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Downstream processing of monoclonal antibodies—Application of Platform Approaches
Abstract
This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.
... The Fc region is dimeric and IgG is the common format used for this bioengineered protein due to its effector function and increased plasma half-life [51]. The crystal structure of the IgG1 Fc region is shown in Fig. 2. The Fc region of this protein interacts with the FcRn receptor on endothelial cells to prevent intracellular degradation and extend its half-life [52]. ...
... After the proteins are produced and harvested, the next step is purification. The principal concern for purification is purity, followed by speed, overall yield, cost, and process throughput [52]. However, although purification will remove most unwanted molecules, heterogeneities will still occur [39]. ...
... Affinity purification are often the choice method for antibodies although its effectiveness is reliant on the antibody's ability to bind to an affinity adsorbent [110]. For process scale purification, Protein A affinity chromatography derived by the cell wall of Staphylococcus aureus is the benchmark as it utilises specific ligand interactions between mAb Fc region and immobilised Protein A to achieve high selectivity at over 95% [52]. For pharmaceutically accepted purity levels, Protein A affinity chromatography is followed up by cation-exchange chromatography and then anion-exchange chromatography [52]. ...
Background
Antibodies have proven to be remarkably successful for biomedical applications. They play important roles in epidemiology and medicine from diagnostics of diseases to therapeutics, treating diseases from incessant chronic diseases such as rheumatology to pandemic outbreaks. With no end in sight for the demand for antibody products, optimizations and new techniques must be expanded to accommodate this.
Methods and Results
This review discusses optimizations and techniques for antibody production through choice of discovery platforms, expression systems, cell culture mediums, and other strategies to increase expression yield. Each system has its own merits and demerits, and the strategy chosen is critical in addressing various biological aspects.
Conclusions
There is still insufficient evidence to validate the efficacy of some of these techniques, and further research is needed to consolidate these industrial production systems. There is no doubt that more strategies, systems, and pipelines will contribute to enhance biopharmaceutical production.
... Although a wide range of technology is available for the downstream processing of proteins, most mAb purification systems are based on the use of Protein A affinity chromatography (3). Protein A affinity chromatography is highly selective for antibodies, allowing the removal of more than 95 % of the impurities from a complex fermentation broth culture in one step (4). However, using Protein A has some disadvantages, which are essentially related to the high cost and sensitivity of Protein A resins and possible leakage of the ligand from the resin matrix (5). ...
... 3 by using low pH and virus filtration steps to ensure an acceptable level of virus clearance (4,7,8). ...
Research background. Protein A affinity chromatography is a well-established method currently used in the pharmaceutical industry. However, the high costs usually associated with Protein A chromatographic separation and its difficulties in continuous operation make the investigation of alternative purification methods of great importance. Experimental approach. In this study, the development and optimization of extraction-back extraction and precipitation-dissolution methods were performed, and they were compared with Protein A and cation exchange chromatographic separations in terms of mAb yield and concentration of residual impurities, such as DNA, host cell proteins, and mAb aggregates. To perform a comprehensive comparison of the different methods, experiments were carried out from the same cell-free fermentation broth containing adalimumab. Results and conclusions. Protein A and cation exchange chromatographic separations resulted in high adalimumab yield and purity. The precipitation-based process resulted in high yield, but with lower purity. The extraction-based purification resulted in low yield and purity. Thus, the precipitation-based method was found to be more promising for direct purification of adalimumab from harvested cell culture fluid compared to the extraction-based method. Novelty and scientific contribution. Although alternative purification methods could offer the advantages of simplicity and low-cost operation, further significant improvements are still required to compete with the performance of the chromatographic separations of adalimumab from real fermentation broth.
... They have showed the potential to widen the available 51 options in the treatment of certain diseases, including also rare conditions [2]. Amongst 52 biopharmaceuticals, immunoglobulin G (IgG) antibodies are widely applied for 53 therapeutic purposes, especially monoclonal antibodies (mAbs), which have shown 54 remarkable contributions in clinical practice for the treatment of oncologic and 55 autoimmune diseases [3]. In turn, polyclonal antibodies (pAbs) derived from human 56 serum are usually applied for the prevention and treatment of infections in 57 immunodeficient patients, and also for the treatment of autoimmune and inflammatory 58 diseases [4]. ...
... Material (Table S5). 611 In what concerns TPP formation, deviations to the behavior observed with human 612 serum, in which TPP was formed regardless of the IL structure and concentration, were process robustness [54]. The use of ILs as adjuvants not only allowed the best mAb recovery and purification 797 balance, with an exceptional ability to remove HCP, but also showed potential to reduce 798 production costs. ...
... reverse-phase and hydrophobic interaction chromatography, which separate molecules according to their hydrophobicity; size-exclusion and ion-exchange chromatography, in which the separation is achieved according to the molecular size and charge, respectively; and mixed-mode chromatography, which is a combination of the previously mentioned approaches (Saraswat et al., 2013;Hanke and Ottens, 2014;Jozala et al., 2016;Tavares et al., 2019). For example, the purification of mAbs generally requires a sequence of three chromatographic steps (Shukla et al., 2007;Azevedo et al., 2009). The mAbs capture step employs Protein A affinity chromatography, a technique based on the specific interaction between the mAbs Fc region and the Protein A ligand-a recombinant protein derived from Staphylococcus aureus-that is immobilized in a resin support (Shukla et al., 2007). ...
... For example, the purification of mAbs generally requires a sequence of three chromatographic steps (Shukla et al., 2007;Azevedo et al., 2009). The mAbs capture step employs Protein A affinity chromatography, a technique based on the specific interaction between the mAbs Fc region and the Protein A ligand-a recombinant protein derived from Staphylococcus aureus-that is immobilized in a resin support (Shukla et al., 2007). Afterwards, two more chromatographic steps are required to polish the final product by removing impurities, such as aggregates of high molecular mass, host cell proteins, clipped species of low molecular mass, DNA, and leached Protein A. ...
The emergence of biopharmaceuticals, including proteins, nucleic acids, peptides, and vaccines, revolutionized the medical field, contributing to significant advances in the prophylaxis and treatment of chronic and life-threatening diseases. However, biopharmaceuticals manufacturing involves a set of complex upstream and downstream processes, which considerably impact their cost. In particular, despite the efforts made in the last decades to improve the existing technologies, downstream processing still accounts for more than 80% of the total biopharmaceutical production cost. On the other hand, the formulation of biological products must ensure they maintain their therapeutic performance and long-term stability, while preserving their physical and chemical structure. Ionic-liquid (IL)-based approaches arose as a promise alternative, showing the potential to be used in downstream processing to provide increased purity and recovery yield, as well as excipients for the development of stable biopharmaceutical formulations. This manuscript reviews the most important progress achieved in both fields. The work developed is critically discussed and complemented with a SWOT analysis.
... The advantages of PEG are relatively low cost, it is no-toxic, noncorrosive, nonflammable, and has low-vapor pressure (Sim, He, Tscheliessnig, Mueller, Tan, Jungbauer 2012a). The presence of PEG has also been reported to exert stabilization effect on protein structures (Bell et al., 1983;Polson et al., 1964;Scopes, 1994). PEG-aided precipitation is a simple method exhibiting fast precipitation kinetics. ...
... The aggregation of mAbs is a potential safety concern in their processing and formulation in pharmaceutical industry. Thus, reducing the aggregate content in the bioproduct is of major importance (Rosenberg, 2006;Shukla et al., 2007). Furthermore, the activity The phenomenon was exploited to reduce av in downstream pools of mAb2 and mAb3 differing in molecular structure and isoelectric point, which were precipitated with PEGs of different types and concentrations. ...
Microheterogeneity of mAbs can impact their activity and stability. Formation of charge variants is considered as the most important source the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variant in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was as a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs. This article is protected by copyright. All rights reserved.
... However, low pH may also promote disruption of antibody secondary structure which can lead to aggregate formation [21][22][23][24][25]. Therefore, suppression or elimination of antibody aggregation, during both upstream and downstream processing, has become a subject of ongoing investigation in bioprocess development [25]. The high antibody concentration present in the cell culture medium increases the frequency of protein self-adsorption events [21,26,27], which, of course, can also lead to aggregation. Several strategies and/or small molecule additives, aimed at minimizing antibody aggregation during column elution, have been tested. ...
... Several strategies and/or small molecule additives, aimed at minimizing antibody aggregation during column elution, have been tested. They have included for example: (a) addition of 0.5-1 M urea [21] or (b) of arginine monomers [26] to the running buffer. Suppressing the formation of antibody oligomers is vital: studies have shown that such aggregates may reduce (i) therapeutic antibody potency; (ii) batch to batch reproducibility; as well as (iii) promoting Abbreviations: DDW, Double distilled water; DLS, Dynamic light scattering; DMSO, Dimethyl sulfoxide; E. coli, Escherichia coli; hIgG, human IgG; IgG, Immunoglobulin G; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis; Tyr, tyrosine; Val, valine. ...
Industrial scale production of therapeutic monoclonal antibodies (mAbs) is commonly achieved with Protein A chromatography, a process that requires exposure of the antibody to strongly acidic conditions during the eluting step. Exposure to acid inactivates virus contaminants but may, in parallel, lead to antibody aggregation that must be eliminated or kept at acceptably low levels. This report seeks to provide a practical method for overcoming a long-standing problem. We show how Brij-O20 detergent micelles, conjugated by the amphiphilic [(bathophenanthroline)3:Fe²⁺] complex in the presence of amino acid monomers: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), isoleucine (Ile) or valine (Val), efficiently capture polyclonal human IgG (hIgG) at neutral pH and allow its recovery by extraction either at pH 4 (85-97% yield) or at pH 6.3 (72-84% yield). Of the five amino acid monomers surveyed, Phe or Tyr produced the highest overall process yield at both pH 4 and 6.3. The monomeric state of the purified hIgG's was confirmed by dynamic light scattering (DLS). Potential advantages of the purification method are discussed.
... The percentage of aggregate area increased with decreasing pH. Protein A affinity chromatography is commonly applied in the purification of antibody products due to its high selectivity and effective removal of impurities (Shukla et al., 2007). Mammalian cell expression systems intrinsically pose a risk of viral contamination. ...
Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone.
... The operation of the capture step as a batch process presents some limitations due to the underutilization of the stationary phase in the column while loading [20], [21], [22]. Due to this, the process may require the use of excessive amounts of solvent. ...
Monoclonal antibodies (mAbs) have emerged as indispensable assets in medicine, and are currently at the forefront of biopharmaceutical product development. However, the growing market demand and the substantial doses required for mAb clinical treatments necessitate significant progress in its large-scale production. Most of the processes for industrial mAb production rely on batch operations, which result in significant downtime. The shift towards a fully continuous and integrated manufacturing process holds the potential to boost product yield and quality, while eliminating the extra expenses associated with storing intermediate products. The integrated continuous mAb production process can be divided into the upstream and downstream processes. One crucial aspect that ensures the continuity of the integrated process is the switching of the capture columns, which are typically chromatography columns operated in a fed-batch manner downstream. Due to the discrete nature of the switching operation, advanced process control algorithms such as economic MPC (EMPC) are computationally difficult to implement. This is because an integer nonlinear program (INLP) needs to be solved online at each sampling time. This paper introduces two computationally-efficient approaches for EMPC implementation, namely, a sigmoid function approximation approach and a rectified linear unit (ReLU) approximation approach. It also explores the application of deep reinforcement learning (DRL). These three methods are compared to the traditional switching approach which is based on a 1% product breakthrough rule and which involves no optimization.
... IgG1-DM1 ADC was supplied by Amgen; the mAb is comprised of two human heavy chains and two human λ light chains, and was expressed in Chinese hamster ovary cells and purified by chromatographic procedures developed at Amgen. 72 Detailed procedures for the preparation of the ADC have been described previously. 12 Briefly, a maytansinoid DM1 payload was conjugated onto primary amines of the naked IgG1 mAb through a noncleavable linker, N-succinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SMCC) ( Figure S1). ...
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-of-principle study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules.
... As opposed to smallmolecule drugs, which are mostly unknown to the human body, a large part of an antibody sequence and structure is known to our immune system. Although many small molecules can be synthesized chemically where the tools for characterization are well-established, antibodies, which are approximately 150 kDa in mass, are synthesized in cell culture and are variable starting from clone selection to final product packaging, and these variables potentially affect safety and efficacy (Birch and Racher, 2006;Shukla et al., 2007). Important factors include primary structure, disulfide linkages, post-translational modifications, and high order structure and stability over the lifetime of the antibody (Beck et al., 2013;Alt et al., 2016). ...
Antigen-antibody interactions are a fundamental subset of protein-protein interactions responsible for the “survival of the fittest.” Determining the interacting interface of the antigen, called an epitope, and that on the antibody, called a paratope, is crucial to antibody development. Because each antigen presents multiple epitopes (unique footprints), sophisticated approaches are required to determine the target region for a given antibody. Although X-ray crystallography, Cryo-EM, and nuclear magnetic resonance can provide atomic details of an epitope, they are often laborious, poor in throughput, and insensitive. Mass spectrometry-based approaches offer rapid turnaround, intermediate structural resolution, and virtually no size limit for the antigen, making them a vital approach for epitope mapping. In this review, we describe in detail the principles of hydrogen deuterium exchange mass spectrometry in application to epitope mapping. We also show that a combination of MS-based approaches can assist or complement epitope mapping and push the limit of structural resolution to the residue level. We describe in detail the MS methods used in epitope mapping, provide our perspective about the approaches, and focus on elucidating the role that HDX-MS is playing now and in the future by organizing a discussion centered around several improvements in prototype instrument/applications used for epitope mapping. At the end, we provide a tabular summary of the current literature on HDX-MS-based epitope mapping.
... On the other hand, acid exposure is a severe issue in the production process of antibody pharmaceuticals. Although treatment with acidic solutions is one of the key steps used in the column purification of antibodies and inactivation of viruses, antibodies are always at risk of acid denaturation due to acid exposure [19,20]. Therefore, acid-induced effects on stability and conformation of antibodies are well studied [21][22][23][24][25][26]. ...
Purpose
Antibody drugs are usually formulated as highly-concentrated solutions, which would easily generate aggregates, resulting in loss of efficacy. Although low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore, knowing the physical properties at low pH under high concentration conditions is important.
Methods
Raman spectroscopy was used to investigate pH-induced conformational changes of antibodies at 50 mg/ml. Experiments in pH 3 to 7 were performed for human serum IgG and recombinant rituximab.
Results
We detected the evident changes at pH 3 in Tyr and Trp bands, which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH range demonstrated that the thermal transition temperature (Tm) was highest at pH 3. Acid-treated and neutralized one showed higher Tm than that of pH 7, indicating that their extent of intermolecular interactions correlated with the Tm values. Onset temperature was clearly different between concentrated and diluted samples. Colloidal analyses confirmed the findings of the Raman analysis.
Conclusion
Our studies demonstrated the positive correlation between Raman analysis and colloidal information, validating as a method for evaluating antibody conformation associated with aggregation propensities.
... Lead molecules should express acceptably well and ideally be amenable to a platform-based purification strategy. 92 While a final lead may be selected based on transiently expressed material, material from stable cell-lines may express at higher purity and yield and have more representative core glycosylation profiles. 93 A final lead should have acceptable yield and purity following all platform purification steps, such as ProA affinity, low pH inactivation, anion-exchange (AEX) flow through, and cation-exchange (CEX) bind and elute processes. ...
Large-molecule antibody biologics have revolutionized medicine owing to their superior target specificity, pharmacokinetic and pharmacodynamic properties, safety and toxicity profiles, and amenability to versatile engineering. In this review, we focus on preclinical antibody developability, including its definition, scope, and key activities from hit to lead optimization and selection. This includes generation, computational and in silico approaches, molecular engineering, production, analytical and biophysical characterization, stability and forced degradation studies, and process and formulation assessments. More recently, it is apparent these activities not only affect lead selection and manufacturability, but ultimately correlate with clinical progression and success. Emerging developability workflows and strategies are explored as part of a blueprint for developability success that includes an overview of the four major molecular properties that affect all developability outcomes: 1) conformational, 2) chemical, 3) colloidal, and 4) other interactions. We also examine risk assessment and mitigation strategies that increase the likelihood of success for moving the right candidate into the clinic.
... This generally occurs during the viral clearance, chromatography and ultrafiltration/diafiltration (UF/DF) steps. Mitigation strategies are focussed on reducing the residence time at low pH conditions and gentle pH shifts in UF/DF can circumvent the inherent critical effects of these processes in the final mAb product [51]. ...
Therapeutic antibodies dominate the biopharmaceutical market with continual innovations being made to provide novel and improved antibody treatment strategies. Speed to-market and cost-efficiency are of increasing importance due to the changing landscape of the biopharmaceutical industry. The increasing levels of competition from biosimilars, the increase in small volume products and political and social pressure to reduce the cost of treatments are some of the challenges currently being faced. Chinese hamster ovary (CHO) cells have been the workhorse in the production of therapeutic antibodies over the last 36 years due to the robust nature and high productivity of these cell lines. However, there are many biomanufacturing challenges remaining. The aim of this review is to examine the current biological, and engineering challenges facing the biomanufacturing of antibodies and to identify the mitigations and emerging technologies that can be employed to overcome them. Developments in cell line engineering, intensified processing, continuous manufacturing, automation and innovations in process analytical technologies and single use technology will be discussed with regard to their ability to improve the current performance of mAb production processes.
... Vicente et al. [54] reported a slightly higher recovery yield of 82 % (vs 76 % obtained in this work using [Si][N 3888 ]Cl), however, with a lower purification factor. Affinity chromatography, namely Protein A affinity, is one of the commonly used techniques for antibody purification due to its high selectivity to IgG (high purity, > 90 %) from complex biological sources [55,56]. However, there is an urgent demand towards the development of novel chromatographic resins with improved stability, which will be essential to decrease the purification costs. ...
Over the past few years, antibodies such as immunoglobulin G, IgG, have increased their market share as alternative therapeutics. However, their production at high purity levels is still costly due to the absence of a cost-effective platform for their recovery and purification from the complex biological media in which they are produced. This work describes, for the first time, that materials modified with ionic liquids (ILs) can be designed for the effective capture and purification of antibodies from complex matrices, allowing both the selective adsorption of IgG or the selective adsorption of other proteins present in the media. The best results correspond to IgG with 59 % of yield and 84 % of purity in the aqueous solution, and IgG with 76 % of yield and 100 % of purity on the surface of one SIL due to the selective adsorption of IgG from human serum. The best conditions and materials were then applied to other IgG-containing matrices, namely rabbit serum and Chinese hamster ovary (CHO) cell culture supernatants, proving the robustness of the developed strategy. Furthermore, it is demonstrated that the secondary structure of IgG is preserved during the purification process and that these antibodies remain biologically active. In summary, it is shown that by only changing the IL chemical structure at the material surface it is possible to selectively adsorb IgG or to adsorb other proteins leaving IgG in solution. These findings prove that SILs are customizable materials with future potential to act in the flow-through or bind-and-elute modes. Therefore, SILs can be envisioned as potential chromatographic columns capable of substituting the high-cost commercial chromatographic columns based on biological ligands currently used to purify IgG.
... Triazine-based ligands, the oldest biomimetic affinity ligands, are considered ideal for the purification of high-value biopharmaceutical proteins because of their economic cost, chemically defined and nontoxic nature, and no cleavable bonds [ 9 , 10 ]. Furthermore, these ligands are sterilizable, resistant to chemical, and biodegradation, can be cleaned in situ, and easily immobilized to produce affinity-selective adsorbents with usable capacities for their complementary proteins [11] . Some of these commercially available ligands offer excellent selectivity, high binding capacity, and chemical stability [12] . ...
Immunoglobulin purification from different biological fluids is considered one of the most critical steps in antibody production for diagnostic, therapeutic, and research purposes. The current study aimed to elucidate the role of the different aryl substituents in triazine-based affinity ligands on the performance of an affinity chromatography purification media to separate immunoglobulin G (IgG). The biomimetic triazine-based affinity ligand was chosen as a varied containing fix spacer and support. The sepharose beads were activated by epichlorohydrin, and five types of aryl substituents were replaced in the triazine ring and covalently immobilized to the resin surface by 1, 4-diaminobutane spacer. All affinity resins with various ligands were characterized and validated using FTIR, SEM, EDX, and microscopic images. The findings revealed that using R1=3-aminophenol and R2=3-aminophenol substituents in the triazine ring, as affinity ligands attached to the sepharose surface with a 10-atom linker CAES-6B-Cl@R1= MAF, R2= MAF (No. 4), leads to better purification of IgG from human and rabbit plasma with 22.8 mg/mL resin binding capacity in 73±5% yield and 95% of purity. All results confirmed that the designed triazine-based affinity ligands could effectively purify IgG compatible with a fast and low-cost approach.
... The mAb proteins bound with the Protein A ligands are then eluted with an acidic buffer such as sodium acetate, glycine, or sodium citrate at pH 3.0-4.0 [13][14][15] . ...
Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.
... Since their introduction in the mid-1980's, monoclonal antibodies (mAbs) have become the largest class of human therapeutics, in terms of market size, due to their broad application in the treatment of cancer, autoimmune diseases, and various infectious diseases. Manufacturing processes for mAbs have evolved dramatically over the past thirty years [1]. The low productivity (<0.1 g/L mAb titer) of early cell culture processes required the use of very selective Protein A affinity chromatography as the initial capture step for mAb purification, and Protein A is still used as part of the platform process for essentially all mAb products [2]. ...
Recent increases in monoclonal antibody titer from Chinese Hamster Ovary cell culture has led to renewed interest in precipitation for the initial capture/purification of these high-value proteins. In this work, we examined the effect of the membrane module geometry on the sustainable flux and fouling mechanisms using human serum Immunoglobulin G (IgG) precipitated with 10 mM zinc chloride and 7 w/v % polyethylene glycol as a model system. The sustainable flux was evaluated using flux stepping experiments for both open channel cassettes and hollow fiber membrane modules. The hollow fiber modules had relatively low sustainable flux due to clogging of the fibers at the module inlet, with this behavior confirmed using both SEM imaging and hydraulic permeability data for the fouled modules. In contrast, the open channel cassettes showed no evidence of channel clogging, enabling continuous operation for at least 24 h at a filtrate flux below the experimentally determined sustainable flux. These results provide important insights into the origin of the sustainable flux during tangential flow filtration of precipitated proteins, greatly facilitating the design of precipitation-filtration processes for continuous purification of monoclonal antibody products.
... On the other hand, acid exposure is a severe issue in the production process of antibody pharmaceuticals. Although treatment with acidic solutions is one of the key steps used in the column purification of antibodies and inactivation of viruses, antibodies are always at risk of acid denaturation due to acid exposure [19,20]. Therefore, acid-induced effects on stability and conformation of antibodies are well studied [21][22][23][24][25][26]. ...
Antibody drugs are usually formulated as highly-concentrated solutions, which would easily generate oligomer and aggregation, resulting in loss of efficacy. Although low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore, knowing the physical properties of antibodies at low pH under high concentration conditions is an important insight into the quality evaluation of high concentration antibodies. Raman spectroscopy is a powerful tool to obtain conformational information derived from amino acid residues and secondary structures without dilution. In this study, Raman spectroscopy was used to investigate pH-induced conformational changes of antibodies at high concentrations. Raman experiments in pH 3 to 7 were performed for human serum IgG and recombinant rituximab. We detected the evident changes at pH 3 in Tyr and Trp Raman bands, which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH range demonstrated that the transition temperature (aggregation temperature, Tagg) was highest at pH 3. Acid-treated and neutralized one showed higher Tagg than that of pH 7, indicating that acid-induced conformational changes were not completely reversible. Colloidal analyses confirmed the findings of the Raman analysis, validating Raman spectroscopy as a method for evaluating antibody conformation associated with colloidal information.
... As such, there is an urgent demand to develop highly efficient separation processes to produce high-quality bioproducts (Smith 2005). To meet the quality requirements, downstream separation costs can account for more than 60% of the total cost of biomanufacturing process (Shukla et al. 2007). As the most important process in downstream separation processing, liquid chromatography (LC) has been commonly used for the separation and purification of biomacromolecules because of its mild process and high resolution. ...
Cellulose microspheres are commonly chromatographic media yet seriously limited in biomacromolecules separation and purification due to the slow mass transfer kinetics resulting from their narrow nanopores. Herein, a macroporous cellulose microsphere (MCM) with enhanced mass transfer ability has been successfully developed by an oil-in-water-in-oil (O1/W/O2) double emulsion strategy. The evolution profile of the double emulsion was tracked and achieved the optimization of interconnected macroporous structure. The macroporous structure not only provide fast mass transfer pathways for proteins but also increase the accessibility of meso/micropores. Benefitting from the macropores, the obtained diethylaminoethyl-modified MCM (DEAE-MCM) exhibits high permeability (3.81 × 10–13 m²), and fast adsorption rate (reaching equilibrium within 40 min) and high adsorption capacity (334.21 mg/g) for bovine serum albumin, far superior to commercially DEAE Sepharose Fast Flow. More importantly, under the high flow rate, DEAE-MCM remains a high dynamic adsorption capacity, promising it for fast protein chromatography.
As the biopharmaceutical industry matures and embraces process intensification methodologies allied to the emergence of newer personalized medicines, a key constant is the regulatory need to purify products that satisfy the criteria of safety, quality, and efficacy in each batch of released product destined for clinical use. Downstream processing operations and in particular chromatographic separations continue to play a key role in manufacturing strategies with the industry being well served by commercially available resins that provide different options to purify a particular target molecule of interest. In recent years, mixed-mode chromatography, a technique based on multimode interactions between ligands and proteins, had attracted much attention. This short review will discuss the concept and benefit of mixed-mode chromatography in purification strategies and specifically look at its application in the purification of IgG subtype monoclonal antibodies, a key product class in today's industry.
Integrated continuous bioprocessing has been identified as the next important phase of evolution in biopharmaceutical manufacturing. Multiple platform technologies to enable continuous processing are being developed. Multi-column counter-current chromatography is a step in this direction to provide increased productivity and capacity utilization to capture biomolecules like monoclonal antibodies (mAbs) present in the reactor harvest and remove impurities. Model-based optimization of two prevalent multi-column designs, 3-column and 4-column periodic counter-current chromatography (PCC) was carried out for different concentrations of mAbs in the feed, durations of cleaning-in-place and equilibration protocols. The multi-objective optimization problem comprising three performance measures, namely, product yield, productivity, and capacity utilization was solved using the Radial basis function optimization technique. The superficial velocities during load, wash, and elute operations, along with durations of distinct stages present in the multi-column operations were considered as decision variables. Optimization results without the constraint on number of wash volumes showed that 3-Column PCC performs better than 4-Column PCC. For example, at a feed concentration of 1.2 mg/mL, productivity, yield and capacity utilization, respectively, were 0.024 mg/mL.s, 0.94, and 0.94 for 3-Column PCC and 0.017 mg/mL.s, 0.87, and 0.83 for 4-column PCC. Similar trends were observed at higher feed concentrations also. However, when the constraint on number of wash volumes is included, 4-Column PCC was found to result in consistent productivity and product yield under different operating conditions but at the expense of reduced capacity utilization.
Protein A affinity chromatography is an important step in the purification of monoclonal antibodies (mAbs) and mAb-derived biotherapeutics. While the biopharma industry has extensive expertise in the operation of protein A chromatography, the mechanistic understanding of the adsorption/desorption processes is still limited, and scaling up and scaling down can be challenging because of complex mass transfer effects in bead-based resins. In convective media, such as fiber-based technologies, complex mass transfer effects such as film and pore diffusions do not occur which facilitates the study of the adsorption phenomena in more detail and simplifies the process scale-up. In the present study, the experimentation with small-scale fiber-based protein A affinity adsorber units using different flow rates forms the basis for modeling of mAb adsorption and elution behavior. The modeling approach combines aspects of both stoichiometric and colloidal adsorption models, and an empirical part for the pH. With this type of model, it was possible to describe the experimental chromatograms on a small scale very well. An in silico scale-up could be carried out solely with the help of system and device characterization without feedstock. The adsorption model could be transferred without adaption. Although only a limited number of runs were used for modeling, the predictions of up to 37 times larger units were accurate.
Continuous crystallization is an area of intense research, with particular respect to the pharmaceutical industry and fine chemicals. Improvements in continuous crystallization technologies offer chemical industries significant financial gains, through reduced expenditure and operational costs, and consistent product quality.
Written by well-known leaders in the field, The Handbook of Continuous Crystallization presents fundamental and applied knowledge, with attention paid to application and scaling up, and the burgeoning area of process intensification. Beginning with concepts around crystallization techniques and control strategies, the reader will learn about experimental methods and computational tools. Case studies spanning fine and bulk chemicals, the pharmaceutical industry, and employing new mathematical tools, put theory into context.
Recent work has shown that aggregates in monoclonal antibody (mAb) solutions may be made up not just of mAb oligomers but can also harbor hundreds of host-cell proteins (HCPs), suggesting that aggregate persistence through downstream purification operations may be related to HCP clearance. We have examined this in a primary analysis of aggregate persistence through processing steps that are typically implemented for HCP reduction, demonstrating that the phenomenon is relevant to depth filtration, protein A chromatography and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy observations show that aggregates compete with the mAb to adsorb specifically in protein A chromatography and that this competitive interaction is integral to the efficacy of protein A washes. Column chromatography reveals that the protein A elution tail can have a relatively high concentration of aggregates, which corroborates analogous observations from recent HCP studies. Similar measurements in flow-through AEX chromatography show that relatively large aggregates that harbor HCPs and that persist into the protein A eluate can be retained to an extent that appears to depend primarily on the resin surface chemistry. The total aggregate mass fraction of both protein A eluate pools (∼ 2.4 - 3.6%) and AEX flow-through fractions (∼ 1.5 - 3.2%) correlates generally with HCP concentrations measured using enzyme-linked immunosorbent assay (ELISA) as well as the number of HCPs that may be identified in proteomic analysis. This suggests that quantification of the aggregate mass fraction may serve as a convenient albeit imperfect surrogate for informing early process development decisions regarding HCP clearance strategies.
Multimodal chromatography resins are becoming a key tool in the purification of biomolecules. The main objective of this research was the establishment of an iterative framework for the rapid development of new multimodal resins to provide novel selectivity for the future purification challenges. A large chemically diverse virtual library of 100 multimodal Capto™ MMC ligand analogues was created, and a broad array of chemical descriptors were calculated for each ligand in silico. Principal component analysis (PCA) was used to map the chemical diversity and guide selection of ligands for synthesis and coupling to the Capto ImpRes agarose base matrix. Twelve new ligands were prepared in two groups: 'group one' consist of L00-L07 and 'group two' consist of L08-L12. These ligands are diverse in the influence of varied secondary interactions such as hydrophobic interactions, H-bonding, etc. Additional resin prototypes were also prepared to look at the chromatographic impact of ligand density variation. High-throughput plate-based studies were performed for parallel resin screening for batch-binding of six model proteins at different chromatographic binding pH and sodium chloride concentration conditions. Principal component analysis of the binding data provided a chromatographic diversity map leading to the identification of ligands with improved binding. Further, the new ligands have improved separation resolution between a monoclonal antibody (mAb1) and product related impurities, a Fab fragment and high molecular weight (HMW) aggregates, using linear salt gradient elutions. To quantify the importance of secondary interactions, analysis of the retention factor of mAb1 on the ligands at various isocratic conditions lead to estimations of (a) the total number of water molecules and counter salt ions released during adsorption, and (b) hydrophobic contact area (HCA). The iterative mapping approach of chemical and chromatography diversity maps described in the paper proves to be a promising method for identifying new chromatography ligands for biopharmaceutical purification challenges.
High-performance and cost-effective purification is necessary for the development of antibody drugs. This study found that nanoparticles of zirconia modified with phosphate groups selectively adsorb immunoglobulin G (IgG) antibodies against serum proteins with high adsorption capacity. The IgG antibodies collected from the zirconia nanoparticle surfaces retain their molecular conformation. Importantly, zirconia nanoparticles have the highest affinity for human IgG antibodies among tested mammalian IgG antibodies. The affinity for human IgG subclasses is in the order IgG3 > IgG1 > IgG2, which contrasts with a conventional ligand (Protein A) that has a lower affinity for IgG3. Because zirconia nanoparticles are chemically and mechanically stable, they can be utilized for the purification of antibody drugs not only in batch methods but also in chromatography as a process upstream or downstream of Protein A chromatography and even as an alternative process.
In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global Regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other proteins expressed in Chinese Hamster Ovary (CHO) cells. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of high pH and minimum hold duration. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (> 4 log10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65 to 3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies. This article is protected by copyright. All rights reserved.
Multimodal chromatography offers an increased selectivity compared to unimodal chromatographic methods and is often employed for challenging separation tasks in industrial downstream processing (DSP). Unfortunately, the implementation of multimodal polishing into a generic downstream platform can be hampered by non-robust platform conditions leading to a time and cost intensive process development. Mechanistic modeling can assist experimental process development but readily applicable and easy to calibrate multimodal chromatography models are lacking. In this work, we present a mechanistic modeling aided approach that paves the way for an accelerated development of anionic mixed-mode chromatography (MMC) for biopharmaceutical purification. A modified multimodal isotherm model was calibrated using only three chromatographic experiments and was employed in the retention prediction of four antibody formats including a Fab, a bispecific, as well as an IgG1 and IgG4 antibody subtype at pH 5.0 and 6.0. The chromatographic experiments were conducted using the anionic mixed-mode resin Capto adhere at industrial relevant process conditions to enable flow through purification. An existing multimodal isotherm model was reduced to hydrophobic interactions in the linear range of the adsorption isotherm and successfully employed in the simulation of six chromatographic experiments per molecule in concert with the transport dispersive model (TDM). The model reduction to only three parameters did prevent structural parameter non-identifiability and enabled an analytical isotherm parameter determination that was further refined by incorporation of size exclusion effects of the selected multimodal resin. During the model calibration, three linear salt gradient elution experiments were performed for each molecule followed by an isotherm parameter uncertainty assessment. Lastly, each model was validated with a set of step and isocratic elution experiments. This standardized modeling approach facilitates the implementation of multimodal chromatography as a key unit operation for the biopharmaceutical downstream platform, while increasing the mechanistic insight to the multimodal adsorption behavior of complex biologics.
Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g., mechanical, or physical stresses, can determine the overall conformational stability of the protein to enhance drug product shelf-life. Several groups have reported surface-induced unfolding and aggregation of monoclonal antibodies and their derivatives during cation exchange chromatography, which results in a two-peak elution behavior of the protein and its species. We have investigated universal influencing factors, like temperature and hold time, on this phenomenon. The formation of the second peak is a kinetic process, which is strongly influenced by temperature during the hold time. However, our main focus was the application of excipients and their influence on the two-peak elution behavior. We compared the on-column screening results with results obtained through a “traditional” in-solution screening using nanoDSF. Mostly, stabilizing excipients, like Sucrose, show their stabilizing abilities in both systems, but some discrepancies, e.g., using Arginine, between the two orthogonal techniques show the potential of the on-column screening system to lead to unexpected results, which would not necessarily be visible in in-solution experiments.
Monoclonal antibodies (mAbs) have become predominant therapeutics by providing highly specific mechanisms of action enabling treatment of complex diseases. However, mAbs themselves are highly complex and require thorough testing and characterization to ensure efficacy and patient safety. In this regard, fragmentation is a degradation product of concern. The biotechnology industry uses capillary gel electrophoresis (CGE) to quantify fragmentation by electrophoretically resolving size variants, such as products resulting from partial reduction of interchain disulfides. However, standard CGE methods may not adequately separate less typical fragments, particularly when there is minimal size difference to the parent molecule. For mAb-1, a degradant only ∼11 kDa smaller than the intact mAb (∼149 kDa) was unable to be resolved under typical non-reducing conditions, preventing an accurate purity assessment and precluding tracking of product purity within stability studies. To address these deficiencies, a subunit-based non-reducing CGE method was developed to employ IdeS protease to produce F(ab')2 and Fc fragments, which resulted in baseline resolution of the clipped subunit species from its parent species. This enabled more accurate trending of purity throughout stability studies. Method characterization ensured that this subunit method monitored expected impurities observed by intact non-reducing CGE and thus could suitably replace non-reducing CGE in the release and stability testing panel. It also has the potential to replace reducing CGE based on its tracking of the deglycosylated Fc species. We believe this approach of utilizing proteases to develop subunit CGE methods for release and stability can be applied to other molecules when in need of resolving analogous fragments.
Recent development in Protein A chromatography has adopted wash buffers with a wide range of pH to improve the removal of process-related impurities, e.g., host cell proteins (HCPs), from Fc-based target biological products. Thus, it is important to investigate the pH effect on the binding behaviors of target products of various properties on Protein A resins. Here we performed column breakthrough experiments for two monoclonal antibodies (mAbs) and one Fc-fusion protein on two Protein A resins from pH 4 to pH 10, and the experimental data was analyzed using a mechanistic model to obtain isotherm and mass transfer parameters. The two mAbs exhibited relatively high dynamic binding capacity (DBC) at 10% breakthrough of 43 – 67 g/L at pH 6–9 followed by a ∼30% decrease from pH 9 to pH 10; while the Fc-fusion protein reached the highest DBC at 10% breakthrough of 10 – 17 g/L at pH 5 and thereafter the capacity gradually reduced. Model analyses revealed that the two mAbs had higher maximum binding capacity by 2 – 5 fold but weaker binding affinity (12 – 64%) than the Fc-fusion protein from pH 5 to pH 9. For the three molecules, similar patterns of the pH impact were observed on the two Protein A resins with the Jetted A50 resin showing generally higher DBC and stronger binding affinity than the MabSelect SuRe LX resin. Additionally, an inverse relationship between the binding affinity and surface diffusivity was observed for both resins. Besides obtaining the isotherm parameters from the column breakthrough data, a direct batch equilibrium measurement showed comparable trend in these parameters with relatively more scattered values due to the inherent uncertainties to accurately determine the initial slope of the isotherm in highly favorable adsorption conditions. Finally, isothermal titration calorimetry (ITC) results revealed that the measured binding affinity using free Protein A ligand was stronger than that obtained from the breakthrough and isotherm results for the resin, possibly due to the reduced accessibility of the immobilized ligand on resin surface. Overall, this work can facilitate future Protein A ligand design and affinity chromatography process optimization for biomanufacturing.
Biologics encompasses a wide variety of therapeutics including monoclonal antibodies, fusion proteins, and enzymes, among others. The biologics market is increasing at a rapid pace and different manufacturing processes, including continuous manufacturing processes, are being increasingly adopted. There is a strong drive to assess the sustainability of such processes. Here, we calculated the process mass intensity (PMI) of a continuous manufacturing process and compared it to the PMI of traditional fed-batch processes for monoclonal antibodies (mAbs). Results show that the PMI of the continuous manufacturing process is comparable to that of batch processes. Sensitivity analysis was performed to identify potential strategies to improve the material efficiency of continuous processes. Although PMI does not account for all sources of environmental impact from a biologics manufacturing process, such as energy consumption, drug substance dosing and demand, it is a powerful benchmarking metric to compare material efficiencies of processes, as well as a tool to emphasize the main impactful areas for environmentally friendly innovation. Comparison of a higher PMI continuous process with a lower PMI batch process operating at the same bioreactor scale shows that since the productivity (in g of drug substance, DS) per unit time is multifold higher for the continuous process, the overall energy consumption per unit of DS produced might be lower leading to a more environmentally sustainable process. This study highlights some of these key aspects that would require additional metrics and models to be developed to assess the overall sustainability of biologics processes.
Therapeutic monoclonal antibodies (mAbs) provide new means for treatments of a wide range of diseases and comprise a large fraction of all new approved drugs. Production of mAbs is expensive compared to conventional drug production, primarily due to the complex processes involved. The affinity purification step is dominating the cost of goods in mAb manufacturing. Process intensification and automation could reduce costs, but the lack of real-time process analytical technologies (PAT) complicates this development. We show a specific and robust fiber optical localized surface plasmon resonance (LSPR) sensor technology that is optimized for in-line product detection in the effluent in affinity capture steps. The sensor system comprises a flow cell and a replaceable sensor chip functionalized with biorecognition elements for specific analyte detection. The high selectivity of the sensor enable detection of mAbs in complex sample matrices at concentrations below 2.5 μg mL-1. In place regeneration of the sensor chips allowed for continuous monitoring of multiple consecutive chromatographic separation cycles. Excellent performance was obtained at different purification scales with flow rates up to 200 mL min-1. This sensor technology facilitates efficient column loading, optimization, and control of chromatography systems, which can pave the way for continuous operation and automation of protein purification steps.
Allogeneic chimeric antigen-receptor T-cell process flows comprise large-scale primary cultures incorporating traditional biologics processing steps such as cell expansion, with novel processing steps such as electroporation. In addition, these process flows begin with donor-derived peripheral blood mononuclear cells and generate single batches of product, which may be used to treat hundreds of patients. The corresponding unit operation-process safety considerations are individually diverse, and in combination are currently unique and rapidly evolving. The use of available regulations and the principles of quality by design are useful in designing appropriate control strategies into the process during development to ensure process safety.
Since the earliest days, occurrence of deadly outbreaks caused by prevalence of different pathogenic virus infections affected human community periodically and caused millions of deaths per year. In order to improve safety and quality of human life against virus infections, it is imperative to do rigorous evaluation of existing virus treatment or elimination processes that would help to understand their potential merits, limitations and further prompts identifying the scope for developing advanced future technologies towards viral elimination. Considering the objective, present review comprehensively analyze present scope, potential and limitations of individual physical, chemical and biological methods in the direction of holistic management (recovery, purification, detection, elimination and disinfection) of viruses from liquid, solid, gaseous and interfacial environments. Among various treatment approaches, membrane filtration technology has been recognized as most promising, cost effective and energy efficient tool to deal with virus elimination from various resources. Membrane-based methods facilitates wide ranges of virus removal and disinfection capacity, mediated by size-exclusion, electrostatic or hydrophobic interactions and entrapment mechanisms. Based on the present demand of developing a complete and fully mature virus disinfection technique, potential of electro-spun nanofibrous membranes in selective capture and inactivation of viruses are clearly evaluated in the present review. Furthermore, it was found out from comprehensive analysis of available membrane mediated virus elimination processes that membranes derived from different forms of abundantly available, renewable and natural polymers such as cellulose are quite effective to capture, separate and inactivate viruses through depth filtration, charge based filtration and adsorption-oriented techniques.
Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation.
Monoclonal antibodies (mAbs) have been served as ‘magic bullet’ to treat autoimmune diseases, cancers, and infectious diseases. Due to the complexity of biological sample and the interference of high abundance impurities, affinity enrichment of mAbs is crucial for downstream process and in vivo analysis. This review mainly focuses on the emerging affinity methods for antibody enrichment in recent years. Alternative affinity ligands for mAb recognition, including engineered staphylococcal protein A (SpA) mimicking ligands, biomimetic peptides, aptamers, and engineered small molecules, will be described in detail. Moreover, recent progress in support materials, such as porous polymers, membranes, magnetic adsorbents, molecularly imprinted polymers, and nanomaterials, will also be highlighted. Finally, these emerging methods for antibody enrichment will be summarized and their future development trends will be discussed.
Nanoparticles are readily coated by proteins in biological systems. The protein layers on the nanoparticles, which are called the protein corona, influence the biological impacts of the nanoparticles, including internalization into cells and cytotoxicity. This study expands the scope of the nanoparticle's protein corona for exogenous artificial nanoparticles to that for exogenous proteinaceous nanoparticles. Specifically, this study addresses the formation of protein coronas on nanoscale human antibody aggregates with a radius of approximately 20-40 nm, where the antibody aggregates were induced by a pH shift from low to neutral pH. The size of the human immunoglobulin G (hIgG) aggregates grew to approximately 25 times the original size in the presence of human serum albumin (HSA). This size evolution was ascribed to the association of the hIgG aggregates, which was triggered by the formation of the hIgG aggregate's protein corona, i.e., protein's protein corona, consisting of the adsorbed HSA molecules. Because hIgG aggregate association was significantly reduced by the addition of 30-150 mM NaCl, it was attributed to electrostatic attraction, which was supported by molecular dynamics (MD) simulations. Currently, the use of antibodies as biopharmaceuticals is concerning because of undesired immune responses caused by antibody aggregates that are typically generated by a pH shift during the antibody purification process. The present findings suggest that nanoscale antibody aggregates form protein coronas induced by HSA and the resulting nanoscale antibody-HSA complexes are stable in blood containing approximately 150 mM salt ions, at least in terms of the size evolution. Mechanistic insights into protein corona formation on nanoscale antibody aggregates are useful for understanding the unintentional biological impacts of antibody drugs.
Process analytical technology (PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance. Currently, biopharmaceutical producers mainly monitor chromatographic processes with Ultraviolet/Visible (UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody (mAb) and host cell proteins (HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration, respectively. Each 0.1 column volume (CV) fraction of UV/Vis chromatogram peak area were calculated, and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high mAb recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs
The safety requirements for vaccines are extremely high since they are administered to healthy people. For that reason, vaccine development is time-consuming and very expensive. Reducing time-to-market is key for pharmaceutical companies, saving lives and money. Therefore the need is raised for systematic, general and efficient process development strategies to shorten development times and enhance process understanding. High throughput technologies tremendously increased the volume of process-related data available and, combined with statistical and mechanistic modeling, new high throughput process development (HTPD) approaches evolved. The introduction of model-based HTPD enabled faster and broader screening of conditions, and furthermore increased knowledge. Model-based HTPD has particularly been important for chromatography, which is a crucial separation technique to attain high purities.
This review provides an overview of downstream process development strategies and tools used within the (bio)pharmaceutical industry, focusing attention on (protein subunit) vaccine purification processes. Subsequent high throughput process development and other combinatorial approaches are discussed and compared according to their experimental effort and understanding. Within a growing sea of information, novel modeling tools and artificial intelligence (AI) gain importance for finding patterns behind the data and thereby acquiring a deeper process understanding.
Still today, high–concentration protein formulations are often developed based on high-throughput experimental screening approaches. Although likely delivering working formulations, these approaches do not lead to a deep / mechanistic understanding of the protein phase behavior in solution. Within this work, we thus optimized and enhanced a recent approach for an initial low effort selection of potential excipients and excipient mixtures to be used in high concentration protein formulations. This approach considers both: molecular interactions and thermodynamic determinants to access the phase behavior of the proteins in solution, as well as pharmaceutical engineering boundaries (such as osmotic pressure and osmolality) to deliver on optimal formulation conditions. Water activity coefficient γW–calculations (used to describe the protein environment in solution), unfolding temperature (conformational stability) and protein–protein interactions (colloidal stability) are used as determinants. Amino acids (20 proteinogenic amino acids), selected amino acid mixtures, as well as mixtures of amino acids and trehalose (l-arginine-trehalose; l-histidine-trehalose) are considered as model excipients. The approach is extends by studying the long-term stability of the predicted formulation conditions for a γ globulin from human blood and denosumab. The results reveal, that by combining protein-specific experiments as well as model-based studies for the selection of excipient mixtures in high concentration protein formulations, the effort as well as the resource requirements can be reduced significantly.
For CHO expressed monoclonal antibodies (mAbs), host cell proteins (HCPs) represent a major class of process-related impurities and their removal is a key focus of downstream process development. HCPs are highly heterogeneous in nature, differing in molecular weight, isoelectric point and hydrophobicity, and some of them can be difficult to remove. Although Protein A affinity chromatography alone can typically remove more than 90% of HCPs in the clarified culture harvest, in many cases reducing HCPs in the final drug product to an acceptable level (e.g., <100 ppm) can still be a challenging task. The relative difficulty of HCP clearance is case dependent and in certain cases a small population of HCPs can persist throughout the downstream process. This article reviews the two major mechanisms that contribute to copurification of CHO HCPs, namely leaching from chromatin heteroaggregates and nonspecific HCP-mAb association. In addition, strategies that can minimize the impact of these two factors are briefly discussed.
Fifty-seven strains of streptococci belonging to groups A, C, and G were tested for γ-globulin Fc-reactivity with sensitized sheep red cells. Twenty-six per cent of the strains were positive. Forty-four per cent of the strains produced a soluble factor with similar γ-globulin reactivity. With a radioimmunologic assay to detect streptococcal reactivity, human IgG was found to be positive, whereas human IgA, IgM, IgD, and IgE were negative. The reactivity was detected in heavy chain preparations but not in light chains, F(ab′)2 fragments nor in Fc fragments. Experiments using isolated myeloma globulins representing the four IgG subclasses indicated that streptococci might react not only with IgG1-, IgG2-, and IgG4-like protein A but also with IgG3 molecules. One of the two IgG3 myeloma globulins studied did react with a group A streptococcus and both myeloma globulins did react with a group G streptococcus. Serum samples from 14 selected animal species also indicated differences between the γ-globulin reactivity of protein A and streptococci. Marsupials as well as the Rhea bird were non-reactive with streptococci. The results obtained with eleven other species indicated that the group A and G streptococcal reactivities were similar but differed significantly from staphylococcal protein A reactivity.
Low-molecular-weight synthetic molecules that mimic the activity of native biological macromolecules have therapeutic potential, utility in large-scale production of biopharmaceuticals, and the capacity to act as probes to study molecular recognition events. We have developed a nonpeptidyl mimic for Staphylococcus aureus Protein A (SpA). The specific recognition and complexation elements between the B domain (Fb) of SpA and the Fc fragment of IgG were identified from the x-ray crystallographic structure. Computer-aided molecular modeling was used to design a series of biomimetic molecules around the Phe132-Tyr133 dipeptide involved in its binding to IgG. One of the ligands binds IgG competitively with SpA in solution and when immobilized on agarose beads, with an affinity constant of 10(5)-10(6) M-1. The immobilized artificial Protein A was used to purify IgG from human plasma and murine IgG from ascites fluid, and to remove bovine IgG from fetal calf serum.
Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s. This increase in volumetric productivity has resulted mainly from improvements in media composition and process control. Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering.
Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.
There are currently 26 modern antibody-based therapeutic agents approved by the US and EU, with 500 more products in the global development pipeline. Total market value of these therapeutics could reach more than $17 billion by the close of 2010. This market overview includes all 26 products (plus two that were withdrawn) and their originators, their therapeutic indications, and their approval records.
Small, non-enveloped viruses can be difficult to inactivate or remove from source plasma. Virus filtration is a useful clearance method for these viruses. High-flux filters promise to be economical.
With West Nile virus in the news, viral inactivation is under public scrutiny. Yet, the culture media, the therapeutic product, and the potential viral contaminants can all affect the inactivation method chosen during production. Viral inactivation techniques - those tried or used in manufacturing or in final biopharmaceuticals, vaccines, and media products - have been gathered from recent scientific literature and organized in this article series as an addition to your process development toolbox.
Thorough process characterization can improve success rates in manufacturing, reduce the number of process related incidents, and significantly improve process yields.
Methodology for the purification of monoclonal antibodies from large-scale mammalian cell culture systems has been investigated. The concentration of monoclonal antibodies in conditioned cell culture media was generally found to be in the milligram per liter range, requiring 100–1,000 liters for production of gram quantities. To reduce the volumes, several ultrafiltration systems, including hollow fiber, plate, and frame, and spiral cartridges were investigated and found to be effective for large-scale work. Once a concentrated product was obtained, several methods including ammonium sulfate precipitation, ion exchange, protein. A agarose, and size exclusion chromatograhy were utilized and compared. The best results were obtained when concentrated conditioned medium was either diluted or diafiltered (to reduce the high ionic strength of conditioned medium) and fractionated by cation exchange chromatography. Variations in isoelectric points for different monoclonal antibodies require that specific pH and ionic strength parameters be determined to optimize binding and elution. Additional purification was achieved by further ion exchange steps or the use of ammonium sulfate precipitation. For particularly difficult purifications, protein A affinity chromatography was used. Once a purity of 90–95% was achieved, size exclusion chromatography was used as a final step to remove aggregates, process chemicals, contaminant proteins, and to exchange the antibody into the formulation buffer.
Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M–1. A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M–1, and ΔH = –7.1 ± 0.4 kcal mol–1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E-domain indicating that the E-domain binding site on Fab resides within VH. E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M–1, and ΔH = –24.6 ± 0.6 kcal mol–1. Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.
Production rate is an important parameter in the design of efficient protein A affinity chromatography processes for purifying recombinant monoclonal antibodies. A simple equation was derived that expresses production rate in terms of flow rate and column length. Changes in flow rate and column length will not affect the antibody and are therefore easily varied for bioprocess applications. In the equation, production rate depends on dynamic capacity, which can be expressed as a function of the load flow rate and column length. The only empirical data needed for production rate optimization is the relationship of dynamic capacity to load flow rate and column length, which was quickly determined by using an on-line assay. The optimal production rate was found at a high flow rate, a low column length, and a low dynamic capacity, which has several implications for using high production rate protein A affinity chromatography for antibody manufacturing.
Tangential flow filtration has traditionally been scaled up by maintaining constant the filtrate volume to membrane surface area ratio, membrane material and pore size, channel height, flow path geometry and retentate and filtrate pressures. Channel width and the number of channels have been increased to provide increased membrane area. Several other parameters, however, have not been maintained constant. A new comprehensive methodology for implementation of linear scale up and scale down of tangential flow filtration processes has been developed. Predictable scale up can only be achieved by maintaining fluid dynamic parameters which are independent of scale. Fluid dynamics are controlled by operating parameters (feed flow rate, retentate pressure, fed batch ratio and temperature), geometry (channel length, height, turbulence promoter and entrance/exit design), materials (membrane, turbulence promoter, and encapsulant compression), and system geometry (flow distribution). Cassette manufacturing procedures and tolerances also play a significant role in achieving scale independent performance. Extensive development work in the aforementioned areas has resulted in the successful implementation of linear scale up of ultrafiltration processes for recovery of human recombinant DNA derived pharmaceuticals. A 400-fold linear scale up has been achieved without intermediate pilot scale tests. Scale independent performance has a direct impact on process yield, protein quality and product economics and is therefore particularly important in the biotechnology industry. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 737-746, 1997.
We describe the performance characteristics of five Protein A affinity-chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host-cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.
A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.
Efficient harvest and recovery of high-purity monoclonal antibodies was achieved using hydrophobic charge induction chromatography (HCIC). Both simple and complex feedstocks were studied, including protein-free cell culture supernatant and the clarified/concentrated milk of transgenic goats. Viral clearance studies demonstrated a 4-log reduction of MVM virus (minute virus of mice), along with substantial reduction of DNA content. Sorbent characterization studies confirmed that HCIC is based on the pH-dependent behavior of a dual-mode, ionizable ligand. Binding, based on hydrophobic interaction, was achieved under near-physiological conditions, and in the absence of lyotropic salt. Desorption was accomplished under mild conditions--pH 4.0. At this pH, both ligand and antibody carry a net positive charge, and desorption occurs on the basis of electrostatic charge repulsion. pH-based control of chromatographic function was demonstrated. Chromatography on this antibody-selective HCIC sorbent was evaluated as a cost-effective, process-compatible alternative to affinity chromatography protein A sorbents.
With the technological advances made during the past decade, antibodies now represent an important and growing class of biotherapeutics. With the potential new targets resulting from genomics and with methods now in place to make fully human antibodies, the potential of antibodies as valuable therapeutics in oncology, inflammation and cardiovascular disease can be fully realised. Systems to produce these antibodies as full-length molecules and as fragments include expression in both mammalian and bacterial cells grown in bioreactors and in transgenic organisms. Factors including molecular fidelity and the cost of goods are critical in evaluating expression systems. Mammalian cell culture and transgenic organisms show the greatest promise for the expression of full-length, recombinant human antibodies, and bacterial fermentation seems most favorable for the expression of antibody fragments.
Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications.
Antibodies and antibody derivatives constitute twenty five percent of therapeutics currently in development, and a number of therapeutic monoclonal antibodies have recently reached the market. All antibodies approved by the US Food and Drug Administration, however, contain mouse protein sequences. These partially murine antibodies, therefore, have the potential to elicit allergic or other complications when used in human patients. Recent developments aim to reduce or eliminate murine components, and fully human antibodies are rapidly becoming the norm. A number of technologies exist which enable the development of 100% human antibodies.
Will we replace oil with wheat or corn as a feedstock for producing natural plastic? The success of biotechnology for bulk product manufacturing will heavily depend on engineering solutions in the downstream processes in which separation and purification have a crucial role with respect to commercial development. Development of efficient bioseparation methods is important for a broad range of business areas including pharmaceuticals, nutrition and health products, bio-based materials and crop protection chemicals. Depending on the value of the end product and the scale of production, the processing required varies significantly. Key factors that have an impact on the choice of separation strategy include process throughput, particle size of the product and impurities and the desired end-product concentration. The development of efficient, economical and selective separation methods will be required for successful commercialization of bioprocesses. Despite this well-recognized need, there are relatively few available methods for commercial implementations. Development of novel mechanical systems for selective separation of solid and liquid mixtures must become a top priority for current research investment to reduce the reliance on expensive chromatographic and thermal separation methods.
Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.
Protein A is a popular generic ligand for purification of monoclonal and recombinant antibodies. The performance of 15 commercially available protein A media was studied. Equilibrium and dynamic binding capacity for human IgG was determined and the capture of IgG from a crude feed-stock was investigated. For initial screening the dynamic binding capacity was determined at small scale. Media with good performance were further tested with increased column height. Comparing the data from the two different column heights it could be shown that the dynamic capacity strongly depends on the residence time. Agarose based media exhibited higher binding capacity at residence times longer than 3 min whereas polymeric media or media based on porous glass showed a lesser dependence on the flow velocity and the residence time. A quantitative description of this behavior was derived by determination of the adsorption isotherms and fitting the breakthrough profiles with the Thomas solution. Agarose based media exhibited higher maximum equilibrium binding capacities and the dissociation constants derived from adsorption isotherms were smaller. The other media exhibited higher apparent rate constants, indicating a faster mass transfer. This can be explained by the smaller particle diameter of these media and it can be assumed that constant pattern conditions are thereby obtained more quickly. Selectivity was tested by performing antibody purification under standardized conditions. Polyclonal human IgG in cell culture supernatant containing 2.5% fetal calf serum was used as a representative feed-stock. Under the applied conditions several sorbents showed very tight binding of IgG and in some cases most of the sample remained on the sorbent. The study can be useful as a guide for optimization of large-scale purification processes.
The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.
Protein A affinity chromatography is often employed as a capture step to meet the purity, yield, and throughput requirements for pharmaceutical antibody purification. However, a trade-off exists between step performance and price. Protein A resin removes 99.9% of feed stream impurities; however, its price is significantly greater than those of non-affinity media. With many therapeutic indications for antibodies requiring high doses and/or chronic administration, the consideration of process economics is critical. We have systematically evaluated the purification performance of cation-exchange, anion-exchange, hydroxyapatite, hydrophobic interaction, hydrophobic charge induction, and small-molecule ligand resins in each step of a three-step chromatographic purification process for a CHO-derived monoclonal antibody. Host cell proteins were removed to less-than-detectable for three processes (cation-exchange-anion-exchange-hydrophobic interaction chromatography, cation-exchange-anion-exchange-mixed cation-exchange chromatography, and cation-exchange-mixed cation-exchange-anion-exchange chromatography). The order of the process steps affected purification performance significantly.
Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.
The past 5 years have seen the commercialization of two recombinant protein products from transgenic plants, and many recombinant therapeutic proteins produced in plants are currently undergoing development. The emergence of plants as an alternative production host has brought new challenges and opportunities to downstream processing efforts. Plant hosts contain a unique set of matrix contaminants (proteins, oils, phenolic compounds, etc.) that must be removed during purification of the target protein. Furthermore, plant solids, which require early removal after extraction, are generally in higher concentration, wider in size range, and denser than traditional bacterial and mammalian cell culture debris. At the same time, there remains the desire to incorporate highly selective and integrative separation technologies (those capable of performing multiple tasks) during the purification process from plant material. The general plant processing and purification scheme consists of isolation of the plant tissue containing the recombinant protein, fractionation of the tissue along with particle size reduction, extraction of the target protein into an aqueous medium, clarification of the crude extract, and finally purification of the product. Each of these areas will be discussed here, focusing on what has been learned and where potential concerns remain. We also present details of how the choice of plant host, along with location within the plant for targeting the recombinant protein, can play an important role in the ultimate ease of recovery and the emergence of regulations governing plant hosts. Major emphasis is placed on three crops, canola, corn, and soy, with brief discussions of tobacco and rice.
A quantitative understanding of how proteins interact with hydrophobic charge induction chromatographic resins is provided. Selectivity on this mode of chromatography for monoclonal antibodies as compared to other model proteins is probed by means of a linear retention vs pH plot. The pH-dependent adsorption behavior on this mode of chromatography for a hydrophobic, charged solute is described by taking into account the equilibrium between a hydrophobic, charged solute and an ionizable, heterocyclic ligand. By analogy, an equation that is seen to adequately describe macromolecular retention under linear conditions over a range of pH is developed. A preparative, nonlinear isotherm that can capture both pH and salt concentration dependency for proteins is proposed by using an exponentially modified Langmuir isotherm model. This model is seen to successfully simulate adsorption isotherms for a variety of proteins over a range of pHs and mobile phase salt concentrations. Finally, the widely differing retention characteristics of two monoclonal antibodies are used to derive two different strategies for improving separations on this mode of chromatography. A better understanding of protein binding to this class of resins is seen as an important step to future exploitation of this mode of chromatography for industrial scale purification of proteins.
In this paper, a wide range of antibodies from various subclasses and subfamilies are employed to evaluate the creation of generic separation processes using Protein A chromatography. The reasons for elution pH differences amongst several IgG1s, IgG2s, antibody fragments, and Fc-fusion proteins during Protein A chromatography are investigated using several complimentary techniques. The results indicate that variable region interactions play a major role in determining elution pH for VH3 subfamily antibodies while using traditional protein A chromatographic materials. On the other hand, experiments with a resin which employs a ligand consisting solely of B domain of Protein A indicate that variable region interactions can be mitigated, enabling the use of a single elution pH for a range of antibodies. Finally, the moderation of elution conditions associated with this engineered ligand are shown to minimize problems associated with low pH induced aggregation. It is expected that the findings reported in this paper will facilitate faster process development cycle times for this important class of human therapeutics.
Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.
Protein recovery from a bacterial lysate was accomplished using microfiltration membranes in a flat crossflow filter and in a cylindrical rotary filter. Severe membrane fouling yielded relatively low long-term permeate flux values of 10(-4)-10(-3) cm/s (where I cm/s = 3.6 x 10(4) L/m(2) - h). The permeate flux was found to be nearly independent of transmembrane pressure and to increase with increasing shear rate and decreasing solids concentration. The flux increased with shear to approximately the one-third power or greater for the flat filter and the one-half power or greater for the rotary filter; the stronger dependence for the rotary filter is thought to result from Taylor vortices enhancing the back transport of debris carried to the membrane surface by the permeate flow. The average protein transmission or sieving coefficient was measured at approximately 0.6, but considerable scatter in the transmission data was observed. The largest sieving coefficients were obtained for dilute suspensions at high shear rate. The rotary filter provided higher fluxes than did the flat filter for dilute suspensions, but not for concentrated suspensions. (c) 1995 John Wiley & Sons, Inc.
The Use of NaOH for CIP of rProtein A Media: a 300 Cycle Study Waterside Conference
- H Johansson
- A Bergenstable
- G Rodrigo
- K Oberg