LC-HRMS-based targeted metabolomics for high-throughput and quantitative analysis of 21 growth inhibition-related metabolites in Chinese hamster ovary cell fed-batch cultures.

Chinese hamster ovary (CHO) cells have been widely used in the biopharmaceutical industry for production of therapeutic proteins. CHO cells in fed-batch cultures produce various amino acid-derived intermediate metabolites. These small molecule metabolic byproducts have proven to be critical to cell growth, culture performance, and, more interestingly, antibody drug productivity. Herein, we developed an LC-HRMS-based targeted metabolomics approach for comprehensive quantification of total 21 growth inhibition-related metabolites generated from 14 different amino acids in CHO cell fed-batch cultures. High throughput derivatization procedures, matrix-matched calibration curves, stable isotope-labeled internal standards, and accurate mass full MS scan were utilized to achieve our goal for a wide range of metabolite screening as well as validity and reliability of metabolite quantification. We further present a novel analytical strategy for extending the assay's dynamic range by utilizing naturally occurring isotope M + 1 ion as a quantification analog in the circumstances where the principal M ion is beyond its calibration range. The integrated method was qualified for selectivity, sensitivity, linearity, accuracy, precision, isotope analysis, and other analytical aspects to demonstrate assay robustness. We then applied this metabolomics approach to characterize metabolites of interest in a CHO cell-based monoclonal antibody (mAb) production process with fed-batch bioreactor culture mode. Absolute quantification combined with multivariate statistical analysis illustrated that our target analytes derived from amino acids, especially from branched-chain amino acids, closely correlated with cell viability and significantly differentiated cellular stages in production process.

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells.

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

Phosphorylation of NLRC4 is critical for inflammasome activation.

NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1ß secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.

Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

PILRα negatively regulates mouse inflammatory arthritis.

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sµ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sµ. These results suggest that the sufficiency of Sµ to mediate IgH rearrangements may be influenced by context-dependent cues.

Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation.

Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.

SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.

Pannexin-1 is required for ATP release during apoptosis but not for inflammasome activation.

Apoptotic cell death is important for embryonic development, immune cell homeostasis, and pathogen elimination. Innate immune cells also undergo a very rapid form of cell death termed pyroptosis after activating the protease caspase-1. The hemichannel pannexin-1 has been implicated in both processes. In this study, we describe the characterization of pannexin-1-deficient mice. LPS-primed bone marrow-derived macrophages lacking pannexin-1 activated caspase-1 and secreted its substrates IL-1ß and IL-18 normally after stimulation with ATP, nigericin, alum, silica, flagellin, or cytoplasmic DNA, indicating that pannexin-1 is dispensable for assembly of caspase-1-activating inflammasome complexes. Instead, thymocytes lacking pannexin-1, but not the P2X7R purinergic receptor, were defective in their uptake of the nucleic acid dye YO-PRO-1 during early apoptosis. Cell death was not delayed but, unlike their wild-type counterparts, Panx1(-/-) thymocytes failed to recruit wild-type peritoneal macrophages in a Transwell migration assay. These data are consistent with pannexin-1 liberating ATP and other yet to be defined "find me" signals necessary for macrophage recruitment to apoptotic cells.

Combining regulated and constitutive protein expression significantly boosts protein expression by increasing productivity without affecting CHO cell growth.

Chinese hamster ovary (CHO) cells are commonly used for the expression of therapeutic proteins. To increase the titer output of CHO production cultures either specific productivity (Qp), growth, or both need to be increased. Generally, Qp and growth are inversely correlated and cell lines with high Qp have slower growth and vice versa. During the cell line development (CLD) process, the faster-growing cells tend to take over the culture and represent the majority of the isolated clones post single cell cloning. In this study, combinations of regulated and constitutive expression systems were used to supertransfect targeted integration (TI) cell lines expressing the same antibody either constitutively or under-regulated expression. Clone screening with a hybrid expression system (inducible + constitutive) allowed identification and selection of higher titer clones under uninduced conditions, without a negative impact on cell growth during clone selection and expansion. Induction of the regulated promoter(s) during the production phase increased the Qp without negatively affecting growth, resulting in approximately twofold higher titers (from 3.5 to 6-7 g/L). This was also confirmed using a 2-site TI host where the gene of interest was expressed inducibly from Site 1 and constitutively from Site 2. Our findings suggest that such a hybrid expression CLD system can be used to increase production titers, providing a novel approach for expression of therapeutic proteins with high titer market demands.

Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling.

During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2-3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells.

Increased targeting of donor switch region and IgE in Sgamma1-deficient B cells.

Ab class switch recombination involves a recombination between two repetitive DNA sequences known as switch (S) regions that vary in length, content, and density of the repeats. Abs expressed by B cells are diversified by somatic hypermutation and class switch recombination. Both class switch recombination and somatic hypermutation are initiated by activation-induced cytidine deaminase (AID), which preferentially recognizes certain hot spots that are far more enriched in the S regions. We found that removal of the largest S region, Sgamma1 (10 kb), in mice can result in the accumulation of mutations and short-range intra-S recombination in the donor Smu region. Furthermore, elevated levels of IgE were detected in trinitrophenol-OVA-immunized mice and in anti-CD40 plus IL-4-stimulated B cells in vitro. We propose that AID availability and targeting in part might be regulated by its DNA substrate. Thus, prominently transcribed S regions, such as Sgamma1, might provide a sufficient sink for AID protein to titrate away AID from other accessible sites within or outside the Ig locus.

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.

Expressing antigen binding fragments with high titers in a targeted integration CHO host by optimizing expression vector gene copy numbers and position: A case study.

Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integration (TI) Chinese Hamster Ovary (CHO) host. Without a need for extensive host or process optimization, as is usually required for E. coli, by simply using different vector configurations, clones with very high Fab expression titers were obtained. In this case, by increasing heavy chain (HC) gene copy numbers, clones with titers of up to 7.4 g/L in the standard fed-batch production culture were obtained. Our findings suggest that having a predetermined transgene integration site, as well as the option to optimize gene copy number/dosage, makes CHO TI hosts an effective system for expression of Fab molecules, allowing Fab expression using platform process and without significant process development efforts.

An arrayed CRISPR screen reveals Myc depletion to increase productivity of difficult-to-express complex antibodies in CHO cells.

Complex therapeutic antibody formats, such as bispecifics (bsAbs) or cytokine fusions, may provide new treatment options in diverse disease areas. However, the manufacturing yield of these complex antibody formats in Chinese Hamster Ovary (CHO) cells is lower than monoclonal antibodies due to challenges in expression levels and potential formation of side products. To overcome these limitations, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based knockout (KO) arrayed screening of 187 target genes in two CHO clones expressing two different complex antibody formats in a production-mimicking set-up. Our findings revealed that Myc depletion drastically increased product expression (>40%) by enhancing cell-specific productivity. The Myc-depleted cells displayed decreased cell densities together with substantially higher product titers in industrially-relevant bioprocesses using ambr15 and ambr250 bioreactors. Similar effects were observed across multiple different clones, each expressing a distinct complex antibody format. Our findings reinforce the mutually exclusive relationship between growth and production phenotypes and provide a targeted cell engineering approach to impact productivity without impairing product quality. We anticipate that CRISPR/Cas9-based CHO host cell engineering will transform our ability to increase manufacturing yield of high-value complex biotherapeutics.

A ribonucleoprotein-based decaplex CRISPR/Cas9 knockout strategy for CHO host engineering.

Chinese hamster ovary (CHO) cell engineering based on CRISPR/Cas9 knockout (KO) technology requires the delivery of guide RNA (gRNA) and Cas9 enzyme for efficient gene targeting. With an ever-increasing list of promising gene targets, developing, and optimizing a multiplex gene KO protocol is crucial for rapid CHO cell engineering. Here, we describe a method that can support efficient targeting and KO of up to 10 genes through sequential transfections. This method utilizes Cas9 protein to first screen multiple synthetic gRNAs per gene, followed by Sanger sequencing indel analysis, to identify effective gRNA sequences. Using sequential transfections of these potent gRNAs led to the isolation of single cell clones with the targeted deletion of all 10 genes (as confirmed by Sanger sequencing at the DNA level and mass spectrometry at the protein level). Screening 704 single cell clones yielded 6 clones in which all 10 genes were deleted through sequential transfections, demonstrating the success of this decaplex gene editing strategy. This pragmatic approach substantially reduces the time and effort required to generate multiple gene knockouts in CHO cells.

DNA targets of AID evolutionary link between antibody somatic hypermutation and class switch recombination.

As part of the adaptive immune response, B cells alter their functional immunoglobulin (Ig) receptor genes through somatic hypermutation (SHM) and/or class switch recombination (CSR) via processes that are initiated by activation induced cytidine deaminase (AID). These genetic modifications are targeted at specific sequences known as Variable (V) and Switch (S) regions. Here, we analyze and review the properties and function of AID target sequences across species and compare them with non-Ig sequences, including known translocation hotspots. We describe properties of the S sequences, and discuss species and isotypic differences among S regions. Common properties of SHM and CSR target sequences suggest that evolution of S regions might involve the duplication and selection of SHM hotspots.

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis, gating pyruvate generation, and increasing pyruvate flux into the TCA cycle.

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.

Concurrent transfection of randomized transgene configurations into targeted integration CHO host is an advantageous and cost-effective method for expression of complex molecules.

Complex recombinant proteins are increasingly desired as potential therapeutic options for many disease indications and are commonly expressed in the mammalian Chinese hamster ovary (CHO) cells. Generally, stoichiometric expression and proper folding of all subunits of a complex recombinant protein are required to achieve the desired titers and product qualities for a complex molecule. Targeted integration (TI) cell line development (CLD), which entails the insertion of the desired transgene(s) into a predefined landing-pad in the CHO genome, enables the generation of a homogeneous pool of cells from which clonally stable and high titer clones can be isolated with minimal screening efforts. Despite these advantages, using a single transgene(s) configuration with predetermined gene dosage might not be adequate for the expression of complex molecules. The goal of this study is to develop a method for seamless screening of many vector configurations in a single TI CLD attempt. As testing vector configurations in transient expression systems is not predictive of protein expression in the stable cell lines and parallel TI CLDs with different transgene configurations is resource-intensive, we tested the concept of randomized configuration targeted integration (RCTI) CLD approach for expression of complex molecules. RCTI allows simultaneous transfection of multiple vector configurations, encoding a complex molecule, to generate diverse TI clones each with a single transgene configuration but clone specific productivity and product qualities. Our findings further revealed a direct correlation between transgenes' configuration/copy-number and titer/product quality of the expressed proteins. RCTI CLD enabled, with significantly fewer resources, seamless isolation of clones with comparable titers and product quality attributes to that of several parallel standard TI CLDs. Therefore, RCTI introduces randomness to the TI CLD platform while maintaining all the advantages, such as clone stability and reduced sequence variant levels, that the TI system has to offer.