Real-time in situ monitoring of CRM-197 and polysaccharide conjugation reaction by fluorescence spectroscopy.

Aims: Process analytical technology (PAT) is increasingly being adopted within the pharmaceutical industry to build quality into a process. Development of PAT that provides real-time in situ analysis of critical quality attributes are highly desirable for rapid, improved process development. Conjugation of CRM-197 with pneumococcal polysaccharides to produce a desired pneumococcal conjugate vaccine is a significantly intricate process that can tremendously benefit from real-time process monitoring. Methods: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time. Results & conclusion: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time.

Process monitoring of polysaccharide deketalization for vaccine bioconjugation development using in situ analytical methodology.

Pneumococcal conjugate vaccines (PCVs) are formed by bioconjugation of a carrier protein to the purified capsular polysaccharide (Ps) from multiple serological strains of Streptococcus pneumoniae. The associated bioconjugation chemistry relies on initial selective modifications to the Ps backbone structure. Among these modifications, removal of a ketal functional group, termed deketalization, is one that is important for pharmaceutical PCV production. Herein, we report a process monitoring investigation into the deketalization of a polysaccharide relevant to PCV process development. We have applied process analytical technology (PAT) for in situ process monitoring to study the deketalization reaction in real time. We find that in situ FTIR spectroscopy elucidates multiple classes of reaction kinetics, one of which correlates strongly with the deketalization reaction of interest. This PAT approach to real time reaction monitoring offers the possibility of improved process monitoring in the pharmaceutical production of PCVs. To our knowledge, this report represents the first PAT investigation into Ps deketalization. Our findings suggest that broader application of PAT to the chemical modifications associated with PCV bioconjugation, as well as other pharmaceutically relevant bioconjugation processes, carries the power to enhance process understanding, control, and efficiency through real time process monitoring.

Utilizing in situ spectroscopic tools to monitor ketal deprotection processes.

The use of protection groups to shield a functional group during a synthesis is employed throughout many reactions and organic syntheses. The role of a protection group can be vital to the success of a reaction, as well as increase reaction yield and selectivity. Although much work has been done to investigate the addition of a protection group, the removal of the protection group is just as important - however, there is a lack of methods employed within the literature for monitoring the removal of a protection group in real time. In this work, the process of removing, or deprotecting, a ketal protecting group is investigated. Process analytical technology tools are incorporated for in situ analysis of the deprotection reaction of a small molecule model compound. Specifically, Raman spectroscopy and Fourier transform infrared spectroscopy show that characteristic bands can be used to track the decrease of the reactant and the increase of the expected products over time. To the best of our knowledge, this is the first report of process analytical technology being used to monitor a ketal deprotection reaction in real time. This information can be capitalized on in the future for understanding and optimizing pharmaceutically-relevant deprotection processes and downstream reactions.

Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: a comparison to conventional approaches.

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale.