Effective temperature shift strategy development and scale confirmation for simultaneous optimization of protein productivity and quality in Chinese hamster ovary cells

Temperature shifts to lower culture temperatures are frequently employed in the manufacturing of protein therapeutics in mammalian cells to improve productivity, viability, or quality attributes. The direction and extent to which a temperature shift affects productivity and quality may vary depending on the expression host and characteristics of the expressed protein. We demonstrated here that two Chinese hamster ovary (CHO) clones expressing different human monoclonal antibodies responded differently to a temperature shift despite sharing a common parental CHO cell line. Within a single CHO line, we observed a nonlinear response to temperature shift. A moderate shift to 35°C significantly decreased final titer relative to the unshifted control while a larger shift to 32°C significantly increased final titer by 25%. Therefore, we proposed a systematic empirical approach to assess the utility of a temperature shift for faster implementation during process development. By testing multiple shift parameters, we identified optimum shift conditions in shake flasks and successfully translated findings to benchtop bioreactors and 1,000-L bioreactor scale. Significant differences in final antibody titer and charge variants were observed with temperature shift increments as small as Δ1.5°C. Acidic charge variants decreased monotonically with decreasing shift temperature in both cell lines; however, final antibody titer required simultaneous optimization of shift day and temperature. Overall, we were able to show that a systematic approach to identify temperature shift parameters at small scales is useful to optimize protein production and quality for efficient and confident translation to large-scale production.     

Addition of Valproic Acid to CHO Cell Fed-Batch Cultures Improves Monoclonal Antibody Titers

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.     

Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.     

Perfusion seed cultures improve biopharmaceutical fed‐batch production capacity and product quality

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof‐of‐concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high‐seed fed‐batch production cultures. First, we optimized the perfusion N‐1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N‐1 duration, reaching >40 × 10⁶ vc/mL at the end of the perfusion N‐1 stage. The cultures were subsequently split into high‐seed (10 × 10⁶ vc/mL) fed‐batch production cultures. This strategy significantly shortened the culture duration. The high‐seed fed‐batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low‐seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N‐1 and high‐seed fed‐batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low‐seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:616–625, 2014     

Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non‐UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long‐term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.     

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene‐of‐interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long‐term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non‐UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one‐step amplification procedure. However, non‐UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long‐term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non‐UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1014–1025, 2015     

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single‐Cell Level

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single‐cell level are still lacking. To address this need, we apply O‐propargyl‐puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single‐cell level. In conclusion, OPP‐labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.     

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

IL‐17F co‐ ;expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL‐17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL‐17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL‐17F expression improves the efficiency of cell line subcloning processes. IL‐17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame. Biotechnol. Bioeng. 2013; 110: 1153–1163. © 2012 Wiley Periodicals, Inc.     

Analysis of Tubespins as a suitable scale‐down model of bioreactors for high cell density CHO cell culture

High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed‐batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab‐scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2‐week 50‐mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell‐specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C‐terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS‐simulated HCD cultures can be effectively used as a simple scale‐down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:490–499, 2017     

CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96–320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.     

Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells

Enhanced product yields, reduction in throughput time, improved cost-effectiveness and product quality are examples of benefits gained by delaying apoptotic cell death in bioreactors. To examine the effect on recombinant protein production, bcl-x(L) was overexpressed in a CHO cell line secreting humanized monoclonal antibody directed against the alpha1beta1 integrin. When cell lines overexpressing bcl-x(L) were compared to the parent, cell viability was increased by 20% and titers by 80%. Total viable cell densities were similar and specific productivities were enhanced by almost two-fold on scale-up to bioreactors. Comparison in a chemically defined media demonstrated an even greater sustained viability in bcl-x(L) expressing cells by 50% and up to 90% increase in titer with no impact on product quality. Caspase 3 activities were monitored as a marker for apoptotic cell death. In the presence of Bcl-x(L), caspase activities were reduced to background levels. The role of Bcl-x(L) in protecting cells from premature death was further examined in studies performed in the presence of NaBu, at concentrations known to trigger cell death. Results demonstrated that cells expressing bcl-x(L) retained 88% cell viability with >2 fold increase in titer. Bcl-x(L) was similarly overexpressed in a different CHO cell line producing a humanized mAb against the chemokine MCP1. Once again, production titer was increased by 80% and viability by 75%. Together the studies have shown that overexpression of bcl-x(L) in production cell lines was able to significantly increase the titer by enhancing both the specific activity and total cell viability while maintaining product quality.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells

Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over‐expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess‐relevant characteristics of Chinese hamster ovary (CHO) cell‐derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell‐cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub‐optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR‐transgenic CHO‐derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four‐fold increase compared to the parental production cell line. mTOR‐based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Biotechnol. Bioeng. 2011; 108:853–866. © 2010 Wiley Periodicals, Inc.     

Recombinase‐mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line

To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in‐market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase‐mediated cassette exchange (RMCE) system to build a site‐specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT‐flanked mAb expression cassette, we generated a clonal cell line with good productivity, long‐term production stability, and low mAb gene‐copy number indicating the vector was located in a ‘hot‐spot.’ A SSI host cell line was made by removing the mAb genes from the ‘hot‐spot’ by RMCE, creating a ‘landing pad’ containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP‐based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened ‘time‐to‐clinic’ for therapeutic mAbs. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1645–1656, 2015     

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.     

Improvement of the efficiency and quality in developing a new CHO host cell line

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.     

Screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed‐batch systems

The major challenge in the selection process of recombinant cell lines for the production of biologics is the choice, early in development, of a clonal cell line presenting a high productivity and optimal cell growth. Most importantly, the selected candidate needs to generate a product quality profile which is adequate with respect to safety and efficacy and which is preserved across cell culture scales. We developed a high‐throughput screening and selection strategy of recombinant cell lines, based on their productivity in shaking 96‐deepwell plates operated in fed‐batch mode, which enables the identification of cell lines maintaining their high productivity at larger scales. Twelve recombinant cell lines expressing the same antibody with different productivities were selected out of 470 clonal cell lines in 96‐deepwell plate fed‐batch culture. They were tested under the same conditions in 50 mL vented shake tubes, microscale and lab‐scale bioreactors in order to confirm the maintenance of their performance at larger scales. The use of a feeding protocol and culture conditions which are essentially the same across the different scales was essential to maintain productivity and product quality profiles across scales. Compared to currently used approaches, this strategy has the advantage of speeding up the selection process and increases the number of screened clones for getting high‐producing recombinant cell lines at manufacturing scale with the desired performance and quality. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:160–170, 2016