Today’s biologics manufacturing practices incur high costs to the drug makers, which can contribute to high prices for patients. Timely investment in the development and implementation of continuous biomanufacturing can increase the production of consistent-quality drugs at a lower cost and a faster pace, to meet growing demand. Efficient use of equipment, manufacturing footprint, and labor also offer the potential to improve drug accessibility. Although technological efforts enabling continuous biomanufacturing have commenced, challenges remain in the integration, monitoring, and control of traditionally segmented unit operations. Here, we discuss recent developments supporting the implementation of continuous biomanufacturing, along with their benefits.

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Depth filtration is widely used in downstream bioprocessing to remove particulate contaminants via depth straining and is therefore applied to harvest clarification and other processing steps. However, depth filtration also removes proteins via adsorption, which can contribute variously to impurity clearance and to reduction in product yield. The adsorption may occur on the different components of the depth filter, that is, filter aid, binder, and cellulose filter. We measured adsorption of several model proteins and therapeutic proteins onto filter aids, cellulose, and commercial depth filters at pH 5-8 and ionic strengths <50 mM and correlated the adsorption data to bulk measured properties such as surface area, morphology, surface charge density, and composition. We also explored the role of each depth filter component in the adsorption of proteins with different net charges, using confocal microscopy. Our findings show that a complete depth filter's maximum adsorptive capacity for proteins can be estimated by its protein monolayer coverage values, which are of order mg/m , depending on the protein size. Furthermore, the extent of adsorption of different proteins appears to depend on the nature of the resin binder and its extent of coating over the depth filter surface, particularly in masking the cation-exchanger-like capacity of the siliceous filter aids. In addition to guiding improved depth filter selection, the findings can be leveraged in inspiring a more intentional selection of components and design of depth filter construction for particular impurity removal targets.

Depth filtration is a commonly-used bioprocessing unit operation for harvest clarification that reduces the levels of process-and product-related impurities such as cell debris, host-cell proteins, nucleic acids and protein aggregates. Since depth filters comprise multiple components, different functionalities may contribute to such retention, making the mechanisms by which different impurities are removed difficult to decouple. Here we probe the mechanisms by which doublestranded DNA (dsDNA) is retained on depth filter media by visualizing the distribution of fluorescently-labeled retained DNA on spent depth filter discs using confocal fluorescence microscopy. The extent of DNA displacement into the depth filter was found to increase with decreasing DNA length with increasing operational parameters such as wash volume and buffer ionic strength. Finally, using 5ethynyl-2′-deoxyuridine (EdU) to label DNA in dividing CHO cells, we showed that Chinese hamster ovary (CHO) cellular DNA in the lysate supernatant migrates deeper into the depth filter than the lysate re-suspended pellet, elucidating the role of the size of the DNA in its form as an impurity. Apart from aiding DNA purification and removal, our experimental approaches and findings can be leveraged in studying the transport and retention of nucleic acids and other impurities on depth filters at a small scale.

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host-cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind-and-elute cation-exchange chromatography (CEX) polishing step. A variant of the bind-and-elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%-1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low-prevalence HCPs resulted in an effectively bimodal-like distribution of HCPs along the length of a multi-column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of lowprevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography. K E Y W O R D S aggregates, continuous chromatography, displacement, frontal chromatography, host-cell proteins, multicolumn chromatography 1 | INTRODUCTION Displacement was classified as one of three forms of chromatography, together with elution and frontal, in 1943 (Tiselius, 1943). Separation via elution chromatography is achieved after multiple components of the sample first bind to the column, then are eluted sequentially in the order of increasing affinity by a buffer of gradually increasing eluent strength. Separation by displacement chromatography, in contrast, uses a high-affinity displacer to displace and separate the transiently bound product. A variant of displacement chromatography, sample-or self-displacement chromatography, refers to the displacement of a lower-affinity product component by a higher-affinity counterpart in a multicomponent system. Similar to self-displacement chromatography, frontal chromatography refers to the separation of components based on their relative affinities. In a variant of frontal chromatography, termed flow-through chromatography, the operational parameters are chosen to let the impurities bind, allowing a purer product to exit the column unbound (Hill, Mace, & Moore, 1990). While elution and flow-through chromatography employ different approaches to achieve purification, displacement simply indicates that a competitor with a stronger affinity replaces the more weakly bound rival. Given these general

Protein mobility at solid–liquid interfaces can affect the performance of applications such as bioseparations and biosensors by facilitating reorganization of adsorbed protein, accelerating molecular recognition, and informing the fundamentals of adsorption. In the case of ion-exchange chromatographic beads with small, tortuous pores, where the existence of surface diffusion is often not recognized, slow mass transfer can result in lower resin capacity utilization. We demonstrate that accounting for and exploiting protein surface diffusion can alleviate the mass-transfer limitations on multiple significant length scales. Although the surface diffusivity has previously been shown to correlate with ionic strength (IS) and binding affinity, we show that the dependence is solely on the binding affinity, irrespective of pH, IS, and resin ligand density. Different surface diffusivities give rise to different protein distributions within the resin, as characterized using confocal microscopy and small-angle neutron scattering (length scales of micrometer and nanometer, respectively). The binding dependence of surface diffusion inspired a protein-loading approach in which the binding affinity, and hence the surface diffusivity, is modulated by varying IS. Such gradient loading increased the protein uptake efficiency by up to 43%, corroborating the importance of protein surface diffusion in protein transport in ion-exchange chromatography.

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Chromatographic workstations can significantly impact column chromatography performance by altering the peak shape and retention time. Inclusion of system contributions can help prevent misinterpretation of process data during column qualification, process scale-up, tech transfer to a different facility, or the implementation of equipment changes over the product life cycle. Holdup volumes, not usually considered, can pose significant risks during process development. Using bench-scale and pilot-scale AKTA chromatography systems, we investigated the impact of individual units within the flow path on ion-exchange chromatography operation. Experimental data was used to build a comprehensive model comprising of dispersive plug flow and ideal continuous stir tank reactors to mimic the holdup volume. Our models, which include a column model and a system model, demonstrate the importance of accounting for system holdup volume in chromatography modeling. These comprehensive models can also predict the impact of foreseeable system variations.