Probing interactions of therapeutic antibodies with serum via second virial coefficient measurements

Larsen, Hayli A.; Atkins, William M.; Nath, Abhinav

doi:10.1016/j.bpj.2021.08.007

Cited by 3 publications

(10 citation statements)

References 47 publications

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“…36 A linear fit to the data provides D 0 = (64 ± 4) μm 2 /s and k D = (4 ± 1) × 10 −3 mL/mg, which is fully consistent with previously reported values for BSA under similar experimental conditions. 37 To enable a direct comparison with the output of the DLS measurements, we calculated for each sample the corresponding hydrodynamic radius using the Stokes−Einstein relation (eq 2). The results are shown in Figure 3c.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Protein Sizing with Differential Dynamic Microscopy

Guidolin,

Heim,

Adams

et al. 2023

Macromolecules

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Introduced more than 50 years ago, dynamic light scattering (DLS) is routinely used to determine the size distribution of colloidal suspensions as well as of macromolecules in solution, such as proteins, nucleic acids, and their complexes. More recently, differential dynamic microscopy (DDM) has been proposed as a way to perform DLS experiments with a microscope, with much less stringent constraints in terms of cleanliness of the optical surfaces but a potentially lower sensitivity due to the use of camera-based detectors. In this work, we push bright-field DDM beyond known limits and show it to be sufficiently sensitive to size small macromolecules in diluted solutions. By considering solutions of three different proteins (bovine serum albumin, lysozyme, and pepsin), we accurately determine the diffusion coefficient and hydrodynamic radius of both single proteins and small protein aggregates down to concentrations of a few milligrams per milliliter. In addition, we present preliminary results showing an unexplored potential for the determination of virial coefficients. Our results are in excellent agreement with those obtained in parallel with a state-of-the-art commercial DLS setup, showing that DDM represents a valuable alternative for rapid, label-free protein sizing with an optical microscope.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Protein Sizing with Differential Dynamic Microscopy

Guidolin,

Heim,

Adams

et al. 2023

Macromolecules

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“…Although the contribution of serum-induced nonideality on mAb efficacy or disposition has not yet been directly investigated, we have observed a case suggesting a potential correlation between net attraction in serum and lower in vivo binding affinity. In our previous study, Carlumab, a discontinued antibody from Janssen Therapeutics, exhibited more attraction (k diff = À11 mL/g) to fetal bovine serum than clinical (tocilizumab) or reference standard (NIST mAb) antibodies (Larsen et al, 2021). Interestingly, Carlumab's failure during clinical trials was attributed to a discrepancy in the in vitro and in vivo binding affinity for its target, CCL2, that resulted in limited therapeutic efficacy (Majety et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Translational diffusion coefficients ( D ) were determined from diffusion times and used to calculate interaction parameters ( k diff ), that are directly proportional to the second osmotic virial coefficient ( B 2 ). The methodology and validation behind these measurements are outlined in our previous publication (Larsen et al, 2021).…”

Section: Methodsmentioning

confidence: 99%

“…Given the long circulating half‐lives of therapeutic antibodies (typically several weeks), blood serum is the biological environment to which they are exposed the longest. For this reason, there is growing interest in understanding how therapeutic antibodies interact with serum proteins, and how serum impacts target or receptor binding (Chaturvedi et al, 2020; Demeule et al, 2009; Kim et al, 2019; Larsen et al, 2021; Wright, Hayes, Stafford, et al, 2018). Human serum is a concentrated, complex fluid whose composition varies with physiological or disease state, from one individual to another; in general, serum contains approximately 60–80 mg/mL protein, comprised of mainly albumin (60%) and immunoglobulin G, IgG (20%) with lower levels of other globulins, lipoproteins, transport proteins, complement factors, and smaller osmolytes (Anderson et al, 2004; Gonzalez‐Quintela et al, 2008; Leeman et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…This work builds on a previously developed and validated in‐serum FCS approach to probe nonideality effects through apparent second virial coefficient ( B 2,app ) measurements (Larsen et al, 2021). Traditional self‐term ( B 22 ) and cross‐term ( B 12 ) second virial coefficients describe weak, nonspecific, pairwise interactions between two of the same or different molecules, respectively.…”

Section: Introductionmentioning

confidence: 99%

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The origins of nonideality exhibited by monoclonal antibodies and Fab fragments in human serum

Larsen,

Atkins,

Nath

2023

Protein Science

Self Cite

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The development of therapeutic antibodies remains challenging, time consuming and expensive. A key contributing factor is a lack of understanding of how proteins are affected by complex biological environments such as serum and plasma. Nonideality due to attractive or repulsive interactions with co‐solutes can alter the stability, aggregation propensity, and binding interactions of proteins in solution. Fluorescence correlation spectroscopy (FCS) can be used to measure apparent second virial coefficient (B2,app) values for therapeutic and model monoclonal antibodies (mAbs) that capture the nature and strength of interactions with co‐solutes in directly in undiluted serum and similar complex biological media. Here, we use FCS‐derived B2,app measurements to identify the components of human serum responsible for non‐ideal interactions with mAbs and Fab fragments. Most mAbs exhibit neutral or slightly attractive interactions with intact serum. Generally, mAbs display repulsive interactions with albumin and mildly attractive interactions with IgGs in the context of whole serum. Crucially, however, these attractive interactions are much stronger with pooled IgGs isolated from other serum components, indicating that the effects of serum nonideality can only be understood by studying the intact medium (rather than isolated components). Moreover, Fab fragments universally exhibited more attractive interactions than their parental mAbs, potentially rendering them more susceptible to nonideality‐driven perturbations. FCS‐based B2,app measurements have the potential to advance our understanding of how physiological environments impact protein‐based therapeutics in general. Furthermore, incorporating such assays into preclinical biologics development may help de‐risk molecules and make for a faster and more efficient development process.This article is protected by copyright. All rights reserved.

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