Antibody Manufacturing Research Articles

Optimization of a platform process operating space for a monoclonal antibody susceptible to reversible and irreversible aggregation using a solution stability screening approach

Platform manufacturing processes are widely adopted to simplify and standardize the development and manufacturing of monoclonal antibodies (mAbs). However, there are mAbs that do not conform to a platform design due to instability or other protein properties leading to a negative impact on product quality or process performance (non-platform mAb). Non-platform mAbs typically require prolonged development times and significant deviations from the platform process to address these issues due to the need to sequentially optimize individual process steps.In this study, we describe an IgG2 mAb (mAb A) that is susceptible to aggregation and reversible self-association (RSA) under platform conditions. In lieu of a sequential optimization approach, we evaluated the solution stability of mAb A across the platform operating space (solution stability screen). This screening design was used to identify interacting parameters that affected the non-platform mAb stability. A subsequent response surface design was found to predict an acceptable operating space that minimized aggregate formation and RSA across the entire process. This information guided the selection of optimal parameters best suited to avoid destabilizing conditions for each process step. Substantial time savings was achieved by focusing development around these factors including protein concentration, buffer pH, salt concentration, and excipient type. In addition, this work enabled the optimization of a cation exchange chromatography step that removed aggregate without yield losses due to the presence of reversible aggregation. The final optimized process derived from this study resulted in an increase in yield of ˜30% over the original process while maintaining the same level of aggregate clearance to match product quality.Solution stability screening is readily adapted to high throughput technologies to minimize material requirements and accelerate analytical data availability. Implementation of high throughput approaches will further expedite process development and enable enhanced selection of candidate drugs by including process development objectives.

Assisting continuous biomanufacturing through advanced control in downstream purification

Aiming to significantly improve their processes and secure market share, monoclonal antibody (mAb) manufacturers seek innovative solutions that will yield improved production profiles. In that space, continuous manufacturing has been gaining increasing interest, promising more stable processes with lower operating costs. However, challenges in the operation and control of such processes arise mainly from the lack of appropriate process analytics tools that will provide the required measurements to guarantee product quality. Here we demonstrate a Process Systems Engineering approach for the design a novel control scheme for a semi-continuous purification process. The controllers are designed employing multi-parametric Model Predictive Control (mp-MPC) strategies and the successfully manage to: (a) follow the system periodicity, (b) respond to measured disturbances and (c) result in satisfactory yield and product purity. The proposed strategy is also compared to experimentally optimized profiles, yielding a satisfactory agreement.

Tandem Immunoaffinity Purification Using Anti-FLAG and Anti-HA Antibodies.

The immunoaffinity purification of target proteins followed by the identification and characterization of associated proteins by mass spectrometry is a widely used technique. An immunoaffinity purification bears resemblance to a standard immunoprecipitation; however, the end product for mass spectrometric analysis in the femtomole (10-15) to attomole (10-18) range is required to be of exceptional purity. This high degree of sensitivity in detection renders it of extreme importance to eliminate most if not all of the nonspecific background proteins and can be achieved by performing a tandem affinity purification (TAP). In TAP, the cDNA of the target protein is engineered to contain at least two different epitope tags, and the target protein is extracted under nondenaturing conditions upon expression using an appropriate protein expression platform (CHO cells, HEK 293 cells, or yeast). The expressed protein is initially immunoprecipitated using an antibody against one epitope tag and is eluted in the presence of excess peptide by competition for antibody-binding sites, before being reimmunoprecipitated using an antibody that specifically recognizes the second epitope. These sequential immunoprecipitations significantly reduce the presence of associated nonspecific proteins. Numerous combinations of epitope tags have been applied for tandem affinity purification, and this protocol illustrates the use of tandem hemagglutinin (HA) and FLAG epitope tags. The first immunoprecipitation uses an anti-FLAG antibody followed by the elution in the presence of a competing FLAG peptide before the reimmunoprecipitation of the protein using an anti-HA antibody. Numerous high-quality antiepitope tag antibodies are commercially available from different antibody manufacturers.

Comparison between Batch and Continuous Monoclonal Antibody Production and Economic Analysis

This work aims to evaluate the benefits of the continuous process in biopharmaceutical manufacturing of monoclonal antibodies (mAbs). An integrated continuous process is designed and built using a process simulator and compared with a fed-batch process production line. The comparisons are based on cost of goods (COG/g) calculation and sensitivity analysis. The fed-batch process results in operating COG/g of $99/g in mAbs production, whereas the continuous process accounts for $51/g. Because of the smaller footprint and fewer storage tanks required in the continuous process, the facility cost reduced by 66%, compared to the fed-batch process. In continuous production, there is a better utilization of the primary capture resin, which leads to a reduction in consumables cost by 68%. Sensitivity analysis is used to evaluate the process in different manufacturing scales (50–1200 kg/yr), upstream titers (1.5–5.5 g/L), and downstream yield (70%–80%). Cost savings can be observed through the entire range.

Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies

High product purity, preserving natural IgG architecture, and excellent production efficiency are highly desirable in bispecific antibody manufacturing. We have reported a platform called Bispecific Antibody by Protein Trans-Splicing (BAPTS) to synthesize BsAbs with natural human IgG structure and no chain mispairing. In the method, two antibody fragments carrying different target-specificities are separately expressed in mammalian cells and subsequently fused to form BsAbs by utilizing the trans-splicing property of the split intein Npu DnaE. The hinge region of antibody, a region with less functional impact, is selected for conjugating the two fragments. The method involves the following steps: (i) constructing five plasmids coding antibody components; (ii) separately expressing and purifying two antibody fragments A and B. Fragment A contains one Fab, “Knobs-into-Holes” mutations in the CH3 domain and NPU DnaEC. Fragment B contains another Fab and NPU DnaEN; (iii) mixing of fragments A and B under permissive reducing conditions in vitro to enable trans-splicing reaction; (iv) removing the reductant to allow re-oxidation of disulfide bonds; (v) isolating BsAb product from unreacted precursors by affinity chromatography. The method allows correct assembly of two heavy and two light chains to form bispecific IgG antibodies in natural structure with no synthetic linkers. No chain mispairing was observed in the product by UPLC-MASS. In addition, the observed kinetics and low reaction activation energy confirmed that the trans-splicing is thermodynamically favored reaction. The BAPTS technology is feasible for industrial applications.

Inactivation of deubiquitinase CYLD enhances therapeutic antibody production in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are promising host engineering cells for industry manufacturing of therapeutic antibodies. However, cell death due to apoptosis remains a huge challenge to augment antibody production, and developing CHO cells with enhanced anti-apoptosis and proliferation ability is fundamental for cell line development and high-yielding bioprocesses. Deubiquitinase cylindromatosis (CYLD) has been proved to be a tumor suppressor by negatively regulating NF-κB and Wnt/β-catenin signaling pathways. Its mutation or deletion is a common chromosome variation in several types of cancers. Here, we engineered CHO CYLD-/- cells by CRISPR-Cas9 editing technology. These cells displayed stronger cell proliferation and anti-apoptosis ability compared to parental cells. Three antibody expression plasmid kits were transiently transfected into these cells. Our data showed that inactivation of CYLD increased the highest titers of rituximab, Herceptin, and one bispecific antibody by 105, 63, and 228%, respectively. Reversely, overexpression of CYLD could promote cell apoptosis, whereas inhibiting cell proliferation and antibody production. Furthermore, inhibition of CYLD in CHO cells stably expressing an IgG antibody (CHO-IgG) achieved about 50% increase in product titer compared to parental cells. Meanwhile, inhibition of CYLD did not affect the quality of antibody. Thus, our data demonstrated that inactivation of CYLD could promote CHO cell proliferation, anti-apoptosis ability, and subsequent antibody production, suggesting that CYLD is a potential functional target for CHO cell engineering.

A pharmacology guided approach for setting limits on product-related impurities for bispecific antibody manufacturing

Introduction: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoβ (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1.Methods: To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling.Results: Here, we show that product-related impurities of bFKB1, in particular, high molecular weight (HMW) impurities and anti-FGFR1-related impurities, when purposefully enriched, stimulate FGFR1 in a KLB-independent manner. By taking this approach, the tolerable levels of product-related impurities were successfully determined.Discussion: Our study demonstrates a general pharmacology-guided approach to setting a product specification for a bispecific antibody whose homomultimer-related impurities could lead to undesired biological effects.

Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered Fc\u03b3Receptor IIIa-Immobilized Column

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A.

The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.

An IRES-Mediated Tricistronic Vector for Efficient Generation of Stable, High-Level Monoclonal Antibody Producing CHO DG44 Cell Lines.

The generation of stable, high-level monoclonal antibody (mAb) producing cell lines remains a major challenge in biopharmaceutical industry. The commonly used plasmid vectors for mAb expression, which express light chain (LC), heavy chain (HC), and selection marker genes on separate vectors or via multiple promoters on a single vector, are not able to accurately control the ratio of LC over HC expression and tend to result in non-expressing clones. To overcome these issues, we have developed a tricistronic vector using two internal ribosome entry sites (IRES) to express the LC, HC, and dihydrofolate reductase (DHFR) selection marker genes in one transcript. In this tricistronic vector, the three genes are under the control of a hapten-modified human cytomegalovirus (hCMV) promoter containing a core CpG island element (IE) to enhance the production stability. The LC gene is arranged as the first cistron followed by a wild-type IRES to control the HC expression. Such design expresses excess LC polypeptides which enhance mAb expression level and reduce aggregate. A mutated IRES with attenuated strength is applied on DHFR to reduce its expression for enhancing the stringency of selection for high producers. This vector allows easy generation of stable, high mAb producing CHO DG44 pools and clones for antibody development and manufacturing.

Vitamin B12 association with mAbs: Mechanism and potential mitigation strategies.

Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in-process pools and drug substances in the antibody manufacturing process. Vitamin B12 is covalently bound to mAbs via a cobalt-sulfur coordinate bond via the cysteine residues. The vitamin B12 was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys-214), and heavy chain (Cys-134, Cys-321, Cys-367, and Cys-425). Prior to attachment to mAbs, the vitamin B12 needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B12 added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11-14 days at room temperature and with room light intensity about 500-1,000 lux) and by short-wavelength visible light (400-550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B12 . Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light-induced color variations in final products.

Insights into the generation of monoclonal antibody acidic charge variants during Chinese hamster ovary cell cultures.

Charge variation is one of the most important heterogeneities during monoclonal antibody (mAb) manufacturing and this study presents insights into the generation of acidic charge variants during cell culture processes. Since acidic variants generate both intracellularly and extracellularly, main charge fraction collected by weak cation exchange chromatography (WCX) was incubated in harvested cell supernatant (HCS) to simulate and investigate the extracellular process firstly. It is found that the main fraction was degraded rapidly into acidic variants rather than basic variants extracellularly, and the degradation sites were located in both Fab and Fc fragments indicated by papain digestion. Besides, certain process parameters were investigated as their potential roles in the extracellular process. As a result, media composition showed significant influence on degradation while culture time point did not, suggesting that the extracellular process was a spontaneous process without enzyme catalysis. Additionally, kinetics study reveals that the extracellular process was a pseudo first-order reaction. The E app value (21.59kcal/mol) estimated from the Arrhenius equation suggests that the extracellular degradation might be mainly attributed to asparagine deamidation. Furthermore, we established an acidic variants generation model, indicating that the extracellular process plays a dominant role in modulating the final acidic variant level. This study provides better understanding for controlling product heterogeneity in mAb manufacturing.

An integrated precipitation and ion-exchange chromatography process for antibody manufacturing: Process development strategy and continuous chromatography exploration

In the past decades, research was carried out to find cost-efficient alternatives to Protein A chromatography as a capture step in monoclonal antibody (mAb) purification processes. In this work, polyethylene glycol (PEG) precipitation has shown promising results in the case of mAb yield and purity. Especially with respect to continuous processing, PEG precipitation has many advantages, like low cost of goods, simple setup, easy scalability, and the option to handle perfusion reactors.Nevertheless, replacing Protein A has the disadvantage of renouncing a platform unit operation as well. Furthermore, PEG precipitation is not capable of reducing high molecular weight impurities (HMW) like aggregates or DNA. To overcome these challenges, an integrated process strategy combining PEG precipitation with cation-exchange chromatography (CEX) for purification of a mAb is presented.This work discusses the process strategy as well as the associated fast, easy, and material-saving process development platform. These were implemented through the combination of high-throughput methods with empirical and mechanistic modeling. The strategy allows the development of a common batch process. Additionally, it is feasible to develop a continuous process. In the presented case study, a mAb provided from cell culture fluid (HCCF) was purified. The precipitation and resolubilization conditions as well as the chromatography method were optimized, and the mutual influence of all steps was investigated. A mAb yield of over 95.0% and a host cell protein (HCP) reduction of over 99.0% could be shown. At the same time, the aggregate level was reduced from 3.12% to 1.20% and the DNA level was reduced by five orders of magnitude. Furthermore, the mAb was concentrated three times to a final concentration of 11.9mg/mL.

In silicoevaluation of the interactions among two selected single chain variable fragments (scFvs) and ESAT-6 antigen ofMycobacterium tuberculosis

Nowadays antibody engineering is an important approach in the design and manufacture of therapeutic and diagnostic antibodies. The study of interactions between antibodies and antigens is the critical step in the design of antibodies with desirable properties. Computational docking is a useful tool for structural characterization of bimolecular interactions. Docking is the process of predicting bound conformations and binding enthalpy of antibody–antigen complexes. In this study, the three-dimensional structures of two ribosome displayed-selected scFv antibodies were constructed by Kotai Antibody Builder. By using ClusPro 2.0 web server, the ESAT-6 antigen (a tuberculosis-specific antigen) structure was docked to both scFv models to obtain the structures of the binding complexes and molecular dynamics (MD) simulations were performed using GROMACS 4.5.3 package. By analyzing of the ESAT-scFv complexes, important amino acids involved in antigen–antibody interactions were identified which were Asn164 in VL3, Ser164 in VL7 and Asn55 in VH7. All three amino acids belonged to the CDRs. In conclusion, results achieved from this bioinformatics study can help in the design and development of novel antibodies with improved affinities for tuberculosis diagnosis.

Choices of capture chromatography technology in antibody manufacturing processes.

The capture process employed in monoclonal antibody downstream purification is not only the most critically impacted process by increased antibody titer resulting from optimized mammalian cell culture expression systems, but also the most important purification step in determining overall process throughput, product quality, and economics. Advances in separation technology for capturing antibodies from complex feedstocks have been one focus of downstream purification process innovation for past 10 years. In this study, we evaluated new generation chromatography resins used in the antibody capture process including Protein A, cation exchange, and mixed mode chromatography to address the benefits and unique challenges posed by each chromatography approach. Our results demonstrate the benefit of improved binding capacity of new generation Protein A resins, address the concern of high concentration surge caused aggregation when using new generation cation exchange resins with over 100mg/mL binding capacity, and highlight the potential of multimodal cation exchange resins for capture process design. The new landscape of capture chromatography technologies provides options to achieve overall downstream purification outcome with high product quality and process efficiency.

A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line

Combinations of different antibodies have been shown to be more effective for managing certain diseases than monotherapy. Co-expression of the antibody mixture in a single cell line is key to reducing complexity during antibody development and manufacturing. However, co-transfection of multiple light and heavy chains into cells often leads to production of mismatched, heterodimeric by-products that are inactive, making the development of co-expression systems that robustly and efficiently produce highly active antibody mixtures a high priority. In this study, we modified the CH3 domain interface of the antibody fragment crystallizable (Fc) region by changing several charge pairs to create electrostatic interactions favoring Fc homodimer formation and disfavoring Fc heterodimer formation. When co-expressed, these modified antibodies with altered charge polarity across the Fc dimer interface preferentially formed homodimers that fully preserved the functions of each component, rather than inactive heterodimers whose formation was reduced because of rationally designed repulsive interactions. We designed eight different combinations and experimentally screened the best one, which enabled us to produce a binary antibody mixture against the EGF receptor with a minimal heterodimer contaminant. We further determined the crystal structure of a triple-mutated Fc variant in the best combination, and we elucidated the molecular interactions favoring Fc homodimer over heterodimer formation, which provided a structural basis for further optimization. The approach presented here demonstrates the feasibility of rational antibody modification for efficient and consistent production of monoclonal antibody mixtures in a single cell line and thus broadens our options for manufacturing more effective antibody-based therapeutic agents.

Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing

Screening and characterization of cell lines for stable production are critical tasks in identifying suitable recombinant cell lines for the manufacture of protein therapeutics. To aid this essential function we have developed a methodology for the selection of antibody expressing cells using fluorescence based ClonePix FL colony isolation and flow cytometry analysis following intracellular staining for immunoglobulin G (IgG). Our data show that characterization of cells by flow cytometry early in the clone selection process enables the identification of cell lines with the potential for high productivity and helps to eliminate unstable cell lines. We further demonstrate a correlation between specific productivity (qP) and intracellular heavy chain (HC) content with final productivity. The unique combination of screening using ClonePix FL and the flow cytometry approaches facilitated more efficient isolation of clonal cell lines with high productivity within a 15week timeline and which can be applied across NS0 and CHO host platforms. Furthermore, in this study we describe the critical parameters for the ClonePix FL colony based selection and the associated calculations to provide an assessment of the probability of monoclonality of the resulting cell lines.

Assessing the Impact of Charge Variants on Stability and Viscosity of a High Concentration Antibody Formulation

Characterizing molecular charge variants or isoforms is essential for understanding safety, potency, and bioavailability of antibody therapeutics. However, there is little information on how they influence stability and viscosity—properties governing immunogenicity and delivery. To bridge this gap, we studied antibody stability as a function of charge variant content generated via bioreactor process. We were able to systematically vary acidic variant levels as a function of bioreactor harvest time. Importantly, we do not observe any impact on aggregation behavior of a formulated antibody at high protein concentration as a function of acidic variant level. Furthermore, we confirm that acidic variants enriched using fractionation do not influence viscosity, colloidal or conformational stability. Interestingly, variants with the most acidic isoelectric points contribute disproportionately to formulation color. We discuss our findings in context of antibody manufacturing processes that may yield increased charge variant content.

Fate of a Stressed Therapeutic Antibody Tracked by Fluorescence Correlation Spectroscopy: Folded Monomers Survive Aggregation.

Antibodies are therapeutic proteins that are becoming indispensable for the treatment of serious diseases. Their efficacies depend on their folded structures, the loss of which, through unfolding or aggregation, should be closely monitored during their manufacture, storage, and dosing for safe usage. In downstream processes, exposure of the crude antibody solution to acid, followed by neutralization, is an established standard protocol for antibody purification and inactivation of viruses in antibody preparations. Nevertheless, this treatment also triggers an unwanted side reaction, i.e., antibody aggregation. The aggregates continuously evolve at neutral pH. It remains unclear whether the aggregates progressively incorporate the native, folded, monomers into themselves, enabling aggregate growth as seen in amyloid fibrils. In the present study, the diffusion of fluorescently labeled monoclonal humanized immunoglobulin G1 monomers in aggregates produced by pH-shift stress was tracked by fluorescence correlation spectroscopy. This method was used in addition to monitoring aggregate formation by dynamic light scattering. The diffusing velocities of the monomers indicated that the folded monomers were not involved in aggregate formation, in contrast with unfolded monomers. On the basis of the results, we propose a bifurcated pathway for the refolding and aggregation of antibodies triggered by a pH shift from acidic to neutral. In the scheme, the refolded monomers survive until their unfolding. The insights in this study will contribute to the manufacture of aggregation-resistant therapeutic antibodies.

Design and operation of a continuous integrated monoclonal antibody production process.

The realization of an end-to-end integrated continuous lab-scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter-based cell-retention, a continuous two column capture process, a virus inactivation step, a semi-continuous polishing step (twin-column MCSGP), and a batch-wise flow-through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity-yield trade-off of classical batch-wise bind-elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight-through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end-to-end integration. The steady-state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303-1313, 2017.