Technical Decision-Making with Higher Order Structure Data: Detecting Reversible Concentration-Dependent Self-Association in a Monoclonal Antibody and a Preliminary Investigation to Eliminate It

Wei, Julie Y.; Bou-Assaf, George M.; Houde, Damian J.; Weiskopf, Andrew

doi:10.1002/jps.24616

Cited by 13 publications

(7 citation statements)

References 23 publications

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“…This agrees with the calculated at these concentrations ( Figure 4C) showing a single major peak at about =50 Å for 1-2 mg/ml, two peaks at 50 Å and 200 Å in 5 mg/ml, and three peaks at 50 Å, 200 Å, and 350 Å in 10 and 20 mg/ml protein solutions. These data also correlate with earlier studies by Wei et al (36).…”

Section: Solution Structures Of Igg Moleculessupporting

confidence: 93%

Utility of Solution X-Ray Scattering for the Development of Antibody Biopharmaceuticals

Inouye

Houde

Temel

et al. 2016

Journal of Pharmaceutical Sciences

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Characterization of immunoglobulin solutions at high concentrations represents a significant challenge. A current trend in the biopharmaceutical industry is to manufacture highly concentrated drug products, which can be used to deliver high doses in small volumes, via subcutaneous injections. Studying a molecule's structure and properties in its final drug product formulation is ideal, but characterization is typically performed under dilute solution conditions with critical stabilizing buffer components removed because of interference effects, which can result in an incomplete understanding of the molecule's properties. Direct study of protein conformation and protein-protein interactions in concentrated solutions is challenging for most biophysical and biochemical techniques; however, X-ray solution scattering offers opportunities. Combined with other biophysical techniques, X-ray scattering has the potential to provide relevant information on both structure and interactions in protein solutions over a broad concentration range. Here, we report X-ray solution scattering of 4 monoclonal antibodies, designated mAb1 (glycosylated and de-glycosylated), mAb2, and mAb3 at concentrations between 0.5 and >168 mg/mL. Data acquired from these measurements are combined with the results from other biophysical measurements to generate a comprehensive profile of their solution behaviors. Our results show that X-ray solution scattering can assess key parameters needed to aid in formulation development.

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Section: Solution Structures Of Igg Moleculessupporting

confidence: 93%

Utility of Solution X-Ray Scattering for the Development of Antibody Biopharmaceuticals

Inouye

Houde

Temel

et al. 2016

Journal of Pharmaceutical Sciences

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“…Reversible self-association of proteins results in changes in sedimentation profiles due to association of the monomers into higher-order species as the concentration is increased. [15][16][17] Similarly, interaction (binding) between two different antibodies is also expected to result in changes in sedimentation profiles as a function of total concentration or ratio of species. To assess interactions in Combo, we compared the AUC sedimentation profiles of mAb-A, Combo (a:b mixtures of mAb-A and mAb-B), and a positive control of intermolecular interactions (a: b mixtures of mAb-A and anti-mAb-A antibody) ( Figure 6(a)).…”

Section: Analytical Ultracentrifugationmentioning

confidence: 99%

“…DLS can detect such protein-protein associations by measuring the hydrodynamic size of the molecules. 16,17 When mAb-A and mAb-B existed as monomers, the theoretical average diameter in Combo was calculated to be 13.4 nm (Table 3). Conversely, if mAb-A and mAb-B associate with each other, the theoretical diameter of the Combo could range from 16.9 nm (minimum) to 26.9 nm (maximum).…”

Section: Dynamic Light Scatteringmentioning

confidence: 99%

Analytical characterization of coformulated antibodies as combination therapy

Kim

Cao

et al. 2020

mAbs

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When two therapeutic agents are combined in a single formulation, i.e., coformulated, the quality and safety of the individual agents must be preserved. Here we describe an approach to evaluate the quality attributes of two individual monoclonal antibodies (mAbs), designated mAb-A and mAb-B, in coformulation. The mAbs were fractionated from heat-stressed coformulated drug product (DP) by hydrophobic interaction chromatography. Each purified mAb fraction was then compared with mAb-A and mAb-B in their individual formulations from the same drug substance sources used to make the coformulated DP lot, which was subjected to the same stress conditions. Product variants were evaluated and compared by using several analytical tests, including high-performance size exclusion chromatography (HPSEC), reducing and nonreducing gel electrophoresis, ion-exchange chromatography, capillary isoelectric focusing, and peptide mapping with mass spectrometry. Intermolecular interactions in coformulated and photostressed DPs were studied by evaluating aggregates fractionated from coformulated DP by HPSEC. Aggregate fractions of coformulated DP contained dimers, but not coaggregates, of mAb-A or mAb-B. Moreover, extensive assays for higher-order structure and biological interactions confirmed that there was no interaction between the two mAb molecules in the coformulation. These results demonstrate that the two coformulated therapeutic mAbs had the same quality attributes as the individually formulated mAb-A and mAb-B, no new quality attributes were formed, and no physicochemical, intermolecular, or biological interactions occurred between the two components. The approach described here can be used to monitor the product quality of other coformulated antibodies. ARTICLE HISTORY

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“…These values are in good agreement with literature results for other mAbs. [13,16,[19][20][21][22] The slopes of the best fit lines in Figure 3…”

Section: Mab Characterizationmentioning

confidence: 99%

The role of intermolecular interactions on monoclonal antibody filtration through virus removal membranes

Billups,

Minervini,

Holstein

et al. 2023

Biotechnology Journal

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The removal of viruses by filtration is a critical unit operation to ensure the overall safety of monoclonal antibody (mAb) products. Many mAbs show very low filtrate flux during virus removal filtration, although there are still significant uncertainties regarding both the mechanisms and antibody properties that determine the filtration behavior. Experiments were performed with three highly purified mAbs through three different commercial virus filters (Viresolve Pro, Viresolve NFP, and Pegasus SV4) with different pore structures and chemistries. The flux decline observed during mAb filtration was largely reversible, even under conditions where the filtrate flux with the mAb was more than 100‐fold smaller than the corresponding buffer flux. The extent of flux decline was highly correlated with the hydrodynamic diameter of the mAb as determined by dynamic light scattering (DLS). The mAb with the lowest filtrate flux for all three membranes showed the largest attractive intermolecular interactions and the greatest hydrophobicity, with the latter determined by binding to a butyl resin in an analytical hydrophobic interaction chromatography (HIC) column. These results strongly suggest that the flux behavior is dominated by reversible self‐association of the mAbs, providing important insights into the design of more effective virus filtration processes and in the early identification of problematic mAbs/solution conditions.

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Technical Decision-Making with Higher Order Structure Data: Detecting Reversible Concentration-Dependent Self-Association in a Monoclonal Antibody and a Preliminary Investigation to Eliminate It

Cited by 13 publications

References 23 publications

Utility of Solution X-Ray Scattering for the Development of Antibody Biopharmaceuticals

Utility of Solution X-Ray Scattering for the Development of Antibody Biopharmaceuticals

Analytical characterization of coformulated antibodies as combination therapy

The role of intermolecular interactions on monoclonal antibody filtration through virus removal membranes

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