Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

Virus clearance validation across continuous capture chromatography

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover.     

A novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution

Charge heterogeneity of monoclonal antibodies is considered a critical quality attribute and hence needs to be monitored and controlled by the manufacturer. Typically, this is accomplished via separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality upon slightest changes in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. In this study, we propose a novel approach for separation of charge variants during continuous CEX chromatography by utilizing a combination of displacement mode chromatography and salt-based step elution. It has been demonstrated that while the displacement mode of chromatography enables control of acidic variants ≤26% in the CEX eluate, salt-based step gradient elution manages basic charge variant ≤25% in the CEX eluate. The proposed approach has been successfully demonstrated using feed materials with varying compositions. On comparing the designed strategy with 2-column concurrent (CC) chromatography, the resin specific productivity increased by 95% and resin utilization increased by 183% with recovery of main species >99%. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was examined in our in-house continuous mAb platform.     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

A novel twin‐column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin‐column continuous chromatography (called N‐rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled‐up batch‐mode cation exchange (CEX) chromatography was conducted. For the N‐rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22‐cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled‐up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N‐rich and scaled‐up CEX processes, the results indicated that the N‐rich method displays a remarkable advantage on the product yield, i.e. 1.46‐fold increment without the loss of productivity and purity. Generally, twin‐column N‐rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.     

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow‐rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log₁₀ of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750–757, 2015     

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non‐infectious retrovirus‐like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO^? and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV. Biotechnol. Bioeng. 2012;109: 157–165. © 2011 Wiley Periodicals, Inc.     

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification,Characterization, and Mitigation

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019     

Quality by design approach for viral clearance by protein a chromatography

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus‐like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X‐MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design‐of‐experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95–103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow-through mode using ion exchange or mixed-mode chromatography. Recent studies have highlighted the presence of “problematic HCP” species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb-producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide-based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many “problematic HCPs” by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next-generation adsorbents for safer and cost-effective manufacturing of biologics.     

Cross-scale quality assessment of a mechanistic cation exchange chromatography model

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

Novel spiking methods developed for anion exchange chromatography operating in a continuous process

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a nonhomogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in-line spiking. This is challenging to be implemented at the contract research organization performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow-through anion exchange chromatography step in a monoclonal antibody (mAb) manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of anion exchange chromatography unit operating in continuous processing.     

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.     

The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation‐exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size‐exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co‐incubating Fab and Fc fragments with their respective full‐length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX‐based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein–protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb‐based therapeutics.     

The Removal of Model Viruses, Poliovirus type 1 and Canine Parvovirus, During the Purification of Human Albumin using Ion-exchange Chromatographic Procedures

The manufacturing process for albumin in Australia is based primarily on ion-exchange chromatography. The capacity of ion-exchange matrices to remove non-enveloped viruses (canine parvovirus and poliovirus type 1) was assessed using a scaled-down chromatographic process which was shown to yield product meeting purity criteria set for the manufacturing process. Poliovirus type 1 and canine parvovirus were added at one tenth the volume of desalted and delipidated Supernatant II+III produced by traditional Cohn Fractionation from human plasma before the material was applied to DEAE and CM ion-exchangers connected in series. Samples were taken at equilibration, wash, elution and regeneration steps and the log clearance and reduction of the viruses calculated. The mean clearance and reduction factors for viral load of poliovirus type 1 were 5·3 logs and 3·2 logs, respectively and 1·8 logs and 1·8 logs for canine parvovirus.     

Magnetic Precipitation: A New Platform for Protein Purification

One of the trends in downstream processing comprises the use of “anything‐but‐chromatography” methods to overcome the current downfalls of standard packed‐bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation is developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit are tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions are obtained when using PEG3350 as precipitant at 4 °C for 1 h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti‐TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies.