The established platform for monoclonal antibody (mAb) purification, centered on Protein A affinity capture and subsequent polishing steps, has enabled large-scale manufacturing of therapeutic antibodies. However, persistent host cell proteins (HCPs), antibody aggregates, and product fragments remain challenging to clear, particularly as novel mAb formats and intensified upstream processes introduce higher impurity loads. In this study, we present transformative mAb purification platforms that combine novel multimodal resins operating in flowthrough mode with conventional Protein A chromatography to address these limitations. Our new platform utilizes LigaGuard for the selective removal of high-risk and persistent HCPs, chromatin, and aggregates by flowthrough affinity chromatography; and a size-exclusion-mixed-mode (SEMM) resin for the efficient removal of product-related low and high molecular weight impurities and residual process-related impurities. Several alternative process configurations were systematically assessed to maximize purification performance and robustness. A novel process configuration comprising an initial impurity-stripping step using LigaGuard followed by a capture step by Protein A chromatography and a polishing step using SEMM resin achieved global yields up to 86 %, with residual product-related impurities <1 %, HCPs <60 ppm, and hcDNA <10 ppb across diverse industrial feedstocks. The proteomic analyses of the polished streams confirmed the depletion of high-risk and persistent HCPs, including hydrolases and polysorbate-degrading enzymes. The use of two flow-through operations flanking a central affinity capture step enhances the performance of the Protein A resin alongside process productivity and robustness to feedstock variability. These results support a new paradigm for mAb purification, offering scalable and robust solutions for both legacy and emerging antibody modalities.

The sustained growth of monoclonal antibody (mAb)-based therapies and the goal of reducing production costs to below $10 per gram of mAb by the end of the 2020 decade pose an urgent need for novel purification technologies that boost process intensification and cost reduction. While upstream advances now routinely deliver high harvest titers and convective Protein A adsorbents provide superior binding capacity, the efficient removal of high and low molecular weight (HMW and LMW) product-related impurities remains a key challenge in downstream bioprocessing. These impurities, often associated with host cell proteins and nucleic acids, compromise product purity and safety. Addressing the need for rapid and disposable bioprocess solutions, our team introduced a single-use adsorbent - SEMM-silica resin - that integrates size-exclusion and mixed-mode mechanisms for antibody polishing in flow-through mode. A panel of industrial mAbs, including IgG1 and IgG4 with pIs ranging between 6.1 and 9.2, was purified by Protein A chromatography and subjected to controlled enzymatic digestion to generate model feedstocks with diverse impurity profiles. The SEMM-silica resin achieved robust removal of LMW species, capturing up to 67 % of F(ab)2, 44 % of Fab/Fc, and 44 % of polypeptide fragments, as well as up to 36 % of aggregates. This resulted in polished pools with monomeric mAb purities up to 99 %, and average LMW and HMW levels below 2 % and 1 %, respectively. Notably, the synergism of size-exclusion and mixed-mode mechanisms enabled product yields above 90 % at a residence time of 6 min, translating into productivities of up to 50 g/L-h.

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25-33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions - namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase - afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.

Genetic Engineering & Biotechnology NewsVol. 38, No. 15 Next-Generation Processing: A Multidisciplinary PursuitMilliporeSigma Advocates Evolutionary Transformation through CollaborationMichael Felo and Michael PhillipsMichael FeloMichael Felo is director, downstream process integration, and Michael Phillips, Ph.D. (E-mail Address: michael.phillips@emdmillipore.com), is director, next-generation process, R&D, at MilliporeSigma. Website: www.emdmillipore.com.Search for more papers by this author and Michael PhillipsMichael Felo is director, downstream process integration, and Michael Phillips, Ph.D. (E-mail Address: michael.phillips@emdmillipore.com), is director, next-generation process, R&D, at MilliporeSigma. Website: www.emdmillipore.com.Search for more papers by this authorPublished Online:1 Sep 2018https://doi.org/10.1089/gen.38.15.16AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 38Issue 15Sep 2018 InformationCopyright © GEN PublishingTo cite this article:Michael Felo and Michael Phillips.Next-Generation Processing: A Multidisciplinary Pursuit.Genetic Engineering & Biotechnology News.Sep 2018.S27-S29.http://doi.org/10.1089/gen.38.15.16Published in Volume: 38 Issue 15: September 1, 2018PDF download

The effect of ligand density was studied on protein adsorption and transport behavior in tentacular cation-exchange sorbents at different ionic strengths. Results were obtained for lysozyme, lactoferrin and a monoclonal antibody (mAb) in order to examine the effects of protein size and charge. The combination of ligand density and ionic strength results in extensive variability of the static and dynamic binding capacities, transport rate and binding affinity of the proteins. Uptake and elution experiments were performed to quantify the transport behavior of selected proteins, specifically to estimate intraparticle protein diffusivities. The observed trend of decreasing uptake diffusivities with an increase in ligand density was correlated to structural properties of the ligand-density variants, particularly the accessible porosity. Increasing the ionic strength of the equilibration buffer led to enhanced mass transfer during uptake, independent of the transport model used, and specifically for larger proteins like lactoferrin and mAb, the most significant effects were evident in the sorbent of the highest ligand density. For lysozyme, higher ligand density leads to higher static and dynamic binding capacities whereas for lactoferrin and the mAb, the binding capacity is a complex function of accessible porosity due to ionic strength-dependent changes. Ligand density has a less pronounced effect on the elution rate, presumably due to ionic strength-dependent changes in the pore architecture of the sorbents.

The Mobius® CellReady bioreactor product platform incorporates novel disposable technologies that provide optimal performance for suspension mammalian cell culture. Here we show the utility of EMD Millipore's 3L and 50L CellReady single use bioreactors for the cultivation of adherent mammalian cells on microcarriers. Cytodex3® and Solohill® collagen microcarriers were first tested in a mixing study to assess feasibility. We evaluated the normalized mixing speed required in the 3L and 50L to achieve a suspension of the microcarriers and enable growth of the cells.

Product . . . . . . . . . 363 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364Virus filtration is one component in the overall strategy to minimize transmission of infectious pathogens for biological products. Recombinant products such as monoclonal antibodies are expressed by mammalian, bacteria or yeast cells in fermentors, and within ascites fluid, or fluids from transgenic mammals. Mammalian cell lines may contain endogenous viruses that are generated in the bioreactor. Endogenous retroviruses are expressed because the retroviral genome is integrated into the cell line and cannot be screened-out during the creation of the Master Cell Bank. This causes retrovirus like particles, or RVLP's, to be produced within the bioreactor. Products can also become contaminated by adventitious viruses which enter the process streams.

Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.