Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes’ size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product’s activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (i) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants, and (ii) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up-to 80% and up-to 50-fold reduction of host cell proteins upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a 2-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of host cell proteins to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blotting, transmission electron microscopy, nanoparticle tracking analysis.

The recent uptick in the approval of ex vivo cell therapies highlight the relevance of Lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification – currently reliant on filtration and anion-exchange or size-exclusion chromatography – suffers from long process times and low yield of transducing particles, which translate in high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50-60% of viral genomes; 40-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·10 9 TU per mL of resin at the residence time of 1 min) and clearance of host cell proteins (up to 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

K. phaffii is a versatile expression system that is increasingly utilized to produce biological therapeutics – including enzymes, engineered antibodies, and gene-editing tools – that feature multiple subunits and complex post-translational modifications. Two major roadblocks limit the adoption of K. phaffii in industrial biomanufacturing: its proteome, while known, has not been linked to downstream process operations and detailed knowledge is missing on problematic host cell proteins (HCPs) that endanger patient safety or product stability; furthermore, the purification toolbox has not evolved beyond the capture of monospecific antibodies, and few solutions are available for engineered antibody fragments and other protein therapeutics. To unlock the potential of yeast-based biopharmaceutical manufacturing, this study presents (i) a secretome survey of K. phaffii cell culture harvests that highlights HCPs with predicted immunogenicity, ability to cause product instability by proteolysis or degradation of excipients, and potential to interfere with purification operations via product association or co-elution; and (ii) a novel affinity adsorbent functionalized with peptide ligands that target the whole spectrum of K. phaffii HCPs – PichiaGuard – designed for the enrichment of therapeutic proteins in flow-through mode. The PichiaGuard adsorbent features high HCP binding capacity (~25 g per liter of resin) and successfully purified a monoclonal antibody and an ScFv fragment from clarified K. phaffii harvests, affording up to 80% product yield, and a >300-fold removal of HCPs. Notably, PichiaGuard outperformed commercial ion exchange and mixed-mode resins in removing high-risk HCPs – including aspartic proteases, ribosomal subunits, and other peptidases – thus demonstrating its value in modern biopharmaceutical processing.

Opportunities for process intensification and increased productivity have made the field of Continuous Biomanufacturing an area of high interest and active research. Within the purification train of producing biologics, Tangential Flow Filtration (TFF) is typically employed after chromatographic separations, to increase drug substance concentration, making the process more economical and further meeting dosage specifications. In a batch operation, concentration occurs via recirculation of the feed material where desired output concentration is attained through multiple pump-passes over the TFF membrane, while steadily excluding the buffer. Single-Pass Tangential Flow Filtration (SPTFF) enables continuity of this process by achieving similar concentration factors through a single – pass over these membranes while operating at low feed flow rates. Our work elucidates the development of a mechanistic process model to predict SPTFF performance across a relatively wide design space using a first principles approach. The developed model is found to be accurate for a range of high feed flow rates but is inaccurate at flow rates below 25 L/m2/hr. At very low flow rates, small differences in the mass transfer coefficient have been observed to significantly alter the prediction of the retentate concentration. We thus describe the challenges in predictive process modeling of SPTFF in antibody biomanufacturing.