Kinetics of Low pH Induced Aggregation of Equine IgG

Lewis, Jeffrey D.; Ju, Robert T.C.; Kim, Alexandra I.; Nail, Steven L.

doi:10.1006/jcis.1997.5187

Cited by 10 publications

(5 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Associationlimited aggregation is characterized by relatively slow assembly steps leading to aggregation (5). In the case of equine IgG, aggregation at pH 3.4 was found to be diffusion limited and strongly dependent on the ionic strength (14). Apparently, the electrostatic repulsion at pH 3.4 was essentially negated already at moderate ionic strength (m = 0.27 M).…”

Section: Aggregation Kinetics Of the Fc And Fab Fragments Ofmentioning

confidence: 99%

“…It is interesting to note that some studies reveal significant antibody aggregation at low pH (14), whereas others fail to observe any measurable aggregation under fairly similar conditions (7). The specific role of the Fc and Fab regions in the low pH aggregation process also has not been fully addressed.…”

mentioning

confidence: 99%

“…Although the effect of acidic conditions on antibody structure, stability, and folding has been thoroughly investigated ,− , there is little information regarding the aggregation of different subclasses of mAbs (IgG1, IgG2, etc.). It is interesting to note that some studies reveal significant antibody aggregation at low pH , whereas others fail to observe any measurable aggregation under fairly similar conditions . The specific role of the Fc and Fab regions in the low pH aggregation process also has not been fully addressed.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Acid-Induced Aggregation of Human Monoclonal IgG1 and IgG2: Molecular Mechanism and the Effect of Solution Composition

et al. 2010

View full text Add to dashboard Cite

The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding C(H)2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated C(H)2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of C(H)2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics.

show abstract

Section: Aggregation Kinetics Of the Fc And Fab Fragments Ofmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Acid-Induced Aggregation of Human Monoclonal IgG1 and IgG2: Molecular Mechanism and the Effect of Solution Composition

et al. 2010

View full text Add to dashboard Cite

show abstract

“…36,37 Antibodies may be exposed to pH as low as 3.5 for a few hours at ambient temperature, which can affect stability of the protein structure and induce aggregation. 36,38 In particular, the C H 2 domains of Fc are likely to be sufficiently destabilized or unfolded to initiate the aggregation process. 11,24 Aggregation of IgG1-A, IgG2-A, and IgG2-B was measured in 100 mM sodium acetate, pH 3.5.…”

Section: Acid-induced Mab Aggregationmentioning

confidence: 99%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

View full text Add to dashboard Cite

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

show abstract

“…There exists a favorable kinetics to drive the equilibrium between monomers and oligomers toward the right direction, when pH of the mAb04 loading solution is decreased. First, it is well-known that the more acidic condition speeds up reversible homogeneous nucleation of mAb molecules to form soluble oligomers mainly consisting of dimers and trimers in the protein solution, − although the increase of their amount due to only one pH unit decrease is not enough to be detected by Raman spectroscopy (Figure ). The association of protein molecules is the result of mAb destabilization due to unfolding of the CH 2 domain of the Fc part or alternatively due to unfolding of the Fab and CH 3 domains .…”

Section: Discussionmentioning

confidence: 99%

Confocal Raman Characterization of Different Protein Desorption Behaviors from Chromatographic Particles

Xiao¹,

Stone²,

Moya³

et al. 2014

Anal. Chem.

View full text Add to dashboard Cite

Confocal Raman spectroscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure protein desorption from chromatographic particles. Monoclonal antibody was loaded onto the Fractogel EMD SO3 (M) cation exchanger at either pH 5 or pH 4. Confocal Raman measurement suggests that only the protein loaded at pH 5 is able to release from chromatographic particles in the elution buffer. Detailed comparison of high-quality spectra indicates that, while proteins loaded at both pH values showed a predominant β-sheet conformation, protein loaded at pH 4 has a broader amide I band with more intensity in the >1680 cm(-1) region. This small but clear and reproducible amide I bandwidth increase is not observed for protein in the solution state at pH 4. No definitive assignment of the increased Raman intensity in the >1680 cm(-1) region could be made, but it might be related to structural changes involved in the association of protein molecules in the adsorbed state, which helps to explain the nearly 100% retention under elution conditions of the monoclonal antibody adsorbed at pH 4 in chromatographic particles.

show abstract

Kinetics of Low pH Induced Aggregation of Equine IgG

Cited by 10 publications

References 15 publications

Acid-Induced Aggregation of Human Monoclonal IgG1 and IgG2: Molecular Mechanism and the Effect of Solution Composition

Acid-Induced Aggregation of Human Monoclonal IgG1 and IgG2: Molecular Mechanism and the Effect of Solution Composition

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

Confocal Raman Characterization of Different Protein Desorption Behaviors from Chromatographic Particles

Contact Info

Product

Resources

About