Molecular modeling of Protein A affinity chromatography

Salvalaglio, Matteo; Zamolo, Laura; Busini, Valentina; Moscatelli, Davide; Cavallotti, Carlo

doi:10.1016/j.chroma.2009.04.035

Cited by 43 publications

(50 citation statements)

References 50 publications

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“…The sterical hindrance induced by the spacer structural complexity reduces the probability of having a ligand preferentially involved in interactions with the support even if characterized by a poor polar/apolar SAA ratio. This feature is evident for the polyLys spacer and confirms the finding of previous modeling studies regarding Protein A, which highlight how the secondary structure of the natural ligand is a determining factor in decoupling ligand-receptor and ligand-support interactions [34].…”

Section: Discussionsupporting

confidence: 86%

“…In the last years, we have placed a considerable effort in the study of the interaction between affinity materials and MABs using molecular dynamics (MD) simulations [15,[31][32][33][34][35]. In particular, we have investigated the interaction between the FC fragment of human IgG, used as a molecular MAB model, and the PAM and A2P affinity ligands, both dispersed in solution and supported on agarose [32,34]. The modeling of the PAM ligand relied on the availability of crystallographic data [15].…”

Section: Introductionmentioning

confidence: 99%

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Molecular modeling to rationalize ligand–support interactions in affinity chromatography

Salvalaglio¹,

Cavallotti²

2011

J of Separation Science

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The development of rational design criteria for synthetic-ligand-based affinity chromatography requires a basic comprehension of all the factors influencing the binding capacity and selectivity of the stationary phase. In this work, molecular dynamics simulations are systematically used to investigate the impact of structural modifications of spacer and ligand on ligand-support interactions. The investigated ligands are characterized by a triazine core bi-functionalized with two amino acid side chains aimed at representing a range of hydrophobic/hydrophilic characters. As spacers both literature (1-2-diaminoethane and 1,4-substituted [1,2,3]-triazole) and speculative oligopeptidic molecules (Gly-[Ala]4-Gly, Gly-[Lys]4-Gly, and Gly-[Glu]4-Gly) have been considered to address the role of charges distribution, rigidity, and structural complexity. In this investigation, the spacer emerged as a key component: on the one hand, the choice of a proper spacer allows improving the hydrophilic character of the ligand-spacer adduct without compromising the structure of the affinity ligand, while on the other hand the use of structurally complex spacers induces spacer-support interactions that enhance the degree of solvation of the ligand regardless of its hydrophobic character. These findings suggest that the use of structured spacers could represent a viable pathway for tailoring the performances of affinity chromatography stationary phases.

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Section: Discussionsupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Molecular modeling to rationalize ligand–support interactions in affinity chromatography

Salvalaglio¹,

Cavallotti²

2011

J of Separation Science

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“…For the binding of biotin analogous to avidin, MM/PBSA and MM/ GBSA outperformed LIE [20,22], whereas for acetylcholinesterase huprine inhibitors, LIE gave better results than MM/PBSA [23]. For the binding of eight hydroxamate inhibitors to gelatinase-A and the binding of fragment B of protein A to the Fc domain of immunoglobulin G, the two methods showed a similar performance [24,25]. Finally, for the binding of primary alcohols to cyclodextrin, LIE was more accurate than MM/GBSA and MM/PBSA, whereas for the binding of guests to cucurbitil [8] uril, LIE was less accurate than MM/GBSA and MM/PBSA [21].…”

Section: Introductionmentioning

confidence: 88%

Binding affinities in the SAMPL3 trypsin and host–guest blind tests estimated with the MM/PBSA and LIE methods

Mikulskis

Genheden

Rydberg

et al. 2011

J Comput Aided Mol Des

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We have estimated affinities for the binding of 34 ligands to trypsin and nine guest molecules to three different hosts in the SAMPL3 blind challenge, using the MM/PBSA, MM/GBSA, LIE, continuum LIE, and Glide score methods. For the trypsin challenge, none of the methods were able to accurately predict the experimental results. For the MM/GB(PB)SA and LIE methods, the rankings were essentially random and the mean absolute deviations were much worse than a null hypothesis giving the same affinity to all ligand. Glide scoring gave a Kendall's τ index better than random, but the ranking is still only mediocre, τ = 0.2. However, the range of affinities is small and most of the pairs of ligands have an experimental affinity difference that is not statistically significant. Removing those pairs improves the ranking metric to 0.4-1.0 for all methods except CLIE. Half of the trypsin ligands were non-binders according to the binding assay. The LIE methods could not separate the inactive ligands from the active ones better than a random guess, whereas MM/GBSA and MM/PBSA were slightly better than random (area under the receiver-operating-characteristic curve, AUC = 0.65-0.68), and Glide scoring was even better (AUC = 0.79). For the first host, MM/GBSA and MM/PBSA reproduce the experimental ranking fairly good, with τ = 0.6 and 0.5, respectively, whereas the Glide scoring was considerably worse, with a τ = 0.4, highlighting that the success of the methods is system-dependent.

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“…Several residues important in the interaction between PAB and IgG1-Fc have been previously identified by mutational experiments [7,11,21-24] and by molecular simulation calculations [25,26]. However, we did not take these positions into particular consideration for our library design in order to test a wide range of mutation positions in PAB.…”

Section: Resultsmentioning

confidence: 99%

Engineered protein A ligands, derived from a histidine-scanning library, facilitate the affinity purification of IgG under mild acidic conditions

et al. 2014

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BackgroundIn antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Herein we describe the development of several types of affinity ligands which enable the purification of antibodies under much milder conditions.ResultsStaphylococcal protein A variants were engineered by using both structure-based design and combinatorial screening methods. The frequency of amino acid residue substitutions was statistically analyzed using the sequences isolated from a histidine-scanning library screening. The positions where the frequency of occurrence of a histidine residue was more than 70% were thought to be effective histidine-mutation sites. Consequently, we identified PAB variants with a D36H mutation whose binding of IgG was highly sensitive to pH change.ConclusionThe affinity column elution chromatograms demonstrated that antibodies could be eluted at a higher pH (∆pH**≧2.0) than ever reported (∆pH = 1.4) when the Staphylococcal protein A variants developed in this study were used as affinity ligands. The interactions between Staphylococcal protein A and IgG-Fab were shown to be important for the behavior of IgG bound on a SpA affinity column, and alterations in the affinity of the ligands for IgG-Fab clearly affected the conditions for eluting the bound IgG. Thus, a histidine-scanning library combined with a structure-based design was shown to be effective in engineering novel pH-sensitive proteins.

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Molecular modeling of Protein A affinity chromatography

Cited by 43 publications

References 50 publications

Molecular modeling to rationalize ligand–support interactions in affinity chromatography

Molecular modeling to rationalize ligand–support interactions in affinity chromatography

Binding affinities in the SAMPL3 trypsin and host–guest blind tests estimated with the MM/PBSA and LIE methods

Engineered protein A ligands, derived from a histidine-scanning library, facilitate the affinity purification of IgG under mild acidic conditions

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