Nitin Yadav

Circular Dichroism Spectroscopy as a Tool for Monitoring Aggregation in Monoclonal Antibody Therapeutics

Aggregation continues to be a critical quality attribute for a monoclonal antibody therapeutic product due to its perceived significant impact on immunogenicity. This paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding protein aggregation. The second application uses thermal scanning CD as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. For establishing far-UV CD as an orthogonal technique, a mAb was incubated in different downstream processing buffers and another mAb in formulation buffers, and they were analyzed by SEC and far-UV CD for aggregate content and conformational stability, respectively. To examine thermal scanning as a high throughput screening tool, ellipticity as a function of the temperature was measured at 218 nm from 20 to 90 °C. Far-UV CD was found to display high sensitivity toward early detection of conformational changes in mAb. CD measurements were also able to elucidate the different aggregation mechanisms. Furthermore, thermal stability scan allowed us to estimate T(onset) which has been found to correlate with aggregation induced by salt, low pH, and buffer species. T(onset) temperature from thermal scanning at 218 nm using CD was correlated successfully to aggregate content measured by SEC. Results from both the studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.

Avoiding antibody aggregation during processing: establishing hold times.

Aggregation of biotech products used therapeutically, such as antibodies, can contribute to potential immunogenicity of the product. Charge‐based heterogeneities may also impact the safety and/or efficacy of a therapeutic. In this study, an approach based on empirical modeling and least squares regression is suggested for establishing hold times for process intermediates during production of monoclonal antibody (Mab) therapeutics. Two immunoglobulins were analyzed with respect to aggregation and charge heterogeneity in buffer conditions that are typically used during downstream processing of Mab products. Size exclusion chromatography, ion exchange chromatography (IEC), and circular dichroism were used. We found that aggregation primarily occurs at pH 3 (buffers used in affinity chromatography) and is higher in citrate buffer compared to acetate and glycine buffers. Aggregation is minimal in buffers used in anion exchange chromatography (Tris–HCl buffer at pH 7.2 and 8) and in cation exchange chromatography (citrate buffer at pH 6, acetate buffer at pH 6, and phosphate buffer at pH 6.5 and 7.5). The behavior is opposite in the case of charged heterogeneities (basic and acidic variants) as measured by IEC. The product is more susceptible to degradation at high pH than at low pH. The data presented here demonstrate that product stability can be a significant issue within the routinely used manufacturing conditions. We suggest that the approach presented needs to be adopted by all manufacturers to ensure product stability during processing.

Localization and implication of oblique whistler wave in the magnetopause region

Nonlinear interaction between highly oblique whistler wave and ion acoustic wave pertinent to magnetopause has been investigated. The density perturbation in whistler wave is supposed to be originated due to the presence of ion acoustic wave in the background. The ponderomotive force components arising due to the high amplitude pump wave, viz., whistler wave are constituted in the nonlinear dynamics of low frequency ion acoustic wave. The coupled nonlinear dynamical equations are then modelled in the form of modified nonlinear Schrodinger equation by considering adiabatic response of low frequency ion acoustic wave. The numerical simulation of this coupled nonlinear equation is performed to study the temporal evolution of nonlinear whistler wave. The obtained simulation results show that the temporal evolution also leads to the cascade of broadband turbulence spectrum at smaller wavelengths. The relevance of the obtained results with the observations of THEMIS spacecraft in the magnetopause region has bee...

Effect of magnetic islands on the localization of kinetic Alfvén wave

Recent studies have revealed an intimate link between magnetic reconnection and turbulence. Observations show that kinetic Alfven waves (KAWs) play a very crucial role in magnetic reconnection and have been a topic of interest from decades in the context of turbulence and particle heating. In the present paper, we study the role that KAW plays in the formation of coherent structures/current sheets when KAW is propagating in the pre-existing fully developed chain of magnetic islands. We derived the dynamical equation of KAW in the presence of chain of magnetic islands and solved it using numerical simulations well as analytic tools. Due to pre-existing chain of magnetic islands, KAW splits into coherent structures and the scale size of these structures along transverse directions (with respect to background magnetic field) comes out to be either less than or greater than ion gyro radius. Therefore, the present work may be the first step towards understanding how magnetic reconnection generated islands may affect the KAW localization and eventually contribute to magnetic turbulence. In this way the present approach may be helpful to understand the interplay between magnetic reconnection and turbulence in ion diffusion region.

Inertial Alfvén wave localization and turbulent spectrum

The localization of pump inertial Alfven wave (IAW) in low β plasmas (β ≪ me/ mi) has been investigated by developing a model based on weak IAW and finite amplitude background density fluctuations. When IAW is perturbed by these fluctuations which are in the form of magnetosonic and ion acoustic waves, its phase velocity gets modified and IAW breaks up into localized structures of very high intensity. Numerical simulation has been carried out to analyze the localized structures and magnetic fluctuation spectrum of pump IAW. We have also developed a simplified model to understand the physical insight into evolution pattern of IAW. Results obtained from simulation reveal that turbulent spectrum up to k⊥λe ≈ 1 follows Kolmogorov power law. Furthermore, at shorter wavelengths, dispersion starts and spectrum steepens with power law index ∼ − 3.8. Energy transport to smaller length scales through this mechanism may be accountable for the observed parallel electron heating in low β plasmas. Results obtained from the numerical simulation are consistent with the observations of various spacecraft like FAST and Hawkeye.

Effect of background density fluctuations on the localized structures of inertial Alfvén wave and turbulent spectrum

In the present paper, we have investigated the nonlinear evolution of three dimensionally (3D) propagating inertial Alfven wave (IAW) in the presence of background density fluctuations for low β plasma. The ponderomotive force, due to IAW and modified background plasma density, has been taken into account in order to develop the model equation. The governing equation has been solved numerically as well as semi-analytically to study the effect of background fluctuations on the evolution of IAW. Results reveal that the IAW gets localized and breaks up into intense localized structures. These localized structures are found to be influenced by the presence of background density fluctuations. To get the physical insight of this proposed interaction, a semi-analytical model based on the paraxial approximation has been developed. Resulting turbulent spectrum has also been studied for different levels of background fluctuations. As a result, it has been found that background density fluctuations affect the nonlin...

Density cavity formation through nonlinear interaction of 3‐D inertial Alfvén wave and ion acoustic wave

Density cavities having transverse-scale size of the order of electron inertial length have been observed in auroral ionosphere by Freja spacecraft. In the present study, density cavity formation in auroral regions has been investigated using numerical simulation techniques. Nonlinear interaction of inertial Alfven waves and ion acoustic waves has been suggested as a possible mechanism to apprehend density cavity formation in auroral regions. In the proposed mechanism, strong modification in the density profile takes place due to the ponderomotive force of inertial Alfven waves and accounts for these density depleted regions. In literature also, it has been demonstrated that these cavities are associated with the ponderomotive forces of inertial Alfven wave. Our presented model also attempts to understand the salient features of auroral density cavities which are reported in literature from the analysis of data available from FAST spacecraft. Relevance of obtained results with observations in auroral ionosphere has also been discussed.

Nonlinear interaction of slow Alfvén wave with ion acoustic wave and applications to space plasma

The paper is concerned with the analytical study of nonlinear coupling of slow Alfven wave (SW) with ion acoustic waves (IAWs) in high-β and low-β plasmas. Here the pump wave (SW) number density gets perturbed in the presence of IAW. The model equations of IAW and SW turn out to be the modified Zakharov system of equations when the ponderomotive nonlinearities are incorporated in the IAW and SW dynamics. Growth rate of modulational instability has been calculated. The relevance of these investigations for solar wind plasma and solar coronal plasma has also been discussed.

Nonlinear evolution of 3D whistler waves in space plasmas

The present model is proposed to study the nonlinear effects related to quasi-transverse 3D whistler waves in three different regions of space plasmas which are Earths radiation belt, magnetopause, and solar wind plasma at 1 A.U. We have analysed the effect of considering the third dimension in the dynamics of whistler waves on Localization of whistler waves and associated magnetic field power spectra. Three dimensionally propagating oblique whistler waves get localized due to background density perturbation. This background density modulation is supposed to be originating due to the propagation of low frequency kinetic Alfven waves in the background. The ponderomotive nonlinearity originating due to high amplitude whistler waves has been taken into account to develop the model equations. Next, these coupled model equations have been solved numerically using the pseudo-spectral method. Simulation results are investigated to study the process of field localization and magnetic field power spectrum. The re...

Nonlinear effects associated with kinetic Alfvén wave in magnetic islands

Observations show that kinetic Alfven waves (KAWs) play a very important role in turbulence as well as in magnetic reconnection and have also been a topic of debate in the context of particle heating. In this paper, we aim to investigate the effect of magnetic islands on the dynamics of the nonlinear KAW. The formation of current sheet/coherent structures has been explored due to the propagation of the nonlinear KAW through the chain of pre-existing fully developed chain of magnetic islands. It is found that the field localization is more intense when the density perturbation due to nonlinearity and the field perturbation due to background islands are taken into account in comparison to the perturbation created by both the sources separately. Thus, it is expected that both nonlinearity and islands (reconnection generated) will aid the field localization and hence turbulence. Based on these coherent structures, we have evaluated the power spectrum and found the scaling after first breakpoint to be consiste...