Monoclonal antibody interactions with micro- and nanoparticles: adsorption, aggregation, and accelerated stress studies

Abstract

Therapeutic proteins are exposed to various wetted surfaces that could shed subvisible particles. In this work we measured the adsorption of a monoclonal antibody (mAb) to various microparticles, characterized the adsorbed mAb secondary structure, and determined the reversibility of adsorption. We also developed and used a front-face fluorescence quenching method to determine that the mAb tertiary structure was near-native when adsorbed to glass, cellulose, and silica. Initial adsorption to each of the materials tested was rapid. During incubation studies, exposure to the air-water interface was a significant cause of aggregation but acted independently of the effects of microparticles. Incubations with glass, cellulose, stainless steel, or Fe(2)O(3) microparticles gave very different results. Cellulose preferentially adsorbed aggregates from solution. Glass and Fe(2)O(3) adsorbed the mAb but did not cause aggregation. Adsorption to stainless steel microparticles was irreversible, and caused appearance of soluble aggregates upon incubation. The secondary structure of mAb adsorbed to glass and cellulose was near-native. We suggest that the protocol described in this work could be a useful preformulation stress screening tool to determine the sensitivity of a therapeutic protein to exposure to common surfaces encountered during processing and storage.

Figure 1

SEM images of microparticles and nanoparticles used in mAb adsorption studies. The images were all collected at 5000× magnification. The scale bars are 5 μm.

Figure 2

Adsorption of mAb to particles (numerical footprint values listed in Table 1) Panel A: glass vials (); glass syringes (); sulfate-washed glass vials (△). Panel B: stainless steel microparticles (■); stainless steel microparticles after passivation treatment (□); Fe₂O₃ microparticles (). Panel C: silica microparticles (); silica nanoparticles (◇); alumina microparticles (); alumina nanoparticles (). Panel D: cellulose microparticles (); titania microparticles (). We also collected an additional data point, where more microparticles than necessary to adsorb 100% of the mAb were added, to confirm that excess microparticles would remove all of the protein from solution (this data point was not used in the footprint calculation). Data points are means ± SD for separate triplicate samples and lines are regressions of the average data. Error bars may be obscured by symbols. The color version of this figure can be accessed in the online article.

Figure 3

Decrease in mAb monomer percentage of all soluble species (non-adsorbed mAb) vs. amount of added stainless steel after a 30 minute incubation with 0.1 mg/mL mAb. Legend: stainless steel (■); stainless steel after passivation treatment (□). Data points are means ± SD for separate triplicate samples. Error bars may be obscured by symbols.

Figure 4

Zoomed-in view of SEC chromatograms showing the increase in soluble aggregates at ~ 6.3 mL elution volume as a function stainless steel added after a 30 minute incubation with 0.1 mg/mL mAb. The y-axis scale of UV counts was normalized to the control protein monomer peak height. The arrows show the trend in peak height/area as the concentration of stainless steel was increased. This mAb initially contained ~ 2% dimer. The elution of mAb monomer was at ~ 8.7 mL, dimer/soluble aggregates at ~ 7.7 mL and soluble aggregates at ~ 6.3 mL. The baselines were flat from 0 to 5 mL elution volume. Legend: control, black line; 100 mg/mL steel, violet line; 200 mg/mL steel, blue line; 300 mg/mL steel, green line; 400 mg/mL steel, gold line; 500 mg/mL steel, red line. The color version of this figure can be accessed in the online article.

Figure 5

Zoomed-in view of SEC chromatograms showing the decrease in soluble aggregates when an initially aggregated sample was incubated with cellulose. The y-axis scale of UV counts was normalized to the initial mAb monomer peak height. The arrow shows the trend in peak height/area. Legend: aggregated mAb without added cellulose, solid red line; aggregated mAb after 30 min incubation with cellulose, short-dashed green line; aggregated mAb after 24 hr incubation with cellulose, long-dashed blue line. The color version of this figure can be accessed in the online article.

Figure 6

Incubation of mAb with microparticles in overfilled vials without headspace (method A). Panels A, B and C show mAb monomer as a function of time. Panel D shows soluble aggregates from incubations of mAb with stainless steel. Common to all panels: un-agitated control (μ); agitated control (ν). Panel A: mAb agitated with glass vials (); mAb agitated with glass syringes (); mAb agitated with sulfate-washed glass vials (). Panel B: mAb agitated with cellulose (). Panel C: mAb agitated with stainless steel (■); mAb agitated with stainless steel after passivation treatment (□), mAb agitated with Fe₂O₃ (). Panel D: soluble aggregates after agitation with stainless steel (■); soluble aggregates after agitation with stainless steel after passivation treatment (□). Data points are means ± SD for separate triplicate samples. Error bars may be obscured by symbols. Some symbols may overlay. The color version of this figure can be accessed in the online article.

Figure 7

Incubation of mAb with silica, alumina, and titania. Incubations were performed in vials with headspace (method B). Additional incubations were performed with silica in vials overfilled to minimize the air-water interface (method A). Common symbols in each panel: un-agitated control (μ); agitated control with headspace (◆). Panel A: agitated control without headspace (ν); mAb agitated with silica microparticles with headspace (); mAb agitated with silica microparticles without headspace (○). Panel B: mAb agitated with alumina microparticles with headspace (); Panel C: mAb agitated with titania microparticles with headspace (). Data points are means ± SD for separate triplicate samples. Error bars may be obscured by symbols. The color version of this figure can be accessed in the online article.

Figure 8

Second-derivative transmission infrared spectra of mAb adsorbed to particles. Representative spectra from three replicates are shown. Legend: reference native mAb at 23 mg/mL, solid black line; boiled aggregated mAb, gray dot-dash line; mAb adsorbed to glass vials, green long-dash line; mAb adsorbed to cellulose, red medium-dash line. The color version of this figure can be accessed in the online article.

Figure 9

Stern-Volmer plots for the acrylamide quenching of mAb when free in solution and when bound to microparticles. Legend: native control mAb (◇), with solid line; mAb unfolded in 9 M urea (□), with short dash line; mAb adsorbed to ground glass vials (); mAb adsorbed to ground glass syringes (); mAb adsorbed to cellulose (); mAb adsorbed to silica (). Data points are mean ± SD for 3 separate experiments. Error bars may be obscured by data points, and some data symbols overlay. The color version of this figure can be accessed in the online article.