Optimizing performance of semi‐continuous cell culture in an ambr15™ microbioreactor using dynamic flux balance modeling

The ambr bioreactors are single‐use microbioreactors for cell line development and process optimization. With operating conditions for large‐scale biopharmaceutical production properly scaled down, microbioreactors such as the ambr15? can potentially be used to predict the effect of process changes such as modified media or different cell lines. While there have been some recent studies evaluating the ambr15? technology as a scale‐down model for fed‐batch operations, little has been reported for semi‐continuous or continuous operation. Gassing rates and dilution rates in the ambr15? were varied in this study to attempt to replicate performance of a perfusion process at the 5 L scale. At both scales, changes to metabolite production and consumption, and cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15? bioreactor that produced metabolic shifts and specific metabolic and protein production rates that are characteristic of the corresponding 5 L perfusion process. A dynamic flux balance (DFB) model was employed to understand and predict the metabolic changes observed. The DFB model predicted trends observed experimentally, including lower specific glucose consumption and a switch from lactate production to consumption when dissolved CO₂ was maintained at higher levels in the broth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:420–431, 2018     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Use of an anti‐apoptotic CHO cell line for transient gene expression

Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti‐apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro‐apoptotic factors, Bax and Bak, in a CHO‐K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO‐K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ～3‐ to 4‐fold higher antibody titers than CHO‐K1 cells. As evidence of their anti‐apoptotic properties post‐transfection, DKO cells maintained higher viability and had reduced levels of active caspase‐3 compared to CHO‐K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post‐transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae‐ and clathrin‐mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection‐induced apoptosis and not differences in endocytosis mechanism. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1050–1058, 2013     

Development of a generic transient transfection process at 100 L scale

We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7–8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L⁻¹ IgG.     

100-liter transient transfection

This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2–4% of DNA in transfection cocktail)were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.     

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.     

Application of high‐throughput mini‐bioreactor system for systematic scale‐down modeling,process characterization,and control strategy development

High‐throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high‐throughput mini‐bioreactor system viz. the Advanced Microscale Bioreactor (ambr15^TM), to perform process characterization in less than a month and develop an input control strategy. As a pre‐requisite to process characterization, a scale‐down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO₂ profiles as well as several other process and product quality parameters. The scale‐down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15^TM system is capable of replacing the bench scale bioreactor system for routine process development and process characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1623–1632, 2015     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis

Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-x(L)) regulators of programmed cell death. We previously identified the mitochondrial potassium channel Kv1.3 as a novel target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects on Kv1.3 and the induction of apoptotic changes in mitochondria. These data indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the mitochondrial changes typical for apoptosis. To gain insight into the mechanism of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x(L) (corresponding to K128 in Bax) to lysine. This substitution turned Bcl-x(L) proapoptotic. Transfection of double knockout (Bax(-/-)/Bak(-/-)) mouse embryonic fibroblasts (DKO MEFs) with either wild-type Bax, BaxK128E, or Bcl-x(L)E158K showed that apoptosis induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax. Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon incorporation into planar lipid bilayers. Our results point to a physiologically relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a distinct lysine in determining the proapoptotic character of Bcl2-family proteins.     

Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak

M11L, a 166-amino-acid antiapoptotic protein of myxoma virus, was previously shown to bind to the peripheral benzodiazepine receptor by hydrophobic interactions at the outer mitochondrial membrane. Here we demonstrate that an additional property of M11L is the ability to constitutively form inhibitory complexes with the proapoptotic Bcl-2 family member Bak in human cells. This binding interaction was identified by both FLAG-tagged pull-down assays and tandem affinity purification from transfected and virus-infected human cells. M11L binds constitutively to human Bak and, under some inducible conditions, to human Bax as well, but not to the other Bcl-2 family members (Bad, Bid, Bcl-2). When stably expressed in human embryonic kidney (HEK293) cells, M11L effectively protects these cells from Fas ligand-induced apoptosis, thereby blocking release of cytochrome c, activation of caspase 9, and cleavage of poly(ADP-ribose) polymerase. We also demonstrate in coexpression studies that M11L can interact with Bak independently of any involvement with Bax. Furthermore, cells stably expressing M11L function to prevent apoptosis that is induced by overexpression of Bak. We conclude that M11L inhibits, in a species-independent fashion, apoptotic signals mediated by activation of Bak.     

Bax and Bak knockout apoptosis-resistant Chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed-batch culture process

In the field of therapeutic protein production, process intensification strategies entailing higher starting cell seeding densities, can potentially increase culture productivity, lower cost of goods and improve facility utilization. However, increased cell densities often trigger apoptotic cell death at the end of the cell culture process and thus reduce total viable cell count. Apoptosis-resistant Chinese hamster ovary cell lines may offer the possibility to diminish this undesired outcome of the intensified production process. In this study, we have generated and tested Bax/Bak double-knock-out (DKO) apoptosis resistant hosts to express standard and bispecific antibodies, as well as complex molecules in intensified production processes both as pools and single cell clones, and at different scales. In all cases, therapeutic proteins expressed from clones or pools generated from the Bax/Bak DKO hosts showed not only better viability but also enabled extended productivity in the later stages of the 14-day intensified production process. The product qualities of the produced molecules were comparable between Bax/Bak DKO and wild type cells. Overall, we showed that Bax/Bak DKO apoptosis-resistant host cell lines significantly improve viability and volumetric productivity of the intensified production cultures without altering product qualities.     

The physical characterisation of a microscale parallel bioreactor platform with an industrial CHO cell line expressing an IgG4

There is a growing body of evidence that the ambr™ workstation from TAP Biosystems performs well in terms of helping to select appropriate clones for scale-up studies. Here we have investigated the physical characteristics of this microscale bioreactor system and found that these are quite different from those that exist in larger scale stirred bioreactors. For example, the flow regime in the ambr™ vessel is transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low whilst the specific power input is much higher (~400 W/m³) when compared to that used at larger scales (typically ~20 W/m³). This specific power input is necessary in order to achieve k_La values sufficiently high to satisfy the oxygen demand of the cells and control of dO₂. In line with other studies, we find that the culture of CHO cells in a 15 mL ambr™ bioreactor gave similar cell growth and productivity to that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different. Given the differences in physical characteristics between the ambr™ and larger stirred bioreactors, we suggest that this similarity in biological performance is due to their similar control capabilities and the ‘equivalence of the stress parameters’ across the scales when compared with shake flasks.     

Chelerythrine induces apoptosis through a Bax/Bak-independent mitochondrial mechanism

Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (DeltaPsi(m)) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.     

High‐level protein expression in scalable CHO transient transfection

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14‐day fed‐batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed. Biotechnol. Bioeng. 2009;103: 542–551. © 2009 Wiley Periodicals, Inc.     

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 10¹¹ vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 10¹¹ vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.     

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 10⁷ cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.     

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.     

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.     

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed‐batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled‐up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale‐up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58–68, 2018     

Identification of upstream culture conditions and harvest time parameters that affect host cell protein clearance

During early stage bioprocess development, characterizing interactions between unit operations is a key challenge. Such interactions include the release of host cell enzymes early in the process causing losses in product quality downstream. Using a CHO-expressed IgG1 system, the impact of cell culture duration was investigated using a 50 L bioreactor and performing scale-down protein A purification. While antibody titer doubled during the last week of culture, the post-protein A host cell protein (HCP) levels increased from 243 to 740 ppm. Effects of pH and temperature were then explored using fed-batch ambr250 bioreactors, and parameters enabling higher titers were linked to a decrease in post-protein A product purity. These trade-offs between titer and product quality were visualized using a window of operation. The downstream space was explored further by exposing shake flask material to shear representative of disc stack centrifugation, prior to purification, and by adding polishing chromatography. While product quality decreased with progressing cultivation, cells became more shear resistant. Polishing chromatography resulted in product fragmentation which increased fourfold from Day 10 to 24, adding constraint to achieving both efficient HCP clearance as well as high monomer purities. These examples highlight the importance of adopting integrated approaches to upstream and downstream development strategies to enable whole process optimization.