@article{pham_linova_smith_brown_elhanafi_fan_lavoie_woodley_carbonell_2024, title={Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant}, volume={1718}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2024.464682}, abstractNote={A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83%. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 minute, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20% loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83% to approximately 92-95%.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Pham, Dan N. and Linova, Marina Y. and Smith, William K. and Brown, Hunter and Elhanafi, Driss and Fan, Jinxin and Lavoie, Joseph and Woodley, John M. and Carbonell, Ruben G.}, year={2024}, month={Mar} } @article{lavoie_fan_pourdeyhimi_boi_carbonell_2023, title={Advances in high-throughput, high-capacity nonwoven membranes for chromatography in downstream processing: A review}, volume={5}, ISSN={["1097-0290"]}, url={https://doi.org/10.1002/bit.28457}, DOI={10.1002/bit.28457}, abstractNote={AbstractNonwoven membranes are highly engineered fibrous materials that can be manufactured on a large scale from a wide range of different polymers, and their surfaces can be modified using a large variety of different chemistries and ligands. The fiber diameters, surface areas, pore sizes, total porosities, and thicknesses of the nonwoven mats can be carefully controlled, providing many opportunities for creative approaches for the development of novel membranes with unique properties to meet the needs of the future of downstream processing. Fibrous membranes are already finding use in ultrafiltration, microfiltration, depth filtration, and, more recently, in membrane chromatography for product capture and impurity removal. This article summarizes the various methods of manufacturing nonwoven fabrics, and the many methods available for the modification of the fiber surfaces. It also reviews recent studies focused on the use of nonwoven fabric devices in membrane chromatography and provides some perspectives on the challenges that need to be overcome to increase binding capacities, decrease residence times, and reduce pressure drops so that eventually they can replace resin column chromatography in downstream process operations.}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Lavoie, Joseph and Fan, Jinxin and Pourdeyhimi, Behnam and Boi, Cristiana and Carbonell, Ruben G.}, year={2023}, month={May} } @article{fan_sripada_pham_linova_woodley_menegatti_boi_carbonell_2023, title={Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane}, volume={317}, ISSN={1383-5866}, url={http://dx.doi.org/10.1016/j.seppur.2023.123920}, DOI={10.1016/j.seppur.2023.123920}, abstractNote={A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate.}, journal={Separation and Purification Technology}, publisher={Elsevier BV}, author={Fan, Jinxin and Sripada, Sobhana A. and Pham, Dan N. and Linova, Marina Y. and Woodley, John M. and Menegatti, Stefano and Boi, Cristiana and Carbonell, Ruben G.}, year={2023}, month={Jul}, pages={123920} } @article{fan_barbieri_shastry_menegatti_boi_carbonell_2022, title={Purification of Adeno-Associated Virus (AAV) Serotype 2 from Spodoptera frugiperda (Sf9) Lysate by Chromatographic Nonwoven Membranes}, volume={12}, ISSN={2077-0375}, url={http://dx.doi.org/10.3390/membranes12100944}, DOI={10.3390/membranes12100944}, abstractNote={The success of adeno-associated virus (AAV)-based therapeutics in gene therapy poses the need for rapid and efficient processes that can support the growing clinical demand. Nonwoven membranes represent an ideal tool for the future of virus purification: owing to their small fiber diameters and high porosity, they can operate at high flowrates while allowing full access to target viral particles without diffusional limitations. This study describes the development of nonwoven ion-exchange membrane adsorbents for the purification of AAV2 from an Sf9 cell lysate. A strong anion-exchange (AEX) membrane was developed by UV grafting glycidyl methacrylate on a polybutylene terephthalate nonwoven followed by functionalization with triethylamine (TEA), resulting in a quaternary amine ligand (AEX-TEA membrane). When operated in bind-and-elute mode at a pH higher than the pI of the capsids, this membrane exhibited a high AAV2 binding capacity (9.6 × 1013 vp·mL−1) at the residence time of 1 min, and outperformed commercial cast membranes by isolating AAV2 from an Sf9 lysate with high productivity (2.4 × 1013 capsids·mL−1·min−1) and logarithmic reduction value of host cell proteins (HCP LRV ~ 1.8). An iminodiacetic acid cation-exchange nonwoven (CEX-IDA membrane) was also prepared and utilized at a pH lower than the pI of capsids to purify AAV2 in a bind-and-elute mode, affording high capsid recovery and impurity removal by eluting with a salt gradient. To further increase purity, the CEX-IDA and AEX-TEA membranes were utilized in series to purify the AAV2 from the Sf9 cell lysate. This membrane-based chromatography process also achieved excellent DNA clearance and a recovery of infectivity higher that that reported using ion-exchange resin chromatography.}, number={10}, journal={Membranes}, publisher={MDPI AG}, author={Fan, Jinxin and Barbieri, Eduardo and Shastry, Shriarjun and Menegatti, Stefano and Boi, Cristiana and Carbonell, Ruben G.}, year={2022}, month={Sep}, pages={944} } @article{fan_boi_lemma_lavoie_carbonell_2021, title={Iminodiacetic Acid (IDA) Cation-Exchange Nonwoven Membranes for Efficient Capture of Antibodies and Antibody Fragments}, volume={11}, ISSN={2077-0375}, url={http://dx.doi.org/10.3390/membranes11070530}, DOI={10.3390/membranes11070530}, abstractNote={There is strong need to reduce the manufacturing costs and increase the downstream purification efficiency of high-value therapeutic monoclonal antibodies (mAbs). This paper explores the performance of a weak cation-exchange membrane based on the coupling of IDA to poly(butylene terephthalate) (PBT) nonwoven fabrics. Uniform and conformal layers of poly(glycidyl methacrylate) (GMA) were first grafted to the surface of the nonwovens. Then IDA was coupled to the polyGMA layers under optimized conditions, resulting in membranes with very high permeability and binding capacity. This resulted in IgG dynamic binding capacities at very short residence times (0.1–2.0 min) that are much higher than those achieved by the best cation-exchange resins. Similar results were obtained in the purification of a single-chain (scFv) antibody fragment. As is customary with membrane systems, the dynamic binding capacities did not change significantly over a wide range of residence times. Finally, the excellent separation efficiency and potential reusability of the membrane were confirmed by five consecutive cycles of mAb capture from its cell culture harvest. The present work provides significant evidence that this weak cation-exchange nonwoven fabric platform might be a suitable alternative to packed resin chromatography for low-cost, higher productivity manufacturing of therapeutic mAbs and antibody fragments.}, number={7}, journal={Membranes}, publisher={MDPI AG}, author={Fan, Jinxin and Boi, Cristiana and Lemma, Solomon Mengistu and Lavoie, Joseph and Carbonell, Ruben G.}, year={2021}, month={Jul}, pages={530} }