Real-Time Monitoring of Biopharmaceutical Crystallization: Chord Length Distribution to Crystal Size Distribution for Lysozyme, rHu Insulin, and Vitamin B12

Abstract

This investigation is focused on designing an effective strategy for the real-time monitoring of biopharmaceutical crystallization using focused beam reflectance measurement (FBRM). Lysozyme, rHu Insulin and Vitamin B12 were selected as model biopharmaceutical molecules for the study. Under selected experimental conditions, Lysozyme exhibits polymorphism with tetragonal and needle-shaped crystals, rHu Insulin forms rhombohedral crystals, whereas Vitamin B12 forms polyhedral shaped crystals. Focused beam reflectance measurements were performed to obtain the real time chord length distribution (CLD) of crystals obtained using static and dynamic conditions. Mathematical models were developed for converting CLD data obtained using FBRM measurements into the useful crystal size distribution (CSD) data for low (tetragonal, rhombohedral and polyhedral) and high aspect ratio crystals (needle). For high aspect ratio crystals, both width-based and length-based models were developed for capturing the CSD. The model validation was performed by comparing the model CSD with the CSD obtained using microscopic image analysis. Developed models effectively capture the CSD for variable shape crystals of Lysozyme, rHu Insulin and Vitamin B12. It was demonstrated that in case of high aspect ratio crystals, there is an intrinsic limitation in using FBRM for lengthbased crystal size measurement.

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

“…The FBRM–SED model approximates the geometry of crystals based on the CLD of FBRM. A tetragonal lysozyme crystal was shown as an example for translating CLD to SED shown in Figure a. (ii) FBRM–CLD inversion: by applying comprehensive algorithm , on the CLD of FBRM to search for the global best solution by finding the optimal aspect ratio for non-spherical crystals.…”

“…Process analytical technology (PAT) tools can be applied to observe real-time solute concentration, crystal counts, and crystal size distribution (CSD). , Focused beam reflectance measurement (FBRM), Coulter counter, laser diffraction, ultrasonic attenuation spectroscopy, and microscope image analysis were employed in tracking the crystal size change in the crystallization process. − The rotating laser beam is able to record the counts and the chord length distribution (CLD) using its intrinsic algorithm, and the CLD will be translated to CSD. In recent years, the methods for translating from CLD to CSD in the crystallization process have included the spherical equivalent diameter (SED) model and the crystal size database based on experimental works. , It is common to couple FBRM with particle vision and measurement (PVM) to visualize the crystallization process and calibrate the CSD in the system. , UV/vis and IR spectroscopies using a distinctive spectrum of a chemical compound and the characteristic wavelength peak can be used to detect the concentrations of different compounds in the solution based on Beer–Lambert law . Until now, very few research on the protein crystallization process with PAT tools hinders the understanding of the crystallization process for scaling up and optimizing the protein crystallization process.…”

Parameter Estimation of the Stochastic Primary Nucleation Kinetics by Stochastic Integrals Using Focused-Beam Reflectance Measurements

“…The CLD was measured with focused beam reflectance measurement (FBRM, made by Mettler Toledo International Inc., Columbus, OH, USA). The FBRM technique is often applied to obtain the size distribution [19][20][21]. However, the CLD obtained with FBRM is different from CSD, and hence, the CLD needs to be converted into the CSD [22,23].…”

“…where the subscript "m" means that the number of crystals reaches a predetermined threshold in this study. Equation ( 21) can be derived from Equation (20) as follows [17]: First, it is assumed from t = 0 to the threshold that C and W s are constant, µ 3 is proportional to µ 0 , and that µ 0,seed is negligibly small compared with rµ 0,seed . Secondly, B 2 is substituted for B* into Equation ( 20) at i = 0, and this equation is integrated from t = 0 to the threshold.…”

Partial Seeding Policy for Controlling the Crystal Quality in Batch Cooling Crystallization

“…The FBRM measuring range was 0-1000 μm and was divided into 100 equal parts by a linear scale. Not the CSD but the chord length distribution (CLD) can be directly determined by FBRM [17][18][19]. However, the CLD can be converted into the CSD with statistical methods via a mapping matrix based on the crystal shape [20][21][22].…”