ArticleLiterature Review

Downstream processing of monoclonal antibodies—Application of Platform Approaches

Authors:
  • Independent Researcher
  • Independent Researcher
To read the full-text of this research, you can request a copy directly from the authors.

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.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Some decisions will affect not only the current step but will have important effects in later unit operations. After the depth filtration (DF), one to three polishing chromatography steps are usually performed, however the sequence of the chromatography steps as well as the type of chromatography exist practically in all possible permutations (Shukla et al., 2007;Kelley et al., 2008a;Liu et al., 2019;Shukla et al., 2007). A flow-through chromatography step [Anion Exchange Chromatography (AEX) or Mixed Mode Chromatography (MMC)] is often optimized by increasing the pH to bind an increasing amount of impurities or by decreasing the conductivity to achieve the same goal (Kelley et al., 2008a;Kelley et al., 2008b). ...
... Some decisions will affect not only the current step but will have important effects in later unit operations. After the depth filtration (DF), one to three polishing chromatography steps are usually performed, however the sequence of the chromatography steps as well as the type of chromatography exist practically in all possible permutations (Shukla et al., 2007;Kelley et al., 2008a;Liu et al., 2019;Shukla et al., 2007). A flow-through chromatography step [Anion Exchange Chromatography (AEX) or Mixed Mode Chromatography (MMC)] is often optimized by increasing the pH to bind an increasing amount of impurities or by decreasing the conductivity to achieve the same goal (Kelley et al., 2008a;Kelley et al., 2008b). ...
... However, if the interaction is weak, it is difficult to understand how some proteins still persist at the end of the bioprocess, when the standard process takes days and uses pH ranges from strongly acidic (virus inactivation, typically pH 3.2-3.8) to slightly basic (usually impurity wash step or similar flow-through chromatography steps) (Shukla et al., 2007). It is therefore reasonable to assume that these proteins behave, at least in part, similarly to antibodies in the polishing steps and that complete separation is probably not feasible. ...
Article
Full-text available
Polysorbates, in particular polysorbate (PS) 20 and 80, are the most commonly used surfactants for stabilising biotherapeutics produced by biotechnological processes. PSs are derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids of varying chain length and degree of saturation. In the past, these surfactants have been reported to have specific liabilities. Chemical (oxidations and hydrolyses) and enzymatic degradations have been reported to affect the stability of PS in drug products. Specifically, the presence of trace amounts (sub-ppm) of certain host cell proteins (HCPs) can induce enzymatic PS degradation, which can lead to the release of free fatty acids during storage over time. Enzymatic polysorbate degradation may impair the functionality of the surfactant in stabilising therapeutic proteins, leading to the formation of visible and/or sub-visible particles in biopharmaceutical drug products. This review summarises the enzymes currently known to be involved in the degradation of polysorbate in mammalian biotechnological processes for therapeutic proteins. In recent years, advanced analytical methods have been developed to qualify and quantify the PS-degrading enzymes. Most of these assays are based on mass spectrometry with a preceding HCP enrichment approach. Efforts were made to measure the enzyme activity and correlate it with observed PS degradation. The impact on drug product quality attributes, including fatty acid solubility and phase separation, up to the formation of visible particles, and the potential induction of protein and protein/fatty acid mixed particles as well as the sensitivity of specific PS quality towards enzymatic degradation, was considered. Various drug substance (DS) mitigation strategies related to the occurrence of PS degrading enzymes are discussed as amongst them the generation of stable HCP knockout cell lines, which are also carefully analysed. The underlying opinion article reflects the undergoing discussions related to PS degrading enzymes and focusses on (i) impact on drug product, (ii) analytics for identification/quantification (characterisation) of the PS degrading enzymes, (iii) enzyme activity (iv) currently identified enzymes, and (v) potential mitigation strategies to avoid enzymatic PS degradation during DS manufacturing.
... In contrast, other components-such as cell debris or host cell proteins (HCP)-flow through the chromatographic unit (CU) unimpeded [16]. Additional chromatographic steps (e.g., ion exchange and hydrophobic interaction) are typically applied to remove remaining impurities like HCPs, residual nucleic acid, and aggregates [17,18]. ...
... All elution samples (time point of greatest peak height) were analyzed by HPLC-SEC, and an average purity of 95.46% with concentrations up to 13 mg·mL −1 was observed. This means that the obtained purity levels match traditional batch standards, even though the product concentration exceeded the observed value of 8 mg·mL −1 for our in-house established batch process [18]. As no HCPs were present in the analyzed samples, minor reductions in the purity were exclusively traced back to the detected aggregate content-an issue that also occurs in similar processes [35]. ...
... The initial capture step is usually followed by polishing steps, which aim to remove the remaining impurities from the acquired antibody solution. A typical method for this polishing step is CEX, which is very useful for the removal of aggregates and HCPs [18]. In the presented PCCC system, the chromatography columns from the previous step are easily replaced by MAs for CEX (also refer to Figure 2). ...
Article
Full-text available
Continuous chromatography has emerged as one of the most attractive methods for protein purification. Establishing such systems involves installing several chromatographic units in series to enable continuous separation processes and reduce the cost of the production of expensive proteins and biopharmaceuticals (such as monoclonal antibodies). However, most of the established systems are bulky and plagued by high dead volume, which requires further optimization for improved separation procedures. In this article, we present a miniaturized periodic counter-current chromatography (PCCC) system, which is characterized by substantially reduced dead volume when compared to traditional chromatography setups. The PCCC device was fabricated by 3D printing, allowing for flexible design adjustments and rapid prototyping, and has great potential to be used for the screening of optimized chromatography conditions and protocols. The functionality of the 3D-printed device was demonstrated with respect to the capture and polishing steps during a monoclonal antibody purification process. Furthermore, this novel miniaturized system was successfully used for two different chromatography techniques (affinity and ion-exchange chromatography) and two different types of chromatographic units (columns and membrane adsorbers). This demonstrated versability underscores the flexibility of this kind of system and its potential for utilization in various chromatography applications, such as direct product capture from perfusion cell cultures.
... Most mAb purification processes include two or three chromatography steps. Each step applies a different mode of separation (Ghose et al., 2010;Liu et al., 2010;Shukla et al., 2007). This orthogonality is essential to achieve strong removal of process-and product-related impurities. ...
... After virus inactivation, two chromatography polishing steps are usually carried out. However, the sequence of the chromatography steps, as well as the type of chromatography, exists in almost all possible permutations (Fahrner et al., 2001;Ghose et al., 2010;Kelley, 2009;Liu et al., 2010;Shukla et al., 2007Shukla et al., , 2017. Anion Exchange Chromatography (AEX), typically operated in flow-through mode, is optimized by increasing the pH to bind more impurities or by decreasing the conductivity to achieve the same goal Kelley, Tobler, et al., 2008). ...
Article
Full-text available
Polysorbates (PS) are commonly used as stabilizers of biopharmaceuticals such as monoclonal antibodies (mAbs). However, they are prone to chemical and enzymatic degradation. The latter can be caused by residual host cell proteins (HCPs) in the drug substance. Degradation affects the functionality of the PS surfactant which can lead to formation of particles. An increasing number of publications describe enzymatic PS degradation. Significant efforts have been made to characterize HCP removal during Downstream Processing (DSP) of mAbs and to develop mitigation strategies. Here we describe the use of glycine buffer for acidic elution in Protein A affinity chromatography compared to acetate buffer, which is more commonly used in the biopharmaceutical industry. Increased turbidity was observed during pH re‐adjustment after low pH virus inactivation when using glycine buffer. Analytical data suggests that this turbidity is caused by the formation of precipitates which include HCP and DNA impurities. Additionally, as a zwitterion, glycine does not contribute to conductivity; this further enhances HCP removal during anion‐exchange flow‐through chromatography. Although glycine is well known as a possible elution buffer for Protein A affinity chromatography, its positive impact on HCP removal and PS stability have not yet been described in literature.
... The broth is separated from the cells via a centrifuge, and additional filtration steps take place for enhanced purification. The primary capture of the peptidic construct from the broth is performed by Protein A chromatography, through which impurities, such as host cell proteins, are removed 19 . The columns required were sized based on the Kozeny-Blake equation (Eq. ...
... In Eq. (3), ΔΡ is the pressure drop inside the packed column in (psi), μ is the viscosity of the mobile phase in (cP), K 0 is an empirical constant, d p is the particle diameter of the resin beads (in the case of porous membranes, the porous characteristic size) in (μm), ε is the porosity, L is the bed height in (cm), and u is the mobile phase linear velocity in (cm/s). Batch chromatographies demonstrate poorer column utilization than continuous ones, and for a given resin capacity 19 , a 95% functional yield was evidently assumed. ...
Article
Full-text available
Vaccine manufacturing fosters the prevention, control, and eradication of infectious diseases. Recombinant DNA and in vitro (IVT) mRNA vaccine manufacturing technologies were enforced to combat the recent pandemic. Despite the impact of these technologies, there exists no scientific announcement that compares them. Digital Shadows are employed in this study to simulate each technology, investigating root cause deviations, technical merits, and liabilities, evaluating cost scenarios. Under this lens we provide an unbiased, advanced comparative technoeconomic study, one that determines which of these manufacturing platforms are suited for the two types of vaccines considered (monoclonal antibodies or antigens). We find recombinant DNA technology to exhibit higher Profitability Index due to lower capital and starting material requirements, pertaining to lower Minimum Selling Price per Dose values, delivering products of established quality. However, the potency of the mRNA, the streamlined and scalable synthetic processes involved and the raw material availability, facilitate faster market penetration and product flexibility, constituting these vaccines preferable whenever short product development cycles become a necessity.
... Affinity purification is typically used as a primary capture step followed by different polishing steps using anion/cation exchange or hydrophobic interaction chromatography [27]. Affinity capture using Protein A or G is commonly used as the first step in the downstream purification process of mAbs [28,29]. This purification step enables the separation of IgG from host cell proteins (HCPs), DNA, and many other contaminants present in the harvest supernatant. ...
... This purification step enables the separation of IgG from host cell proteins (HCPs), DNA, and many other contaminants present in the harvest supernatant. Aggregates formed in previous bioprocess steps or in the capture step due to low elution pH are usually present in the elution pool together with monomers and must typically be removed using other means of chromatography [28]. However, aggregates can also be separated and removed from monomers directly in the affinity capture step using a pH gradient elution [30]. ...
... It is desired to have a purification step that can both concentrate and purify the product to a good extent, further reducing the volumes to be handled downstream. Affinity chromatography is usually the preferred option, and Protein A (ProA) ligands' specificity to mAbs and robustness makes it a very attractive process choice [3,4]. Even accounting for some disadvantages of ProA chromatography (expensive ligand, leaching of ProA) [5], efforts to dethrone this ligand as the first purification step of mAbs have been unsuccessful. ...
... for ( 4 ) for ( 5 ) where the axial position, and L the length of the column. The column was modelled as a loading-elution operation, with given by: ...
... For macromolecules of similar structure, superficial hydrophobicity correlates well with their molecular weight. This technique has been applied both for monoclonal antibodies purification and product polishing [24][25][26]. Polishing is a final step in downstream processing aimed at removing the last contaminants (which are usually in small quantities) and is characterized by having high selectivity but low capacity. As most HIC resins have low binding capacities, they are better suited for this latter use. ...
... Cationic Exchange (CEX), a technique based on differences in net surface charges between macromolecules under defined pH conditions, is also used as a polishing step for aggregate removal. The differences in net charge between these molecules are exploited in bind/elute mode, i.e. the monomer and aggregates are initially bound to the column under low conductivity and a pH below their pI, and then they are resolved by applying a linear gradient of increasing conductivity (the monomer eluted first) [24,27,28]. Hydroxyapatite is another alternative for aggregate removal [29]. ...
... In large-scale purification of mAbs, use of platform purification processes has become a well-established practice. 2,3 Use of platform purification processes for molecules with similar properties leverages platform knowledge to reduce downstream bioprocess development work, mitigate development risks, ensure process fit in manufacturing facilities, and accelerate development timelines. Assessment of chromatographic binding behavior aims to identify atypical molecules that might not readily fit within platform purification processes, evaluate available binding mechanisms and process levers, guide resin selection, and ascertain robust separation process conditions. ...
Article
Full-text available
In early-stage development of therapeutic monoclonal antibodies, assessment of the viability and ease of their purification typically requires extensive experimentation. However, the work required for upstream protein expression and downstream purification development often conflicts with timeline pressures and material constraints, limiting the number of molecules and process conditions that can reasonably be assessed. Recently, high-throughput batch-binding screen data along with improved molecular descriptors have enabled development of robust quantitative structure-property relationship (QSPR) models that predict monoclonal antibody chromatographic binding behavior from the amino acid sequence. Here, we describe a QSPR strategy for in silico monoclonal antibody purification process fit assessment. Principal Component Analysis is applied to extract a one-dimensional basis for comparison of molecular chromatographic binding behavior from multi-dimensional high-throughput batch-binding screen data. Kernel Ridge Regression is used to predict the first principal component for new molecular sequences. This workflow is demonstrated with a set of 97 monoclonal antibodies for five chromatography resins in two salt types across a range of pH and salt concentrations. Model development benchmarks four descriptor sets from biophysical structural models and protein language models. The investigation illustrates the value QSPR models can provide to purification process fit assessment, and selection of resins and operating conditions from sequence alone.
... Protein A and Protein L affinity chromatographies are widely used in mAb and bispecific antibody (bsAb) purification (they are also routinely used for Fc-fusion protein and antibody fragment purification, respectively) [1][2][3]. While the Protein A ligand mainly binds to the Fc region of IgG1, IgG2, and IgG4, the Protein L ligand binds to the variable region of kappa light chains that belong to subtypes 1, 3, and 4 [4][5][6][7][8][9]. ...
Article
Full-text available
Protein A and Protein L affinity chromatographies are extensively used in mAb and bispecific antibody (bsAb) purification. In addition to product capture, they are both capable of separating certain product-related by-products and aggregates under appropriate conditions. For both types of chromatography, previous studies suggested that adding a salt additive to the mobile phase can significantly improve the resolution between product and by-products/aggregates. Nevertheless, the effects of different salt additives on antibody elution in Protein A and Protein L chromatography have not been compared. In the current study, we compared the effects of three salt additives, sodium chloride (NaCl), calcium chloride (CaCl2), and arginine hydrochloride (Arg·HCl), on antibody elution in Protein A and Protein L chromatography. Interestingly, while NaCl suppressed antibody elution in both types of chromatography, CaCl2, and Arg·HCl promoted antibody elution in Protein A chromatography but suppressed antibody elution in Protein L chromatography. In addition, we evaluated the effect of each salt gradient on aggregate removal by Protein L chromatography. The information provided by the current study should be useful to the selection of conditions/additives for improving by-product removal by Protein A and Protein L chromatography.
... As a result, the industry converged on a drug substance manufacturing platform: fed-batch CHO cell culture for production using chemically defined media, clarification by centrifugation, Protein A capture followed by one or two polishing chromatography steps and virus filtration, ultrafiltration and diafiltration to provide assurance of the viral safety of the product and concentrate and formulate the drug substance. [4][5][6] The increase in the production culture titers early in this period led to concern over whether the purification process could handle the increased mass generated upstream. Multiple papers and conference sessions focused on the 'purification bottleneck'. ...
Article
Full-text available
Therapeutic monoclonal antibody (mAb) development and the processes for manufacturing drug substance have evolved since the first approval of the mAb in 1986. As the past is often the prologue to the future, the history of these technologies has been classified here into three eras, leading to speculation about what the next era may hold with regard to development and manufacturing strategies, as well as the potential impacts to patients. The substantial increase in production culture titers and bioreactor production volumes and the availability of large-scale contract manufacturing facilities could translate into improved global access for these therapies and an expansion of indications for therapeutic antibodies.
... Most therapeutic mAbs are typically being purified using protein A chromatography followed by 1-2 polishing steps. These polishing steps may comprise a cation-exchange (CEX) chromatography step, an anion-exchange (AEX)-chromatography, mixed-mode chromatography or a hydrophobic interaction chromatography [2]. As most antibodies have basic isoelectric points in the range of 7-8, with exceptions on multispecific antibodies and antibody-drug conjugates, an AEX-chromatography step is widely used as a polishing step. ...
Article
Full-text available
While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process‐related manufacturability, that is, how easily can a protein be purified. Process‐related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1‐receptor‐agonist‐Fc‐domain‐FGF21‐fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.
... Second, Monoclonal antibodies can be mass-produced as monoclonal antibodies are generated via hybridoma expertise. A further exclusive benefit of the hybridoma approach is that a mixture of analytes/targets can be used to produce selective antibodies [7][8][9][10]. Different chromatography methods are employed in pharmaceutical companies for the separation and purification of antibodies. ...
Article
Full-text available
Antibodies play an important role in health areas such as diagnostic, therapeutic, vaccination, and prevention. Antibodies were explored by various researchers for various purposes. Monoclonal antibodies have some important characteristic features that make them promising candidates for various applications such as being directed against single epitopes, being highly specific, and can be mass-produced. Diagnostic of various diseases relies on this molecule for the specificity and sensitivity of tests. Various diagnostic tests exploit the antibody for various applications such as enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry (IHC), Immunofluorescent antibody test (IFAT), Radioimmunoassay (RIA), Western immunoblotting. Researchers are increasingly using antibodies in an emerging field, called biosensors, to detect various analytes. Monoclonal antibodies were immobilized on the biosensor surface as a biological recognition element, and using such an effective biorecognition element in a biosensor makes detection more selective and sensitive to its target. Several immunosensors were developed that demonstrated a wide variety of detection limits with a satisfactory LOD (limit of detection). In this review, we will summarize the aspects and techniques of antibody purification, as well as their integration with various biosensors, emphasizing their importance in developing the healthcare industry.
... To accelerate process development, what is known as a "platform process" has been introduced. A platform process works for a family of biopharmaceutical products and has been implemented mainly with antibody drugs (Shukla et al., 2007). In a platform process, the individual steps and their sequence are identical. ...
Article
Full-text available
Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models, and global strategies to make a COVID‐19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low‐ and middle‐income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody‐drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics‐like cell and gene therapies. The industry must develop a model‐based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work‐life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.
... [1][2][3] In others, protein precipitation can result in product loss and/or provide impurity removal, depending on the composition of the precipitates. [4][5][6][7] An example of the latter is the formation of insoluble precipitates during the pH neutralization step of a protein A chromatography elution pool after a low-pH viral inactivation (VI) step in mAb purification. 7 An increase in turbidity during this step has been attributed to the growth of insoluble precipitates, comprised largely of host-cell proteins (HCPs) and DNA, 4,6 the removal of which can be optimized by adjusting parameters such as the neutralization pH . ...
Article
Full-text available
Precipitation during the viral inactivation, neutralization and depth filtration step of a monoclonal antibody (mAb) purification process can provide quantifiable and potentially significant impurity reduction. However, robust commercial implementation of this unit operation is limited due to the lack of a representative scale‐down model to characterize the removal of impurities. The objective of this work is to compare isoelectric impurity precipitation behavior for a monoclonal antibody product across scales, from benchtop to pilot manufacturing. Scaling parameters such as agitation and vessel geometry were investigated, with the precipitate amount and particle size distribution (PSD) characterized via turbidity and flow imaging microscopy. Qualitative analysis of the data shows that maintaining a consistent energy dissipation rate (EDR) could be used for approximate scaling of vessel geometry and agitator speeds in the absence of more detailed simulation. For a more rigorous approach, however, agitation was simulated via computational fluid dynamics (CFD) and these results were applied alongside a population balance model to simulate the trajectory of the size distribution of precipitate. CFD results were analyzed within a framework of a two‐compartment mixing model comprising regions of high‐ and low‐energy agitation, with material exchange between the two. Rate terms accounting for particle formation, growth and breakage within each region were defined, accounting for dependence on turbulence. This bifurcated model was successful in capturing the variability in particle sizes over time across scales. Such an approach enhances the mechanistic understanding of impurity precipitation and provides additional tools for model‐assisted prediction for process scaling.
... filtration/diafiltration.17 The adsorption steps employ different mechanisms to remove HCPs.For positively charged anion-exchange (AEX) column binding in flow-through mode, operation at a pH below that of the mAb pI (typically >7.5) leaves the mAb positively charged and unbound, while HCPs with lower pIs will be negatively charged and bound.129 Conductivity levels also need to be kept low to keep the electrostatic binding strength high and avoid charge passivation by salts. ...
Article
Full-text available
Host cell proteins (HCPs) are process‐related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta‐analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of “high‐risk” HCPs lead to more robust manufacturing processes and higher‐quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.
... In the downstream process, protein A affinity chromatography is the preferred option for mAb capture due to the high selectivity and high binding affinity of the protein A ligand for mAbs (Hober et al., 2007;Łącki & Riske, 2020;Shukla et al., 2007). At the same time, most process-related impurities, like host cell proteins (HCP) and DNA, are not retained and flow through the column. ...
Article
Full-text available
Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi‐column chromatography (RC‐PMCC) process and the rapid cycling simulated moving bed (RC‐BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L⁻¹ mAb‐containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L⁻¹ d⁻¹ (RC‐PMCC) and 574 g L⁻¹ d⁻¹ (RC‐BioSMB). At the same time, high removal of process‐related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC‐BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.
... Protein A chromatography with its high selectivity for antibodies is the gold standard for the initial capture of mAbs and Fc-fusion proteins. It efficiently removes >90% of HCP mass in a single step (Follman & Fahrner, 2004;Shukla et al., 2007). Different resins, post-load washes using different salts and additive combinations have been investigated to optimize HCP clearance (Aboulaich et al., 2014;Chollangi et al., 2015;Shukla & Hinckley, 2008;Sisodiya et al., 2012;Tarrant et al., 2012). ...
Article
Full-text available
Host cell proteins (HCPs) are process‐related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc‐based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co‐purification of HCPs with the therapeutic protein are either HCP‐drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc‐based proteins by LC‐MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co‐eluted with less than three Fc‐based proteins indicating a drug‐specific co‐purification for most HCPs. Only ten HCPs co‐purified with over 50% of the 23 Fc‐based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co‐elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein‐protein interaction networks. Here, we propose an additional mechanism for HCP co‐elution involving protein‐protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.
... To overcome some of the packed-bed chromatography limitations, alternative non-chromatographic downstream processes were investigated, namely precipitation, crystallization [47], membrane-based processes [48], magnetic fishing [49], and aqueous biphasic systems [50,51]. The optimization of an efficient and cost-effective purification process is a crucial part of IFN production [52]. ...
Article
Full-text available
Interferon alpha-2b (IFN-α2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-α2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C 2C 1Im][C 4F 9SO 3] and [N 1112(OH)][C 4F 9SO 3]) vs. mere fluoro-containing IL ([C 4C 1Im][CF 3SO 3]), in combination with sucrose or [N 1112(OH)][H 2PO 4] (well-known globular protein stabilizers), or high-charge-density salt K 3PO 4 were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-α2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-α2b high in the enriched IFN-α2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-α2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-α2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics.
... Early in the purification process, the target molecule concentration is often low and IEC can be a very efficient concentration step, in addition to purifying the product. In most monoclonal antibody (mAb) purification platform processes, IEC contributes to at least one of two polishing steps following the Protein A affinity step [57,58]. For mAb polishing, CIEC is commonly used in bind-and-elute mode for the target molecule, which allows negatively charged impurities such as residual DNA, RNA, some host cell proteins (HCPs), leached Protein A, and endotoxin to be removed during loading or in the wash fraction. ...
Article
Full-text available
Separations are key aspects of many modern analytical methods. Real world samples contain many analytes. Many analytical methods do not offer sufficient selectivity to be able to speciate all the analytes that might be present. Separation isolates analytes. Most separation methods involve separation of the analytes into distinct chemical species, followed by detection. Instrumental methods may be used to separate samples using chromatography, electrophoresis or field flow fractionation. The development of the pharmaceuticals brought a revolution in human health. These pharmaceutical would serve their intended only if they are free from impurities and administered in appropriate amount. To make drugs serve their purpose various chemical and instrumentation method were developed at regular intervals which are invo lved in the estimation of drugs. For this analytical instrumentation and methods plays an important role. This review highlights the role of the analytical instrumentation and analytical method in assessing the quality of the drug. The review highlights variety of analytical techniques such as chromatographic, spectroscopic, electrophoretic, titrimetric, microscopic and electrochemical and their corresponding methods that has been applied in the analysis of pharmaceuticals. Quality of pharmaceutical product largely depends upon the environment controls during its storage and handling. Each pharmaceutical product should be handled and stored under specified storage condition labelled on product information data sheet or product pack. Ion chromatography was based on the use of a low-capacity anion exchange separator column used with a basic eluent and a suppressor column. IC is a part of high-performance liquid chromatography used to separate and determine anions and cations.
... Most therapeutic mAbs are typically being purified using protein A chromatography followed by 1-2 polishing steps. These polishing steps may comprise a cation-exchange (CEX) chromatography step, an anion-exchange (AEX)-chromatography, mixed-mode chromatography or a hydrophobic interaction chromatography (Shukla et al., 2007). As most antibodies have basic isoelectric points, an AEXchromatography step is the first choice for a polishing step. ...
Preprint
Full-text available
While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Many biotherapeutics are typically expressed in mammalian cells. Process-related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure safety for the patients. Therefore, during molecule design, an isoelectric point of the target molecule should preferably differ sufficiently from the isoelectric points of the impurities to enable an efficient and straightforward purification strategy. In this feasibility study we have evaluated the possibility to improve manufacturability by increasing the isoelectric point of the target protein. We have generated several variants of a GLP1-receptor-agonist-Fc-domain -FGF21 fusion protein and demonstrate that the critical anion exchange chromatography step can be run at high pH values with maximal product recovery theoretically allowing removal of HCP and viruses. Addressing the isoelectric point can be useful for an efficient process for removing HCP and viruses and this topic should be considered early in the research phase to ensure that other important molecule properties, e.g. safety, efficacy and expression yield are not impacted.
... One of the major goals of the downstream process is to bring down these process-related impurities to acceptable limits in the final drug substance to avoid the unwanted immune response in the patients [39]. This can be achieved by the development of careful monitoring process for the depletion of these impurities along the in-process steps [40,41]. ...
Article
Full-text available
The rising global prevalence of diabetes and increasing demand for insulin, calls for an increase in accessibility and affordability of insulin drugs through efficient and cost-effective manufacturing processes. Often downstream operations become manufacturing bottlenecks while processing a high volume of product. Thus, process integration and intensification play an important role in reducing process steps and time, volume reduction, and lower equipment footprints, which brings additional process efficiencies and lowers the production cost. Manufacturers thrive to optimize existing unit operation to maximize its benefit replacing with simple but different efficient technologies. In this manuscript, the typical property of insulin in forming the pH-dependent zinc-insulin complex is explored. The benefit of zinc chloride precipitation/crystallization has been shown to increase the in-process product purity by reducing the product and process-related impurities. Incorporation of such unit operation in the insulin process has also a clear potential for replacing the high cost involved capture chromatography step. Same time, the reduction in volume of operation, buffer consumption, equipment footprint, and capabilities of product long time storage brings manufacturing flexibility and efficiencies. The data and capabilities of simple operation captured here would be significantly helpful for insulins and other biosimilar manufacturer to make progresses on cost-effective productions.
... The importance of developing an efficient downstream process (DSP) for the recovery and purification of monoclonal antibodies (mAbs) is underlined by its significant proportion of the total manufacturing costs (Hummel et al., 2019;Shukla et al., 2007). Generally, commercial production of mAbs is based on a platform process that includes affinity capture on ProteinA-based chromatography resin followed by purification and polishing steps by cation exchange (CEX) and anion exchange (AEX) or mixed mode (MM) chromatography. of tryptophan with lysine in the H-CDR3 was shown to have an impact on chromatographic behavior in CEX (Saleh et al., 2021). ...
Article
Full-text available
Flowthrough (FT) anion exchange (AEX) chromatography is a widely used polishing step for the purification of monoclonal antibody (mAb) formats. To accelerate downstream process development, high throughput screening (HTS) tools have proven useful. In this study, the binding behavior of six monovalent mAbs (mvAbs) was investigated by HTS in batch binding mode on different AEX and mixed‐mode resins at process‐relevant pH and NaCl concentrations. The HTS entailed the evaluation of mvAb partition coefficients (Kp) and visualization of results in surface‐response models. Interestingly, the HTS data grouped the mvAbs into either a strong‐binding group or a weak‐binding/FT group independent of theoretical Isoelectric point. Mapping the charged and hydrophobic patches by in silico protein surface property analyses revealed that the distribution of patches play a major role in predicting FT behavior. Importantly, the conditions identified by HTS were successfully verified by 1 mL on‐column experiments. Finally, employing the optimal FT conditions (7–9 mS/cm and pH 7.0) at a mini‐pilot scale (CV = 259 mL) resulted in 99% yield and a 21–23‐fold reduction of host cell protein to <100 ppm, depending on the varying host cell protein (HCP) levels in the load. This work opens the possibility of using HTS in FT mode to accelerate downstream process development for mvAb candidates in early research.
... To fully exploit the possible benefits of such a continuous USP, an intensified downstream process (DSP) is required. The routinely applied protein A affinity chromatography step as first mAb capture, offers great potential for improvement due to its limited loading capacity and high resin cost (Shukla et al., 2007). As mitigating strategy many companies cycle the capture columns several times during a batch, resulting in a smaller volume of the resin required for the same amount of mAb. ...
Article
Full-text available
The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield. This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi‐column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow‐through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement.
... 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 (Holzer et al., 2008;Shukla et al., 2007;Mahajan et al., 2012). Due to this, the process may require the use of excessive amounts of solvent. ...
Article
Full-text available
Platforms have long been implemented for downstream process development of monoclonal antibodies (mAbs) to streamline development and reduce timelines. These platforms are also increasingly being used for other complex biologics modalities. While development has traditionally been conducted at the lab bench scale in a sequential manner, automated miniaturized and parallelized approaches like RoboColumns and resin plates have also been implemented for chromatographic screening. Additionally, mechanistic modeling for chromatographic separations has also seen increased use for development applications. In this manuscript, we propose a workflow with elements of both high‐throughput screening and modeling that provides a streamlined roadmap for early process development. The workflow utilizes automated resin plate screens to both narrow screening conditions and calibrate binding isotherm parameters. Mechanistic models are then used to characterize a robust range of conditions suitable for an early manufacturing process. Miniaturized RoboColumns then confirm the process space, thus completing the development without the use of any traditional lab‐scale columns. Case studies demonstrate the utility of this workflow for both cation‐exchange (CEX) and multimodal cation‐exchange (MMCEX) processes. Process parameter sensitivities across process ranges for the models are compared with typical design‐of‐experiment (DOE) statistical models. The models are able to predict the mAb product as well as aggregate impurities. This workflow provides a practical method to enable increased process understanding while also reducing timeline and material requirements for development.
Article
Adeno‐associated virus (AAV) is a versatile viral vector technology that can be engineered for specific functionality in vaccine and gene therapy applications. One of the major challenges in AAV production is the need for a GMP‐ready platform‐based approach to downstream processing, as this would lead to a standardized method for multiple products. Chromatography has huge potential in AAV purification, as it is a scalable method that would enable manufacturing to a high degree of purity, potency, and consistency. Multiple factors need to be considered when developing a chromatography platform, including the chromatography type, format, and mode of operation, along with other commercial considerations that have not been comprehensively reviewed until now. In addition to chromatography factors, this review will also consider the current understanding of AAV characteristics: this will include net surface charge, structural properties, and size, as well as their interactions with metal ions or receptors, and how this impacts the development of a chromatography platform.
Article
The process of antibody purification using Fc affinity ligands such as protein A, G, and L faces several challenges including high cost, low stability, and loss of antibody activity due to harsh elution conditions. Here, we describe a chromatographic purification of antibodies utilizing a pH-responsive mixed-charge polymer that mimics the IgG-binding peptide (Z34C) derived from the B domain of protein A. The protein A mimetic resins were prepared by modifying the surface of a TOYOPEARL, methacrylate resin with a polymer that mimics the amino acid sequence of Z34C and the functions of histidine and acidic and neutral amino acids using histamine methacrylamide (HisMA), methacrylic acid, and neutral monomers. The therapeutic monoclonal antibody (mAb), rituximab, was retained on the column at pH 7 and eluted under mildly acidic conditions at pH 5 using a protein A mimetic resin (HisMA20-EEMA) optimized for antibody interaction. The injected antibodies were selectively captured on the column by hydrophobic and electrostatic interactions with the protein A mimetic polymer under neutral conditions and eluted by electrostatic repulsion under acidic conditions. The HisMA20-EEMA column successfully purified mAbs from mixtures with BSA, mouse ascites fluid, and hybridoma cell culture supernatant. In addition, the HisMA20-EEMA column consistently achieved 90% antibody recovery in 100 consecutive purifications from cell culture supernatant. The antibody purification method presented in this study is low cost, highly durable, easy to synthesize, and allows for mild elution conditions. The results demonstrate that the approach of mimicking IgG-binding peptides with mixed-charge polymers is useful for the development of column packing materials for antibody purification.
Chapter
Commercial production of monoclonal antibodies is a pivotal aspect of modern biotechnology industries. Monoclonal antibodies used to be generated by fusing a specific B cell with a myeloma cell to form a hybridoma to produce antibody. Later, the production of monoclonal antibodies utilizing animal cell lines like CHO and HEK293 became cornerstone of biopharmaceutical manufacturing. This process involves animal cell culture in bioreactors under optimized conditions to produce monoclonal antibodies. Antibody-producing stable cell line is generated through transfection and selection processes. These engineered cell lines are then expanded in culture, with medium formulations tailored to support cell growth and antibody production. Bioreactor parameters are carefully controlled to maximize cell productivity. Harvested cell culture supernatants are subsequently purified using chromatography techniques to isolate the antibody. Commercial monoclonal antibody production using animal cell lines enables scalable and efficient manufacturing of therapeutic antibodies for various clinical applications, contributing significantly to the biopharmaceutical industry.
Article
Full-text available
CaptureSelect CH1-XL and Praesto 70 CH1 are two affinity media that specifically bind to the CH1 domain of an antibody. In the current work, we first demonstrated that these two CH1-specific affinity media bound to different monoclonal antibodies (mAbs) with varied strengths under identical conditions. We previously had observed the same on a Protein L-conjugated resin and showed that such a property could facilitate homodimer removal in asymmetric bispecific antibody (bsAb) purification. Next, using Praesto 70 CH1, we showed that a small difference in binding between two mAbs could be significantly exaggerated by adding sodium chloride to the mobile phase, further demonstrating this resin can potentially play a role in bsAb purification. Finally, with a concrete bsAb case study, we showed that, like Protein L, Praesto 70 CH1 could separate the target heterodimer from the homodimer by-product. Homodimers are common product-related impurities associated with the recombinant production of asymmetric bsAbs, which can be difficult to remove. Their removal, even a partial one, at the capture stage is a big advantage as it can alleviate the purification burden on subsequent polishing steps and render the overall process more robust. Therefore, Praesto 70 CH1’s unique property is highly desirable, and this affinity resin can be a better alternative than Protein A for product capture in asymmetric bsAb purification.
Article
Background Protein A resins have been widely used for product capture during mAb, bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification, certain truncated byproducts may contain the same Fc region as the product but fewer numbers of the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for byproduct separation based on the difference in VH3-binding valency. As the ligands of different VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would be interesting to know whether they possess comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain. Objective This study aims to explore the potential of different VH3-binding Protein A resins for separating antibody species with the same Fc region but different numbers of VH3 domain. Methods The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion, the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3, Jetted A50, MabCapture C, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein A resins for separating species having the same Fc region but different numbers of VH3 domain was evaluated using an artificial mixture composed of the product and a truncated byproduct, which contained one and zero VH3 domain, respectively (both species contained the same Fc region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor Fab purification and separation of species containing the same Fc region but different numbers of VH3 domain. Results When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct, which contained the Fc region only without any VH3 domain, from the product, which included one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating these two species. Conclusion Although different VH3-binding Protein A resins showed varied DBCs towards a VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.
Article
Full-text available
While machine learning (ML) has made significant contributions to the biopharmaceutical field, its applications are still in the early stages in terms of providing direct support for quality-by-design based development and manufacturing of biologics, hindering the enormous potential for bioprocesses automation from their development to manufacturing. However, the adoption of ML-based models instead of conventional multivariate data analysis methods is significantly increasing due to the accumulation of large-scale production data. This trend is primarily driven by the real-time monitoring of process variables and quality attributes of biopharmaceutical products through the implementation of advanced process analytical technologies. Given the complexity and multidimensionality of a bioproduct design, bioprocess development, and product manufacturing data, ML-based approaches are increasingly being employed to achieve accurate, flexible, and high-performing predictive models to address the problems of analytics, monitoring, and control within the biopharma field. This paper aims to provide a comprehensive review of the current applications of ML solutions in the design, monitoring, control, and optimisation of upstream, downstream, and product formulation processes of monoclonal antibodies. Finally, this paper thoroughly discusses the main challenges related to the bioprocesses themselves, process data, and the use of machine learning models in monoclonal antibody process development and manufacturing. Moreover, it offers further insights into the adoption of innovative machine learning methods and novel trends in the development of new digital biopharma solutions.
Chapter
Recombinant biopharmaceutical proteins such as growth factors and antibodies are typically produced in microbial or mammalian cells. However, microbial host cells cannot produce complex biopharmaceuticals that require defined oligomerization or specific glycosylation profiles, and mammalian cells are unsuitable for the production of toxic proteins. These issues are readily overcome by plant cell cultures, which have the machinery required for complex protein modifications and can sequester toxic proteins within subcellular compartments. In this chapter, we first compare the features of plant cell cultures and conventional expression systems, including the ability of plant cells to produce complex and/or toxic proteins. We discuss the main benefits of plant cells and whole plants, including high-throughput transient expression for rapid, low-cost process development reflecting the simplicity of upstream production. We then set out current challenges and trends in the upstream production and downstream processing phases of plant molecular farming. Finally, we discuss how these different elements can be combined into an integrated manufacturing facility based on plants and how this might differ from conventional production platforms.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
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.
Article
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.
Article
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.
Article
Thorough process characterization can improve success rates in manufacturing, reduce the number of process related incidents, and significantly improve process yields.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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