Design and In-line Raman Spectroscopic Monitoring of a Protein Batch Crystallization Process

Abstract

Raman spectroscopy was used to design and monitor a lysozyme protein batch crystallization process in a lab scale study to facilitate the design of a pharmaceutical protein manufacturing process. A D-optimal design that consisted of 18 experiments was performed to elucidate the effect of temperature, concentration of the precipitating agent, time of crystallization, and possible interactions between these three factors on the Raman scattering changes. A polynomial mathematical model was calculated relating the scattering of the lysozyme solutions measured at individual Raman shifts to the significant factors obtained in the previous crystallization experiment. The 2,940-cm −1 band provided the highest correlation values indicative of small prediction errors and good predictive ability for the crystallization model. Raman scattering signals obtained during the experiments were used as input to obtain a response surface for the factors studied and elucidate the relationship between the crystallization process conditions and the crystals obtained. The main factors affecting the crystallization process were the sodium chloride concentration and temperature.

Coacervation-Based Method for Constructing a Multifunctional Strain-Stiffening Crystalline Polyvinylamine Hydrogel

“…However, the intensity of peaks at 1600 and 1672 cm –1 (amide CO stretch) increased and the relative intensity of the two peaks at 1446 and 1473 cm –1 (sp 3 C–H bend) changed over time as shown in Figure d. It indicates that the local concentration of the bonds increased and crystalline domain formed over time. , …”

“…It indicates that the local concentration of the bonds increased and crystalline domain formed over time. 34,35 Nucleation is known as a process of forming new thermodynamic phases by thermal fluctuations at the atomic level. It is usually described by classical nucleation theory (CNT), which considers the free energy associated with the formation of a cluster.…”

Protein Nucleation and Crystallization Process with Process Analytical Technologies in a Batch Crystallizer

“…In this work, the concentrations were determined off-line due to the limitations of the PAT tools with the uncertainty and variations of on-line concentration measurements. Real-time monitoring of the crystallization process contributes to better understanding of nucleation and growth kinetics of pharmaceuticals, such as FBRM and PVM, in situ UV/vis spectrometry, and Raman spectroscopy. − However, PVM cannot be efficiently applied in all experiments due to the formation and suspension of a large number of tiny crystals, especially the needle-like crystals in some experiments, and the images captured were difficult to distinguish single crystals (Figure S4 in the Supporting Information). The in situ concentration measurement in the crystallization solution was not possible because of the lack of characterization peaks and interference of crystal suspension in the solution.…”

The role of Raman spectroscopy in biopharmaceuticals from development to manufacturing

“…The latter paper demonstrated Raman’s ability to measure post-translation modifications, aggregation, and fragmentation in forced degradation samples of IgG4 molecules [ 120 ]. Finally, Raman spectroscopy can be used to rapidly optimize protein crystallization conditions, and confirm the molecular structure and purity of a crystallized protein product [ 89 , 122 ]. Raman microscopy of lysozyme under native and denaturing conditions revealed structural changes from the monomer and oligomeric (fibril) states [ 123 ].…”