Biotherapeutics, molecules produced from biological systems, require rigorous purification steps to remove impurities including host cell proteins (HCPs). Regulatory guidelines require manufacturers to monitor process‐related impurities along the purification workflow. Mass spectrometry (MS) has recently been considered as a complementary method to the well‐established ELISA for HCPs quantification, since it has the advantage of unambiguously identifying individual HCP. In this study, we developed an innovative standard dedicated to MS‐based HCP profiling analysis in order to monitor the consistency of viral vaccine intermediate purification samples. This standard, termed the HCP‐PROFILER standard, is composed of a water‐soluble bead (READYBEADS technology) which, after being added into the sample, releases unlabeled peptides in controlled amounts. The standard meets three desired criteria: (1) it is composed of multiple peptides, at different concentration levels, allowing construction of a calibration curve covering the dynamic range of HCPs present in the target sample, ensuring quantification accuracy; (2) it demonstrates high batch‐to‐batch reproducibility, ensuring quantification robustness and consistency over time; and (3) it is easy to use and avoids user‐induced analytical biases. In this study, we present the use of the HCP‐PROFILER standard for vaccine batches comparison and downstream process performance studies.

Long-distance regeneration of the central nervous system (CNS) has been achieved from the eye to the brain through activation of neuronal molecular pathways or pharmacological approaches. Unexpectedly, most of the regenerative fibers display guidance defects, which prevents reinnervation and further functional recovery. Therefore, characterizing the mature neuronal environment is essential to understand the adult axonal guidance in order to complete the circuit reconstruction. To this end, we used mass spectrometry to characterize the proteomes of major nuclei of the adult visual system: suprachiasmatic nucleus (SCN), ventral and dorsal lateral geniculate nucleus (vLGN, dLGN) and superior colliculus (SC)), as well as the optic chiasm. These analyses revealed the presence of guidance molecules and guidance-associated factors in the adult visual targets. Moreover, by performing bilateral optic nerve crush, we showed that the expression of some proteins was significantly modulated by the injury in the visual targets, even in the ones most distal to the lesion site. On another hand, we found that the expression of guidance molecules was not modified upon injury. This implies that these molecules may possibly interfere with the reinnervation of the brain targets. Together, our results provides an extensive characterization of the molecular environment in intact and injured conditions. These findings open new ways to correct regenerating axon guidance notably by manipulating the expression of the corresponding guidance receptors in the nervous system.