Conjugation of protein therapeutics with polymers like polyethylene glycol (PEG) has been shown to increase their therapeutic efficiency. However, manufacturing of PEGylated drugs requires an additional, carefully controlled reaction step after purifying the protein, followed by further purification of over- and under-PEGylated variants. In this work, we have used a combined spectroscopic and statistical approach for monitoring and control of the PEGylation reaction for G-CSF using near infrared spectroscopy (NIRS). An online NIRS probe deployed in the reaction vessel has been used to track conversion of G-CSF into monoPEGylated and multiPEGylated forms using calibrated partial least squares regression models on the NIRS spectra which are collected in real time every 3 s. A pH probe integrated with a peristaltic pump facilitates automated quenching of the reaction at the targeted time. The NIRS spectra have also been used to build a batch evolution model for the reaction from end-to-end, including the addition of the reactants to the reaction vessel, the progress of the reaction for 70 min, and the final quenching with Tris base. Online spectra are compared against the statistical process control charts of the batch evolution model in real time to detect deviations as soon as they occur. The system was demonstrated for four common deviations in the PEGylation process, namely: delayed quenching time, wrong concentration of reducing agent added, wrong PEG to G-CSF ratio, and wrong sequence of addition of reactants. The system was able to identify all four deviations in real time and alert the operator to take control actions. The PAT approach suggested here embraces the quality by design framework and can be generalized for manufacturing scale monitoring and control of different biotechnology reactions with spectroscopic signatures.

Cancer poses a significant threat to human health worldwide, and many therapies have been used for its palliative and curative treatments. Vincristine has been extensively used in chemotherapy. However, there are two major challenges concerning its applications in various tumors: (1) Vincristine\'s antitumor mechanism is cell-cycle-specific, and the duration of its exposure to tumor cells can significantly affect its antitumor activity and (2) Vincristine is widely bio-distributed and can be rapidly eliminated. One solution to these challenges is the encapsulation of vincristine into liposomes. Vincristine can be loaded into conventional liposomes, but it quickly leak out owing to its high membrane permeability. Numerous approaches have been attempted to overcome this problem. Vincristine has been loaded into PEGylated liposomes to prolong circulation time and improve tumor accumulation. These liposomes indeed prolong circulation time, but the payout characteristic of vincristine is severer, resulting in a compromised outcome rather than a better efficacy compared to conventional sphingomyelin (SM)/cholesterol (Chol) liposomes. In 2012, the USA Food and Drug Administration (FDA) approved SM/Chol liposomal vincristine (Marqibo®) for commercial use. In this review, we mainly focus on the drug\'s rapid leakage problem and the potentially relevant solutions that can be applied during the development of liposomal vincristine and the reason for conventional liposomal vincristine rather than PEGylated liposomes has access to the market.