Use of Water Proton NMR to Characterize Protein Aggregates: Gauging the Response and Sensitivity

Abstract

Water proton transverse relaxation rate R 2(1H2O) measurements by NMR stand out as a powerful noninvasive tool to detect protein aggregates, including subvisible particles in biopharmaceutical formulations. To understand the applicability of water proton NMR (wNMR), we studied the response and sensitivity of wNMR to the aggregates of a monoclonal antibody (mAb) within a wide size range at different aggregate levels, for three different physical stresses: freeze–thaw cycling, heating, and agitation. We compared the sensitivity and response of wNMR with those observed by conventional techniques of size exclusion chromatography (SEC), microflow imaging (MFI), and dynamic light scattering (DLS). Our findings showed that wNMR detects mAb aggregates within wide aggregate levels and in a wide range of aggregate sizes. wNMR was sensitive to an increase in soluble protein aggregates in the range of <1.0%. In most cases, wNMR demonstrated linear response toward the aggregate fraction. Nonlinearity of such response potentially points to the presence of larger size aggregates that possibly rearrange and/or dissociate upon dilution. The results demonstrate the potential of wNMR as a quantitative and noninvasive analytical tool for characterizing protein aggregates in biopharmaceutical formulations.

Re-Envisioning Pharmaceutical Manufacturing: Increasing Agility for Global Patient Access

“…Recent publications have highlighted the use of benchtop NMR analysis as a nondestructive tool to analyze both solid and liquid drug products. Benchtop NMR, which has a small footprint and is nondestructive, has been used to analyze contents of tablets, [78] aggregation in monoclonal antibody liquid product [79,80], and adjuvant fill levels for vaccines [81]. These systems have the potential to aid in biophysical characterization, lot release testing, and stability assessment of the drug product at the fill finish facility.…”

Magnetic Resonance Relaxometry for Determination of Protein Concentration and Aggregation

“…In the case of rather large aggregates, R 2 ( 1 H 2 O) might respond to their formation even at relatively low total protein concentrations, e.g., <0.5 mg/ml (Taraban et al, ). In general, R 2 ( 1 H 2 O) is most reliably sensitive to protein aggregate content >1% (Taraban, DePaz, et al., ).…”

“…One way is to vary the level of the stress used to induce aggregation: for example, heating a protein solution to 60°C for 15 min, 30 min, 60 min, and so on. The other is to fix the stress level (e.g., heating to 60°C for 60 min) and then mix the stressed stock solution with different fractions of the unstressed solution of the same protein concentration (Taraban, DePaz, et al., ). The content of protein aggregates in calibration solutions should be measured by SEC.…”

“…In the case of protein aggregation, longer rotational correlation times ( τ c ) of the aggregates compared to those of the monomeric protein accelerate the relaxation of the water protons involved in proton exchange with hydrophilic/exchangeable protons of the aggregates (Taraban, Deredge, et al., ). For very large aggregates containing water cavities/compartments, other mechanisms that synergistically affect the increase in R 2 ( 1 H 2 O) might also come into play (Yu, Feng, & Taraban, ; Taraban, DePaz, Lobo, & Yu, ). Of note, conventionally used analytical techniques for protein aggregate detection have known size limitations, and some of these techniques are not applicable in certain cases, such as in the case of insoluble aggregates.…”

NMR Relaxation of Nuclei of Buffer as a Probe for Monitoring Protein Solutions Including Aggregation Processes

“…Taraban et al have shown that thermally and mechanically produced aggregation of recombinant antibodies could be quantified using spin-spin relaxation times T 2 of water protons [16,21]. The earlier works examined a two-site model that explains the significant increase of water proton relaxation rates, 1/T 2 , due to the exchange with protein protons [14].…”