Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment

Chakroun, Nesrine; Hilton, David; Ahmad, Shahina S.; Platt, Geoffrey W.; Dalby, Paul A.

doi:10.1021/acs.molpharmaceut.5b00387

Cited by 55 publications

(110 citation statements)

References 64 publications

(119 reference statements)

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“…After the expression of designed variants, their conformational stability was assessed through different thermal stability measures. We selected a formulation condition of 1 mg/ml Fab in 20 mM sodium citrate, pH 4, at an ionic strength of 200 mM, as this partially unfolded the wild-type protein at 65 °C by 6% 36 , and to led to aggregation on a practical timescale. These conditions were used for both thermal stability measurement and aggregation kinetics.…”

Section: Thermal Stability Measurement For Tm Ton and Fraction Of Unmentioning

confidence: 99%

“…Previously, the A33 Fab was studied across a wide range of pH, ionic strength and temperature 36 . It was found that the aggregation rates could only be correlated well with thermal transition temperatures (Tm) at an elevated incubation temperature of 65 °C, while the correlations dropped substantially at 4-45 °C, consistent with previous observations on IgG variants 37 .…”

Section: Introductionmentioning

confidence: 99%

“…Of all designs, twelve stable variants and five unstable ones could be expressed sufficiently for study along with the wild type. The conformational stabilities and aggregation rates of the eighteen Fab variants were compared at a condition (pH 4, 200 mM ionic strength, 65 °C), where f65 < 0.05 for wild-type, and that was therefore, close to the limits of any correlation between aggregation rate and Tm or f65 36 . Therefore, even if mutations had increased Tm and decreased f65, they would not be expected to decrease the rate of aggregation significantly through a global unfolding mechanism.…”

Section: Introductionmentioning

confidence: 99%

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Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Zhang

Samad

et al. 2018

Mol. Pharmaceutics

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Computationally guided semirational design has significant potential for improving the aggregation kinetics of protein biopharmaceuticals. While improvement in the global conformational stability can stabilize proteins to aggregation under some conditions, previous studies suggest that such an approach is limited, because thermal transition temperatures ( T) and the fraction of protein unfolded ( f) tend to only correlate with aggregation kinetics where the protein is incubated at temperatures approaching the T. This is because under these conditions, aggregation from globally unfolded protein becomes dominant. However, under native conditions, the aggregation kinetics are presumed to be dependent on local structural fluctuations or partial unfolding of the native state, which reveal regions of high propensity to form protein-protein interactions that lead to aggregation. In this work, we have targeted the design of stabilizing mutations to regions of the A33 Fab surface structure, which were predicted to be more flexible. This Fab already has high global stability, and global unfolding is not the main cause of aggregation under most conditions. Therefore, the aim was to reduce the conformational flexibility and entropy of the native protein at various locations and thus identify which of those regions has the greatest influence on the aggregation kinetics. Highly dynamic regions of structure were identified through both molecular dynamics simulation and B-factor analysis of related X-ray crystal structures. The most flexible residues were mutated into more stable variants, as predicted by Rosetta, which evaluates the ΔΔ G for each potential point mutation. Additional destabilizing variants were prepared as controls to evaluate the prediction accuracy and also to assess the general influence of conformational stability on aggregation kinetics. The thermal conformational stability, and aggregation rates of 18 variants at 65 °C, were each examined at pH 4, 200 mM ionic strength, under which conditions the initial wild-type protein was <5% unfolded. Variants with decreased T values led to more rapid aggregation due to an increase in the fraction of protein unfolded under the conditions studied. As expected, no significant improvements were observed in the global conformational stability as measured by T. However, 6 of the 12 stable variants led to an increase in the cooperativity of unfolding, consistent with lower conformational flexibility and entropy in the native ensemble. Three of these had 5-11% lower aggregation rates, and their structural clustering indicated that the local dynamics of the C-terminus of the heavy chain had a role in influencing the aggregation rate.

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Section: Thermal Stability Measurement For Tm Ton and Fraction Of Unmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Zhang

Samad

et al. 2018

Mol. Pharmaceutics

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“…Differences in accelerated and innate aggregation propensities (Goldberg et al, 2017) may arise because the acceleration method increases the relative flux through certain pathways which are distinct to those traversed during production or upon storage (Chakroun, Hilton, Ahmad, Platt, & Dalby, 2016; Luo et al, 2011; Phillips et al, 2017; van der Kant et al, 2017). There is thus a need to develop stress tests that more closely replicate the conformational ensemble generated during processing and transport.…”

Section: Introductionmentioning

confidence: 99%

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Willis

Kumar

Dobson

et al. 2018

Biotech & Bioengineering

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Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis‐folding, and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation, and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g., shear and extensional flow) to trigger aggregation has remained unclear. To address this question, we previously developed a device that allows the degree of extensional flow to be controlled. Using this device we demonstrated that mAbs are particularly sensitive to the force exerted as a result of this flow‐field. Here, to investigate the utility of this device to bio‐process/biopharmaceutical development, we quantify the effects of the flow field and protein concentration on the aggregation of three mAbs. We show that the response surface of mAbs is distinct from that of bovine serum albumin (BSA) and also that mAbs of similar sequence display diverse sensitivity to hydrodynamic flow. Finally, we show that flow‐induced aggregation of each mAb is ameliorated by different buffers, opening up the possibility of using the device as a formulation tool. Perturbation of the native state by extensional flow may thus allow identification of aggregation‐resistant mAb candidates, their bio‐process parameters and formulation to be optimized earlier in the drug‐discovery pipeline using sub‐milligram quantities of material.

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“…Native protein conformations are only marginally stable, and are highly dynamic, hence they are more realistically described as a native ensemble. There is increasing evidence to suggest that under native conditions, aggregation takes place primarily from partially unfolded native-like states [8–12]. However, little is known about the structures of native conformers that initiate aggregation, or how these are affected by different stress conditions.…”

Section: Introductionmentioning

confidence: 99%

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Codina

Zhang

Chakroun

et al. 2019

Preprint

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23Successful development of protein therapeutics depends critically on achieving stability 24 under a range of conditions, while retaining their specific mode of action. Gaining a deeper 25 understanding of the drivers of instability across different stress conditions, will potentially enable 26 the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we 27 compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic 28 molecular dynamics simulations under two different stresses of low pH and high temperature.29 RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding 30 was initiated through loss of contacts at the constant domain interface (C L -C H 1), prior to C L domain 31 unfolding. By contrast, thermal unfolding began with loss of contacts in both the C L -C H 1 and 32 variable domain interface (V L -V H ), followed by domain unfolding of C L and also of V H , thus 33 revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the C L -34 C H 1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, 35 packing density calculations found these residues to be under-packed relative to other inter-36 domain residues. Two salt bridges were identified that possibly drive the conformational change 37 at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always 38 with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis 39 of existing Fab sequences revealed considerable scope for further engineering, where certain 40 natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the 41 two stress conditions exposed different predicted aggregation-prone regions (APR), which would 42 potentially lead to different aggregation mechanisms. Overall, our results identified the early 43 stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for 44 future protein engineering work aimed at stabilizing to both thermal and pH-stresses 45 simultaneously. 46 3 47 Author Summary 48 49Currently, antibody-based products are the most rapidly growing class of pharmaceuticals 50 due to their high specificity towards their targets, such as biomarkers on the surface of cancer 51 cells. However, they tend to aggregate at all stages of product development, which leads to 52 decreased efficiency and could elicit an immunological response. Improvements in the stability of 53 therapeutic antibodies are generally made during the development phase, by trial and error of the 54 composition of the formulated product, which is both costly and time consuming. There is great 55 demand and potential for identifying the drivers of instability across different stress conditions, 56 early in the discovery phase, which will enable the rational engineering of protein scaffolds. This 57 work elucidated the stability-limiting regions of the ant...

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Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment

Cited by 55 publications

References 64 publications

Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

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