Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Hartmann, Wanda K.; Saptharishi, Nirmala; Yang, Xiao Yi; Mitra, Gautam; Soman, Gopalan

doi:10.1016/j.ab.2003.10.031

Cited by 76 publications

(56 citation statements)

References 45 publications

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“…Despite being completely different techniques, the difference in the decay rate constants are within 5% of each other and in good agreement with those reported previously [211,214] for the temperature range, 55 o C to 70 o C. …”

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confidence: 91%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

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confidence: 91%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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“…After the molecular weight of 150 kDa [46] and a Stokes radius of 4.2 nm [46] were taken into account, q e for g-globulin was 297 mg/mL. As discussed above, the grafting of dextran led to a multilayer adsorption of proteins in dextran-grafted IEC adsorbents, implying that protein adsorption in dextran-grafted IEC adsorbent was greatly limited by the volume of dextran layer rather than surface area.…”

Section: Adsorption Isothermsmentioning

confidence: 99%

“…For a typical IEC adsorbent, the charged ligands were tightly stacked to provide excessive sites for adsorption, and therefore, surface area or pore volume was considered as the limiting factor for protein adsorption [15]. Based on the limitation of surface area, the monolayer coverage for g-globulin with a Stokes radius of 4.2 nm [46] on SP-SEP was about 4.7 mg/m 2 [47] using a phase ratio of $30 m 2 /mL estimated from Q Sepharose FF [45]. As the jamming effect of protein adsorption was taken into account [15,23], the calculated adsorption capacities of SP-SEP for g-globulin were roughly 77.1-127.9 mg/mL.…”

Section: Adsorption Isothermsmentioning

confidence: 99%

Effect of dextran layer on protein uptake to dextran‐grafted adsorbents for ion‐exchange and mixed‐mode chromatography

Shi

Sun

2011

J of Separation Science

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In the current research, a series of dextran-grafted adsorbents were prepared using sulfopropyl and 4-(1H-imidazol-1-yl) aniline as chromatographic ligands for ion-exchange (IEC) and mixed-mode chromatography (MMC) to respectively investigate the influence of dextran layer on adsorption of γ-globulin. Experimental evidences of static adsorption on dextran-grafted IEC adsorbents showed that adsorption capacity of γ-globulin increased with dextran content. It could be attributed to the multilayer adsorption of charged protein in dextran layer and thus further induced a significant electrical potential gradient at the boundary of adsorbed area and its proximity, improving mass transfer in combination with concentration gradient. In contrast to IEC adsorbents, adsorption capacity and effective diffusivity of dextran-grafted MMC adsorbents did not change obviously with dextran grafting. It was considered that hydrophobic ligands immobilized onto dextran-grafted MMC adsorbents were stuck together at pH 8.0, resulting in the collapse of dextran layer. In concert with measured effective porosity for γ-globulin at pH 4.0, it was confirmed that dextran layer in MMC adsorbent was more complicated and influenced significantly by buffer pH. It was also manifested by protein adsorption at different pHs. Thus, it revealed the complexity in intraparticle mass transfer of the protein in dextran-grafted MMC adsorbent.

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“…17 Susceptibility to pH stress varies among different IgG molecules. 68 Regarding chemical stability, formulation pH may play a critical role in controlling many degradation pathways, such as disulfide bond formation/exchange, deamidation, fragmentation, isomerization, etc. Due to variable effects on different degradation pathways, the optimum pH value varies depending on both the sequence of an antibody and the analytical methods used in monitoring the stability.…”

Section: Effect Of Formulation Phmentioning

confidence: 99%