregation of an IgG1 antibody in acidic conditions: part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates. J Pharm Sci 100, 2087-103. Sahin, E., Grillo, A. O., Perkins, M. D., and Roberts, C. J. (2010) Comparative effects of pH and ionic strength on protein-protein interactions, unfolding, and aggregation for IgG1 antibodies. J Pharm Sci 99, 4830-48. Tessier, P. M., Wu, J., and Dickinson, C. D. (2014) Emerging methods for identifying monoclonal antibodies with low propensity to self-associate during the early discovery process. Expert Opin Drug Deliv 11, 461-5. Geng, S. B., Cheung, J. K., Narasimhan, C., Shameem, M., and Tessier, P. M. (2014) Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions. J Pharm Sci 103, 3356-3363.



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Bioconjugate Chemistry Supporting Information

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A facile method for preparing stable antibody-gold conjugates and application to affinity-capture self-interaction nanoparticle spectroscopy Steven B. Geng, Jiemin Wu, Magfur E. Alam, Jason S. Schultz, Craig Dickinson, Carly Seminer and Peter M. Tessier A

T1-R

T1-L T2-R

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0.6 0.4

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Microcentrifuge tube Figure S1. Comparison of the stability of gold nanoparticles after sedimentation in untreated and lowbinding tubes. Stable gold nanoparticles (1 mL) were aliquoted into four types of microcentrifuge tubes, and their stability during sedimentation and resuspension was evaluated. Two of the tubes were untreated polypropylene microcentrifuge tubes from USA Scientific (T1-R) and Sorenson Bioscience (T2-R), and the other two were the corresponding treated (low-binding) tubes from USA Scientific (T1-L) and Sorenson Bioscience (T2-L). Images of gold colloid in the different tubes (A) before sedimentation, (B) after sedimentation and removal of 95% of the supernatant, and (C) after resuspension of sedimented gold with water. (D) Plasmon wavelength and (E) absorbance measurements of gold nanoparticles after sedimentation and resuspension with water. The absorbance values were calculated as the difference between the maximum and minimum absorbances in the ACS Paragon Plus Environment range of 510 to 610 nm. Two independent experiments were performed, the reported data are averages of three replicates from a representative experiment, and the error bars are the corresponding standard deviations.


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0.3

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Polyclonal antibody Figure S2. Evaluation of the stability of antibody-gold conjugates prepared at acidic pH using polyclonal antibodies from different species. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by adsorbing polyclonal (non-specific) antibodies from rat, human, mouse, rabbit and goat on washed (stable) gold particles at pH ~4–4.3. Gold particles (AuNP: S) were washed once with water and mixed with polyclonal antibodies, the latter of which were buffer exchanged to pH 4.3 (20 mM acetate). The final acetate concentration (after mixing the gold and antibody) was 2 mM. The absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. The errors are standard deviations for three independent experiments. ACS Paragon Plus Environment

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A

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Figure S3. Comparison of the stability of anti-Fc conjugates prepared at low and high pH as a function of antibody concentration. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by adsorbing goat anti-human Fc polyclonal antibodies on washed (stable) gold particles at pH ~4–4.3 (2 mM acetate) and pH ~9 (2 mM borate). Gold particles (AuNP: S) were sedimented, the supernatant (95%) was discarded and the particles were resuspended in water (for low pH conjugation) or 2 mM borate (for the high pH conjugation). The washed particles were then mixed with anti-Fc polyclonal antibodies that were either buffer exchanged twice against pH 4.3 (20 mM acetate) or pH 9 (2 mM borate) solutions. The final acetate and borate concentrations (after mixing the gold and antibody) were ~2 mM. The gold particles were not diluted with water prior to use, which leads to higher absorbance values than those reported in Figure 6. The absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. The errors are standard deviations for three independent experiments. ACS Paragon Plus Environment


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Figure S4. Effect of additional purification of anti-Fc conjugates on plasmon shift and absorbance measurements for rabbit and human mAbs. Stable (washed) gold nanoparticles conjugated with goat anti-rabbit Fc or goat anti-human Fc antibodies were washed twice (instead of once for Figure 7) to further remove unbound anti-Fc antibody and incubated with purified (A) rabbit or (B) human mAbs at a range of mAb concentrations. The experiments were performed at pH 6 (10 mM sodium citrate) and 0 mM NaCl (rabbit mAbs) or 50 mM NaCl (human mAbs). The goat anti-Fc antibody concentration during the conjugation reaction was 160 μg/mL, and the conjugates were blocked with thiolated PEG. Nonspecific goat antibody (100 μg/mL final concentration) was added to minimize non-specific interactions. The averaged plasmon shifts (Δλp) were calculated using controls without human antibody. The average absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the data shown are from a representative experiment, and the error bars are the corresponding standard deviations for three ACS Paragon Plus Environment replicates.

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mAb: 405d

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Figure S5. Effect of anti-Fc conjugation pH on plasmon shift and absorbance measurements for human mAbs. Stable (washed) gold nanoparticles were conjugated with goat anti-human Fc antibody at pH ~4.3 (2 mM acetate) or pH ~9 (2 mM borate), and used to evaluate the self-association of human mAbs 405d (top) and CNTO607 (bottom) at pH 6 (10 mM sodium citrate, 50 mM NaCl). The concentration of goat anti-human Fc antibody during the conjugation reaction was 160 μg/mL, and the conjugates were blocked with thiolated PEG. Non-specific goat antibody (100 μg/mL final concentration) was added to minimize non-specific interactions. The averaged plasmon shifts (Δλp) were calculated using controls without human antibody. The average absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the data are from a representative experiment (three replicates), and the error bars are standard deviations. ACS Paragon Plus Environment


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Figure S6. Effect of anti-Fc conjugation pH on plasmon shift and absorbance measurements for rabbit mAbs. Stable (washed) gold nanoparticles were conjugated with goat anti-rabbit Fc antibody at pH ~4.3 (2 mM acetate) or pH ~9 (2 mM borate), and used to evaluate the self-association of rabbit mAbs 1A7 (top) and 1H9 (bottom) at pH 6 (10 mM sodium citrate). The concentration of goat anti-rabbit Fc antibody during the conjugation reaction was 160 μg/mL, and the conjugates were blocked with thiolated PEG. Non-specific goat antibody (100 μg/mL final concentration) was added to minimize non-specific interactions. The averaged plasmon shifts (Δλp) were calculated using controls without rabbit antibody. The average absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the data are from a representative experiment (three replicates), and the error bars are standard deviations. ACS Paragon Plus Environment

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20

Human mAb (μg/mL)

Figure S7. Impact of using different anti-Fc control samples on plasmon shift measurements for human mAbs. Stable (washed) gold nanoparticles were conjugated with goat anti-human Fc antibody at pH ~4.3 (2 mM acetate), and used to evaluate human polyclonal antibody (pAb) as well as human mAbs 405d and CNTO607 at pH 6 (10 mM sodium citrate, 50 mM NaCl). In (B), the plasmon shifts (λp- λo) are calculated using control samples without human polyclonal antibody. In (C), the plasmon shifts are calculated using control samples with human polyclonal antibody. The concentration of goat anti-human Fc antibody during the conjugation reaction was 160 μg/mL, and the conjugates were blocked with thiolated PEG. Non-specific goat antibody (100 μg/mL final concentration) was added to minimize non-specific interactions. The average absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the data are from a representative experiment (three replicates), and the error bars are standard ACS Paragon Plus Environment deviations.


A

No PEG-SH

580

570

CNTO607 λp (nm)

λp (nm)

560 550 540

0

5

550 540

10

520

20

10

20

No PEG-SH

+ PEG-SH

30 25

20

20

Δλp (nm)

25

15 10

15 10

5

5

0

0 5

10

-5

20

Human mAb (μg/mL) C

Absorbance (AU)

0.04

0.02

10

20

Human mAb (μg/mL)

10

20

+ PEG-SH

0.08

0.06

5

5

Human mAb (μg/mL)

No PEG-SH

0.08

0

5

Human mAb (μg/mL)

30

-5

0

Human mAb (μg/mL) B

Δλp (nm)

560

530

530 520

+ PEG-SH

580

405d

570

Absorbance (AU)

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

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0.06 0.04

0.02

0

5

10

20

Human mAb (μg/mL) ACS Paragon Plus Environment

Figure S8. Effect of blocking the anti-Fc conjugates with thiolated PEG on plasmon shift and absorbance measurements for human mAbs. Stable (washed) gold nanoparticles were conjugated with goat anti-human Fc antibody at pH ~4.3 (2 mM acetate), and the conjugates were either blocked with thiolated PEG or used without blocking. Next, the conjugates were used to evaluate the human mAbs 405d and CNTO607 at pH 6 (10 mM sodium citrate, 50 mM NaCl). The concentration of goat anti-human Fc antibody during the conjugation reaction was 160 μg/mL. Non-specific goat antibody (100 μg/mL final concentration) was added to minimize non-specific interactions. The average plasmon shifts were calculated using control samples without human antibody. The average absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the data are from a representative experiment (three replicates), and the error bars are standard deviations.

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A 560

25

Tecan Safire SpectraMax M5 Biotek Synergy 4 2

20 15

Δλp (nm)

550

λp (nm)

540

10 5

530 0 520

-5

Control

405d CNTO607 Human mAb


C 0.08

Absorbance (AU)

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

0.06

0.04

0.02

0

Control


Figure S9. Comparison of plasmon wavelength and absorbance measurements for anti-human Fc conjugates prepared at low pH using three different plate readers. (A) Plasmon wavelength, (B) plasmon shift and (C) absorbance measurements for purified human mAbs (5 μg/mL final concentration) bound to anti-Fc gold conjugates that were initially prepared at pH 4.3 (2 mM acetate) and blocked with thiolated PEG. The measurements were obtained using three different plate readers (Tecan Safire2, SpectraMax M5 and Biotek Synergy 4). The spectra were measured from 450–650 nm (1 nm intervals), and the absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. The final conditions were pH 6, 10 mM sodium citrate, 50 mM NaCl and 100 μg/mL of nonspecific goat antibody. The concentration of goat anti-Fc antibody during the conjugation reaction was 160 μg/mL. In (A) and (C), the controls are anti-Fc conjugates without human antibody. In (B), the plasmon shifts were calculated using controls without human antibody. The data are for two independent experiments ACS Paragon Plus Environment (three replicates per experiment), and the error bars are standard deviations.


A

B

AuNP + anti-Fc

530

529

528 527

0

2

4

AuNP + anti-Fc

0.5

Absorbance (AU)

531

λp (nm)

6

0.4 0.3 0.2 0.1 0

8

0

2

Storage time (day)

4

6

8

Storage time (day)

C

AuNP + anti-Fc + mAb

30

CNTO607

25 20

Δλp (nm)

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

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15 10 5

405d

0 -5

0

2

4

6

8

Storage time (day)

Figure S10. Effect of storage time on the stability of anti-human Fc conjugates prepared at low pH. (A) Plasmon wavelength and (B) absorbance measurements for washed (stable) gold particles that were conjugated to goat anti-human Fc antibody (pH 4.3, 2 mM acetate) after storage at 4 °C for 0–7 days. (C) Plasmon shift measurements for purified human mAbs (5 μg/mL final concentration) bound to anti-Fc gold conjugates after initial storage of the conjugates at 4 °C for 0–7 days. The final conditions for the measurements with mAbs were pH 6, 10 mM sodium citrate, 50 mM NaCl and 100 μg/mL non-specific goat antibody. The concentration of goat anti-human Fc antibody during the conjugation reaction was 160 μg/mL. In (A) and (B), the particles were not blocked with thiolated PEG or sedimented to remove free anti-Fc antibody. In (C), the conjugates were blocked with thiolated PEG and non-specific goat antibody (100 μg/mL final concentration) to minimize non-specific interactions. The plasmon shifts were calculated using controls without human antibody. Two independent experiments were performed, the data are ACSreplicates), Paragon Plus from a representative experiment (three andEnvironment the error bars are standard deviations.

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Figure 1. Evaluation of the stability of gold nanoparticles and antibody-gold conjugates for multiple batches of gold colloid. Gold nanoparticles (AuNP) S (Stable) and U1 (Unstable, batch 1) are two batches of gold particles (nominal radii of 10 nm) obtained from the manufacturer. (A) Dynamic light scattering measurements of unwashed and washed gold colloid. The particle radii are reported on the primary y-axis and the sum of squares (a measure of the goodness of fit of the light scattering data) are reported on the secondary y-axis. (B) pH, (C) plasmon wavelength and (D) absorbance measurements of unwashed and washed gold colloid before and after conjugation to goat anti-human Fc polyclonal antibodies. The anti-Fc antibodies were buffer exchanged to pH 4.3 (20 mM acetate), and the final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 2 mM, respectively. The washed gold particles were washed once with water. The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments. 177x169mm (300 x 300 DPI)



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

Figure 2. Effect of pH on the stability of gold particles conjugated to anti-Fc antibodies. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by adsorbing goat anti-human Fc antibodies on gold particles at various pH values. Stable gold particles (AuNP: S) were washed once with water and mixed with anti-Fc antibodies, the latter of which were buffer exchanged to pH 4–5.5 (200 mM acetate). The pH values for the antibody-gold conjugates were estimated using pH paper, and the final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 20 mM, respectively. The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments. 150x268mm (300 x 300 DPI)


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Figure 3. Effect of pH on the stability of gold particles conjugated to anti-Fc antibodies. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by absorbing goat anti-human Fc antibodies on gold particles at various pH values. Stable gold particles (AuNP: S) were washed once with water and mixed with anti-Fc antibodies (final concentration of 40 µg/mL), the latter of which were buffer exchanged to pH ~4.3–10 (40 mM buffer). The buffers used were acetate (pH 4.3 and 5.5), phosphate (pH 6.5, 7.4 and 8), and borate (pH 9 and 10). The pH values for the antibody-gold conjugates were estimated using pH paper, and the final buffer concentration (after mixing the gold and antibody) was 20 mM. The antibody-gold conjugates were resuspended in the corresponding buffer used for conjugation (final buffer concentration of 20 mM). The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from two independent experiments. 150x266mm (300 x 300 DPI)



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 ACS Paragon Plus Environment

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Figure 4. Effect of citrate concentration on the stability of antibody-gold conjugates. (A) Plasmon wavelength and (B) absorbance measurements for conjugates prepared by adsorbing goat anti-human Fc antibodies on gold particles at different citrate concentrations (0–10 mM final concentration; pH 4.3). Stable gold particles (AuNP: S) were washed once with water and mixed with anti-Fc antibodies, the latter of which were buffer exchanged to 0–100 mM citrate (10 fold higher than the final citrate concentrations) at a constant concentration of 20 mM acetate (pH 4.3). The final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 2 mM, respectively. The absorbance values were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments. 147x257mm (300 x 300 DPI)



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

Figure 5. Evaluation of the stability of antibody-gold conjugates prepared using gold particles that were synthesized in-house via citrate reduction of chloroauric acid. Gold particles (~12 nm in radius; U2, Unstable batch 2) were synthesized by reducing chloroauric acid with sodium citrate. (A) pH, (B) plasmon wavelength and (C) absorbance measurements of unwashed and washed gold colloid before and after conjugation to goat anti-human Fc polyclonal antibodies. The anti-Fc antibodies were buffer exchanged to pH 4.3 (20 mM acetate), and the final antibody and acetate concentrations (after mixing the gold and antibody) were 40 µg/mL and 2 mM, respectively. The washed gold particles were sedimented once and resuspended with water. The absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. The standard deviations are calculated from three independent experiments. 196x457mm (300 x 300 DPI)


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Figure 6. Effect of polyclonal antibody concentration on the stability of conjugates prepared using gold colloid synthesized via different methods. Plasmon wavelength and absorbance values for antibody-gold conjugates prepared using (A) unwashed stable (S), (B) washed stable (S) and (C) washed unstable (U2) gold particles. Gold particles were incubated overnight with a range of goat anti-human Fc antibody concentrations. Next, the conjugates were mixed with 0.1 µM thiolated PEG (final concentration) for 1 h. The plasmon wavelength and absorbance values of the conjugates were measured prior to sedimentation as well as after sedimentation and resuspension (2 mM acetate, pH 4.3). In (A), the plasmon wavelength of the 2.5 µg/mL sample could not be measured after sedimentation and resuspension due to the low conjugate concentration. The absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the results are averages of three replicates from a representative experiment, and the error bars are the corresponding standard deviations.


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278x435mm (300 x 300 DPI)



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Figure 7. Effect of mAb concentration on plasmon shift and absorbance measurements for anti-Fc antibody conjugates. Washed (stable) gold nanoparticles were conjugated with either goat anti-rabbit Fc or goat antihuman Fc antibody and incubated with purified (A) rabbit or (B) human mAbs at a range of mAb concentrations. The experiments were performed at pH 6 (10 mM sodium citrate) and 0 mM NaCl (rabbit mAbs) or 50 mM NaCl (human mAbs). The concentration of goat anti-Fc antibody during the conjugation reaction was 160 µg/mL and the conjugates were blocked with thiolated PEG. Non-specific goat antibody (100 µg/mL final concentration) was added to minimize non-specific interactions. The averaged plasmon shifts (∆λp) were calculated using controls without human antibody. The average absorbance values were calculated as the difference between the maximum and minimum absorbances in the range of 510 to 610 nm. Two independent experiments were performed, the results are averages of three replicates from a representative experiment, and the error bars are the corresponding standard deviations. 175x173mm (300 x 300 DPI)


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Figure 8. Comparison of plasmon wavelength shifts for mAbs assayed using anti-Fc conjugates that were prepared with different batches of gold colloid. Unwashed and washed stable (S) gold particles as well as washed unstable (U1) particles were conjugated to goat anti-rabbit Fc or goat anti-human Fc antibodies and blocked with thiolated PEG. The conjugates were then incubated with purified (A) rabbit or (B) human mAbs at 5 µg/mL (final mAb concentration). The experiments were performed at pH 6 (10 mM sodium citrate) and 0 mM NaCl (rabbit mAbs) or 50 mM NaCl (human mAbs). The concentration of goat anti-Fc antibody during conjugation was 160 µg/mL. Non-specific goat antibody (100 µg/mL final concentration) was added to minimize non-specific interactions. The averaged plasmon shifts (∆λp) were calculated using controls without human antibody. Three independent experiments are reported (three replicates per experiment) and the error bars are the corresponding standard deviations. 169x341mm (300 x 300 DPI)




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Figure 9. Evaluation of plasmon wavelength shifts and absorbance values for unpurified human mAbs. Washed (stable) gold particles coated with goat anti-human Fc antibody (conjugated at 160 µg/mL, blocked with thiolated PEG) were incubated with unpurified human mAbs at 10 µg/mL (final concentration). The experiments were performed at final conditions of pH 6, 10 mM sodium citrate, 50 mM NaCl and 18% cell culture media. Non-specific goat antibody (100 µg/mL final concentration) was added to minimize nonspecific interactions. In (A), the averaged plasmon shifts (∆λp) were calculated using controls without human antibody. In (B), the absorbances were calculated as the difference between the maximum and minimum absorbance values in the range of 510 to 610 nm. Three independent experiments are reported (three replicates per experiment) and the error bars are the corresponding standard deviations. 173x356mm (300 x 300 DPI)


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