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.

Polymer surfactants are key components of cell culture media as they prevent mechanical damage during fermentation in stirred bioreactors. Among cell-protecting surfactants, Pluronics are widely utilized in biomanufacturing to ensure high cell viability and productivity. Mono-dispersity of monomer sequence and length is critical for the effectiveness of Pluronics - since minor deviations can damage the cells - but is challenging to achieve due to the stochastic nature of polymerization. Responding to this challenge, this study introduces Peptonics, a novel family of peptide and peptoid surfactants whose monomer composition and sequence are de-signed to achieve high cell viability and productivity at a fraction of chain length and cost of Pluronics. A designed ensemble of Peptonics was initially characterized via light scattering and tensiometry to select sequences whose phase behavior and tensioactivity align with those of Pluronics. Selected sequences were evaluated as cell-protecting surfactants using Chinese hamster ovary (CHO) cells expressing therapeutic monoclonal antibodies (mAb). Peptonics IH-T1010, ih-T1010, and ih-T1020 afforded high cell density (up to 3·107 cells·mL-1) and viability (up to 95% within 10 days of culture), while reducing the accumulation of ammonia (a toxic metabolite) by ~10% compared to Pluronic F-68. Improved cell viability afforded high mAb titer (up to 5.5 mg·mL-1) and extended the production window beyond 14 days; notably, Peptonic IH-T1020 decreased mAb fragmentation and aggregation ~5%, and lowered the titer of host cell proteins by 16% compared to Pluronic F-68. These features can improve significantly purification of mAbs, thus increasing their availability at lower cost to patients.

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. The AAV purification pipeline, in particular, hinges on protein ligands for the affinity-based capture step: while featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product’s activity and stability; additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable – yet equally effective – affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, while (ii) enabling product elution under near-physiological conditions (pH 6.0) and (iii) granting extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (~1014 vp per mL) and product yields (~60-80%) on par with commercial adsorbents, and purified AAV2 from HEK293 and Sf9 cell lysates affording high recovery (up to 78%) and reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%) of the purified vectors.

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands – typically camelid antibodies – that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (K D ~ 10 -5-10 -6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC 10% > 10 13 vp per mL of resin) and product yields (~50-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50-80%), 80-to-400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.

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.

This study presents the chromatographic purification of immunoglobulin G (IgG) from human plasma using a two-column process integrating the peptide-based adsorbents LigaGuardTM, which captures non-Ig plasma proteins in flow-through mode, and LigaTrapTM, which isolates IgG in bind-and-elute. Buffer composition and column loading were optimized for both adsorbents. Two process configurations were evaluated. In the first design, plasma was fed to a LigaGuardTM column to capture plasma proteins, the effluent was loaded on the LigaTrapTM column, and the bound IgG was eluted with 63.8% global recovery and 99.7% purity; in comparison, Protein G agarose afforded ~67% recovery and 97.2% purity. In the alternative design, the LigaGuardTM column was utilized to polish the LigaTrapTM elution stream, affording 82.3% global recovery and 98.8% purity. Collectively, these results demonstrate the potential of a fully chromatographic process for purifying polyclonal IgG from plasma feedstocks.