A cytotoxic leachable compound from single‐use bioprocess equipment that causes poor cell growth performance

A current trend in the production of biopharmaceuticals is the replacement of fixed stainless steel fluid‐handling units with disposable plastic bags. Such single‐use systems (SUS) offer numerous advantages, but also introduce a new set of materials into the production process and consequently expose biomanufacturers to a new set of risks related to those materials, not to mention reliance on an entirely new supply chain. In the course of developing and conducting a cell‐growth‐based test for suitability of disposable plastic components destined for use in cell culture operations, we discovered that the cytotoxic compound bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP) leaches out of certain bags and into cell culture media in concentrations that are deleterious to cell growth. Specifically, media held in certain bags for several days at 37°C was found to contain bDtBPP, and use of those held‐media samples in cell growth experiments provides data that overlap neatly with cell growth experiments using media spiked directly with bDtBPP, proving that bDtBPP leaching is responsible for the reduced growth attributable to those SUS bags. Overall, this issue represents a risk to the production of biopharmaceuticals in SUS, a risk that must be managed by diligent collaboration among companies along the entire supply chain for SUS components. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:332–337, 2014     

Process‐relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics

The biopharmaceutical industry has invested considerably in the implementation of single‐use disposable bioreactors in place of or in addition to their stainless steel‐counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene‐film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035–0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label‐free LC‐MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547–1558, 2016     

Sensitivity of a PER.C6® cell line to bis(2,4-di-tert-butylphenyl)phosphate and evaluation of a new,biocompatible single-use film

Single-use technologies have brought numerous advantages to the biopharmaceutical industry. In particular, single-use bags made from multi-layered polymeric films have been adopted for cell culture and liquid handling operations in place of traditional stainless-steel systems. Despite the advantages, leachable compounds originating from the film's materials of construction present a new challenge. In 2013, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) was identified as a common leachable from several single-use bags that is detrimental to the growth and viability of many Chinese hamster ovary (CHO) cell lines. While much work has been completed to characterize CHO cell sensitivity to bDtBPP, little has been done to characterize its impact on other important production cell lines, particularly PER.C6®. This publication investigates inconsistent cell growth observed in a PER.C6® cell line during bioprocess development. The growth inhibition was linked to leachable migration from Bioclear™ 10, a single-use film from Cytiva (formerly GE Healthcare) that was used for cell expansion. It was shown that the PER.C6® cells displayed a sensitivity to bDtBPP, comparable to that observed in sensitive CHO cell lines. Finally, biocompatibility of PER.C6® with Cytiva's new Fortem film was evaluated, demonstrating that Fortem™ film is a suitable single-use technology for culturing PER.C6® cells.     

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

On‐demand killing of adherent cells on photo‐acid‐generating culture substrates

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro‐projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro‐projection systems currently available, we introduced the assist of the photo‐responsive culture substrates functionalized with a photo‐acid‐generating polymer. In clear contrast to the existing laser‐based methods requiring point scanning, areal micro‐prjection of blue light with the wavelength 436 nm killed many CHO‐K1 cells at a time in the irradiated area on the substrate. The effect of the photo‐generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo‐selective killing of the adherent cells after preliminarily patterning through the photo‐induced removal of cell adhesion‐inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348–352. © 2012 Wiley Periodicals, Inc.     

Verification of a new biocompatible single‐use film formulation with optimized additive content for multiple bioprocess applications

Single‐use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single‐use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein‐free cell culture medium that had been used to extract leachables from freshly gamma‐irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma‐irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma‐irradiation, have been established to ensure lot‐to‐lot consistency. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:1171–1176, 2014     

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high‐yielding Chinese hamster ovary (CHO) transient expression system. Suspension‐adapted CHO‐K1 host cells were engineered to express the gene encoding Epstein‐Barr virus (EBV) nuclear antigen‐1 (EBNA‐1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA‐1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA‐1‐only transfectants. This was confirmed with the retransfection of an EBNA‐1‐only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA‐1‐only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:132–141, 2014     

VCD studies on cyclic peptides assembled from L‐α‐amino acids and a trans‐2‐aminocyclopentane‐ or trans‐2‐aminocyclohexane carboxylic acid

The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans‐2‐aminocyclohexane carboxylic acid (Achc) or trans‐2‐aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three‐dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6‐31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR‐based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Vibrational circular dichroism as a probe of solid‐state organisation of derivatives of cyclic β‐amino acids: Cis‐ and trans‐2‐aminocyclobutane‐1‐carboxylic acid

Peptide models built from cis‐ and trans‐2‐aminocyclobutane‐1‐carboxylic acids (ACBCs) are studied in the solid phase by combining Fourier‐transform infrared spectroscopy (FTIR) absorption spectroscopy, vibrational circular dichroism (VCD), and quantum chemical calculations using density functional theory (DFT). The studied systems are N‐tert‐butyloxycarbonyl (Boc) derivatives of 2‐aminocyclobutanecarboxylic acid (ACBC) benzylamides, namely Boc?(cis‐ACBC)?NH?Bn and Boc?(trans‐ACBC)?NH?Bn. These two diastereomers show very different VCD signatures and intensities, which of the trans‐ACBC derivative being one order of magnitude larger in the region of the ν (CO) stretch. The spectral signature of the cis‐ACBC derivative is satisfactorily reproduced by that of the monomer extracted from the solid‐state geometry of related ACBC derivatives, which shows that no long‐range effects are implicated for this system. In terms of hydrogen bonds, the geometry of this monomer is intermediate between the C6 and C8 structures (exhibiting a 6‐ or 8‐membered cyclic NH?O hydrogen bond) previously evidenced in the gas phase. The benzyl group must be in an extended geometry to reproduce satisfactorily the shape of the VCD spectrum in the ν (CO) range, which qualifies VCD as a potential probe of dispersion interaction. In contrast, reproducing the IR and VCD spectrum of the trans‐ACBC derivative requires clusters larger than four units, exhibiting strong intermolecular H‐bonding patterns. A qualitative agreement is obtained for a tetramer, although the intensity enhancement is not reproduced. These results underline the sensitivity of VCD to the long‐range organisation in the crystal.     

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

In recent years, the number of complex but clinically effective biologicals such as multi‐specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult‐to‐express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next‐generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult‐to‐express proteins. In a previous study we identified miR‐143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR‐143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR‐143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen‐activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR‐143‐3p in CHO cells. Finally, siRNA‐mediated knock‐down of MAPK7 could be demonstrated to phenocopy pro‐productive effects of miR‐143. In summary, our data suggest that miR‐143 might represent a novel genetic element to enhance production of difficult‐to‐express proteins in CHO cells which may be partly mediated by down‐regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046–1058, 2017     

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody‐dependent cellular cytotoxicity, we disrupted α‐1, 6 fucosyltransferase (FUT8 ) gene in CHO‐S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO‐S (FUT8 ^?/?) cells was comparable with wild type CHO‐S cells. FUT8 catalyzes the transfer of a fucose residue from GDP‐fucose to N‐glycans residue. Defucosylated IgG1 antibodies produced by FUT8 ^?/? cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody‐dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.     

Anti‐adhesive property of P‐selectin glycoprotein ligand‐1 (PSGL‐1) due to steric hindrance effect

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is an adhesive molecule that is known to be a ligand for P‐selectin. An anti‐adhesive property of PSGL‐1 has not been previously reported. In this study, we show that PSGL‐1 expression is anti‐adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL‐1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL‐1. PSGL‐1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL‐1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL‐1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL‐1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O‐glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL‐1 has Janus‐faced functions, being both adhesive and anti‐adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.     

Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO‐based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell‐specific productivity (Q_p) over time for recombinant CHO cells developed using the glutamine synthetase (GS)‐based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze‐thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze‐thaw did not result in any significant impact beyond the initial post‐thaw passages. Production cultures sourced from cryopreserved cells and their non‐cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Q_p at increased cell age: 17 of the 24 cell lines displayed ≥20% Q_p decline after ～2–3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Q_p decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze‐thaw as a cause for Q_p decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463–477, 2018     

Strategy for selecting disposable bags for cell culture media applications based on a root‐cause investigation

The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non‐disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry's needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application‐specific parameters. It also led to the development of application‐specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture‐based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air‐quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1535–1549, 2013     

Anti‐cell death engineering of CHO cells: Co‐overexpression of Bcl‐2 for apoptosis inhibition,Beclin‐1 for autophagy induction

Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti‐apoptosis engineering. Recently, autophagy has received attention as a new anti‐cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti‐apoptosis and pro‐autophagy in CHO cells (DG44) was attempted by co‐overexpressing an anti‐apoptotic protein, Bcl‐2, and a key regulator of autophagy pathway, Beclin‐1, respectively. Co‐overexpression of Bcl‐2 and Beclin‐1 exhibited a longer culture period as well as higher viability during serum‐free suspension culture, compared with the control (without co‐overexpression of Bcl‐2 and Beclin‐1) and Bcl‐2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl‐2 overexpression, Beclin‐1 overexpression successfully induced the increase in the autophagic marker protein, LC3‐II, and autophagosome formation with the decrease in mTOR activity. Co‐immunoprecipitation and qRT‐PCR experiments revealed that the enforced expression of Beclin‐1 increased Ulk1 expression and level of free‐Beclin‐1 that did not bind to the Bcl‐2 despite the Bcl‐2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co‐overexpression of Bcl‐2 and Beclin‐1 also protected the cells from cell death more efficiently than Bcl‐2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro‐autophagy engineering together with anti‐apoptosis engineering yields a synergistic effect and successfully enhances the anti‐cell death engineering of CHO cells. Biotechnol. Bioeng. 2013; 110: 2195–2207. © 2013 Wiley Periodicals, Inc.     

Synthesis and Properties of a Novel Pyridineoxazoline Containing Optically Active Helical Polymer as a Catalyst Ligand for Asymmetric Diels–Alder Reaction

A novel pyridineoxazoline (PyOx) containing helical polymer, poly{(–)‐(S)‐4‐tert‐butyl‐2‐[5‐(4‐tert‐butylphenyl)‐3‐vinylpyridin‐2‐yl]‐oxazoline} ( PA ), was designed and synthesized to approach the effect of chain conformation on the catalytic property. Its complex with Cu(OTf)₂, i.e., Cu(II)-PA , was employed to catalyze the homogeneous Diels–Alder (D–A) reaction of alkenoyl pyridine N‐oxides with cyclopentadiene in tetrahydrofuran. Compared with the previously reported copper complex, Cu(II)-P1 (RSC Advances, 2015, 5 , 2882), which was derived from a nonhelical poly[(–)‐(S)‐4‐tert‐butyl‐2‐(3‐vinylpyridin‐2‐yl)‐oxazoline], Cu(II)-PA exhibited a remarkably enhanced enantioselectivity and reaction rate. However, its enantioselectivity was lower than the Cu(II) complex of (–)‐(S)‐4‐tert‐butyl‐2‐[5‐(4‐tert‐butylphenyl)‐3‐vinylpyridin‐2‐yl]‐oxazoline ( Cu(II)-A ), a low molar mass model compound. Chirality 27:523–531, 2015. © 2015 Wiley Periodicals, Inc.     

Proteomics in biomanufacturing control: Protein dynamics of CHO‐K1 cells and conditioned media during apoptosis and necrosis

Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D‐LC‐MS^E discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO‐K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control.     

An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

The Cre‐loxP system is frequently used for site‐specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site‐specific gene integration system using Cre recombinase and mutated loxPs in which the Cre‐mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms. Biotechnol. Bioeng. 2010;105: 1106–1114. © 2009 Wiley Periodicals, Inc.