Evaluation of a Non-Arrhenius Model for Therapeutic Monoclonal Antibody Aggregation

Kayser, Veysel; Chennamsetty, Naresh; Voynov, Vladimir; Helk, Bernhard; Forrer, Kurt; Trout, Bernhardt L.

doi:10.1002/jps.22493

Cited by 103 publications

(61 citation statements)

References 85 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82].…”

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

View full text Add to dashboard Cite

a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

show abstract

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

View full text Add to dashboard Cite

show abstract

“…(26). In most cases, upward curvature is observed for temperature-dependent protein aggregation, as seen for different types of monoclonal antibodies (26,57,58). Upward curvature for lnk obs vs. 1/T leads to an overestimation of E a values when extrapolated to low temperatures, and thus to an overestimation of the shelf life that will be gained by cooling to low-temperature conditions (26).…”

Section: Protein Aggregation and Its Temperature Dependencementioning

confidence: 97%

“…One is for a single-domain four-helix bundle protein, bovine granulocyte-colony stimulating factor (bG-CSF) (55). The other is for a monoclonal antibody (MAb) (57). The main panel shows the data over the full set of temperatures reported, which include both accelerated (high-T) and real-time conditions (room T and below).…”

Section: Possible Solutions For Non-arrhenius Aggregation Kineticsmentioning

confidence: 99%

Non-Arrhenius Protein Aggregation

Wang

Roberts

2013

AAPS J

121

109

View full text Add to dashboard Cite

Abstract. Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

show abstract

“…20 Briefly, different functions were used for multi-peak fitting with the number of peaks for fitting being gradually increased. The best fit with the least number of peaks and fit parameters was achieved with 'exponential modified Gaussian functions'.…”

Section: Kinetics Of Surfactant Induced Virus-splitting With Fluorescmentioning

confidence: 99%

Quantitative determination of the surfactant-induced split ratio of influenza virus by fluorescence spectroscopy

Lee

Sahin

Neeleman

et al. 2016

Human Vaccines & Immunotherapeutics

Self Cite

View full text Add to dashboard Cite

The majority of marketed seasonal influenza vaccines are prepared using viruses that are chemically inactivated and treated with a surfactant. Treating with surfactants has important consequences: it produces 'split viruses' by solubilizing viral membranes, stabilizes free membrane proteins and ensures a low level of reactogenicity while retaining high vaccine potency. The formulation stability and potency of split influenza vaccines are largely determined by the specifics of this 'splitting' process; namely, the consequent conformational changes of proteins and interactions of solubilized particles, which may form aggregates. Robust methods to quantitatively determine the split ratio need to be developed before optimal splitting conditions can be investigated to streamline production of superior influenza vaccines.Here, we present a quantitative method, based on both steady-state and time-resolved fluorescence spectroscopy, to calculate the split ratio of the virus after surfactant treatment. We use the lipophilic dye Nile Red (NR) as a probe to elucidate molecular interactions and track changes in molecular environments. Inactivated whole influenza viruses obtained from a sucrose gradient were incubated with NR and subsequently treated with increasing concentrations of the surfactant Triton X-100 (TX-100) to induce virus splitting. NR's emission spectra showed that the addition of TX-100 caused~27 nm red-shifts in the emission peak, indicative of increasingly hydrophilic environments surrounding NR. The emission spectra of NR at different surfactant concentrations were analyzed with multi-peak fitting to ascertain the number of different micro-environments surrounding NR and track its population change in these different environments. Results from both the emission spectra and fluorescence lifetime spectroscopy revealed that NR showed presence in 3 distinct molecular environments. The split ratio of the virus was then calculated from the percentages of NR in these environments using both fluorescence emission and lifetime data. This study can pave the way for the development of robust methods to rapidly quantify splitting extent during vaccine manufacturing. KEYWORDS fluorescence emission; fluorescence lifetime; influenza split virus vaccine; influenza virus; membranesurfactant interactions; Nile Red; partitioning of dye in membranes; protein aggregation; split ratio quantification; surfactant; Triton X-100; vaccine formulation; vaccine manufacturing; virus splitting; virus-surfactant interactions

show abstract

Evaluation of a Non-Arrhenius Model for Therapeutic Monoclonal Antibody Aggregation

Cited by 103 publications

References 85 publications

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Non-Arrhenius Protein Aggregation

Quantitative determination of the surfactant-induced split ratio of influenza virus by fluorescence spectroscopy

Contact Info

Product

Resources

About