Department of Chemical Engineering and Biotechnology, University of Cambridge, Philippa Fawcett Drive, Cambridge, CB3 0AS, UK.
Conjugation Group, Spirogen Ltd, QMB Innovation Centre, 42 New Road, London, E1 2AX, 20878, UK.
Pharm Res. 2019 Mar 22;36(5):71. doi: 10.1007/s11095-019-2591-x.
Dynamic in-situ proton (H) magnetic resonance imaging (MRI) and H T-relaxometry experiments are described in an attempt to: (i) understand the physical processes, that occur during the reconstitution of lyophilized bovine serum albumin (BSA) and monoclonal antibody (mAb) proteins; and (ii) objectify the reconstitution time.
Rapid two-dimensional H MRI and diffusion weighted MRI were used to study the temporal changes in solids dissolution and characterise water mass transport characteristics. One-shot T relaxation time measurements were also acquired in an attempt to quantify the reconstitution time. Both MRI data and T-relaxation data were compared to standard visual observations currently adopted by industry. The H images were further referenced to MRI calibration data to give quantitative values of protein concentration and, percentage of remaining undissolved solids.
An algorithmic analysis of the H T-relaxation data shows it is possible to classify the reconstitution event into three regimes (undissolved, transitional and dissolved). Moreover, a combined analysis of the 2D H MRI and H T-relaxation data gives a unique time point that characterises the onset of a reconstituted protein solution within well-defined error bars. These values compared favourably with those from visual observations. Diffusion weighted MRI showed that low concentration BSA and mAb samples showed distinct liquid-liquid phase separation attributed to two liquid layers with significant density differences.
T relaxation time distributions (whose interpretation is validated from the 2D H MR images) provides a quick and effective framework to build objective, quantitative descriptors of the reconstitution process that facilitate the interpretation of subjective visual observations currently adopted as the standard practice industry.
描述了动态原位质子（H）磁共振成像（MRI）和 H T 弛豫测量实验，旨在：（i）了解在冻干牛血清白蛋白（BSA）和单克隆抗体（mAb）蛋白质复溶过程中发生的物理过程；（ii）客观化复溶时间。
使用快速二维 H MRI 和扩散加权 MRI 研究固体溶解的时间变化，并描述水质量传输特性。还进行了单次 T 弛豫时间测量，试图定量测量复溶时间。将 MRI 数据和 T 弛豫数据与行业目前采用的标准视觉观察进行比较。H 图像进一步参考 MRI 校准数据，以给出蛋白质浓度和未溶解固体的剩余百分比的定量值。
对 H T 弛豫数据的算法分析表明，将复溶事件分为三个阶段（未溶解、过渡和溶解）是可能的。此外，二维 H MRI 和 H T 弛豫数据的综合分析给出了一个独特的时间点，该时间点可在明确定义的误差范围内表征复溶蛋白质溶液的起始时间。这些值与视觉观察结果相当。扩散加权 MRI 显示，低浓度 BSA 和 mAb 样品显示出明显的液-液相分离，归因于两层具有显著密度差异的液体。
T 弛豫时间分布（其解释从二维 H MR 图像得到验证）提供了一种快速有效的框架，用于构建复溶过程的客观、定量描述符，有助于解释目前作为行业标准实践采用的主观视觉观察。
