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Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a “caninized” version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. Mol Cancer Ther; 13(7); 1777–90. ©2014 AACR.

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The production potential of recombinant monoclonal antibody (mAb) expressing cell lines depends, among other factors, on the intrinsic antibody structure determined by the amino acid sequence. In this study, we investigated the influence of somatic mutations in the V(D)J sequence of four individual, mature model mAbs on the expression potential. Therefore, we defined four couples, each consisting of one naturally occurring mAb (2G12, Ustekinumab, 4B3, and 2F5) and the corresponding germline-derived cognate mAb (353/11, 554/12, 136/63, and 236/14). For all eight mAb variants, recombinant Chinese hamster ovary (CHO) cell lines were developed with mAbs expressed from a defined chromosomal locus. The presented workflow investigates critical parameters including productivity, intra- and extracellular product profile, XBP1 splicing, thermal stability, and in silico hydrophobicity. Significant differences in productivity were even observed between the germline-derived mAbs which did not undergo somatic mutagenesis. Accordingly, back-to-germline mutations of mature mAbs are not necessarily reflecting improved expression and stability but indicate opportunities and limits of mAb engineering. From our studies, we conclude that germinalization represents a potential to improve mAb properties depending on the antibody’s germline family, highlighting the fact that mAbs should be treated individually. Electronic supplementary material The online version of this article (10.1007/s00253-019-09998-3) contains supplementary material, which is available to authorized users.

Protein-free media are gaining more and more interest in mammalian cell culture technology. However, the range of commercially available protein-free media is wide, but lack of serum causes the lack of various substances (Keenan et al. in Cytotechnology, 50(1-3):49-56, 2006) which must be substituted case by case. Details on the composition of protein-free media are often unavailable or inaccessible in some cases, and as a consequence, there is an obvious need for testing procedures in order to evaluate the various commercialised products for their performance. Additionally, negative effects of tryptic meat digests on product quality have been reported in the literature (Gu et al. in Biotech Bioeng 56 (4):353-341, 1997). In the present studies of comparing various protein-free media for their suitability in propagation of recombinant CHO cells expressing human growth hormone (hGH), we have found somatotropin to be an excellent candidate for detection of protease activity. Somatotropin contains protease recognition sites for numerous proteases located around amino-acid residues 134-150. In this study, we demonstrate highly specific cleavage of recombinant hGH during batch cultivation. Analysis of the digested molecule was then performed by convergent methods like SDS-PAGE, HPLC and mass spectroscopy, and the results indicate hGH to be an ideal candidate for media and component screening in mammalian cell culture.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of site-directed integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa-Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was co-localised to the target gene.

Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel “DUKX-B11 F3/F” cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as “promoter trap”. The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available “DUKX-B11 F3/F” cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6011-1) contains supplementary material, which is available to authorized users.

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5911-4) contains supplementary material, which is available to authorized users.

Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulum-stress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical.

Electrofusion and EBV transformation were studied by immortalizing human PBLs from blood of HIV-1-positive volunteers. A panel of 33 cell lines producing human monoclonal antibodies (Hu-MAbs) against HIV-1 was established by cell fusion or EBV transformation. For the first fusion experiments the source of B lymphocytes was peripheral blood of HIV-1-infected donors in CDC stages II or III with CD4 cell counts higher than 500/mm3. Later on, from these patients only, those with high anti-HIV titers were chosen as blood donors. By that means the yield of stable specific hybridomas was increased twofold. In our experiments electrofusion turned out to be a more efficient immortalization method than EBV transformation, due to a high and constant immortalization rate. The hybridomas were stable after intensive subcloning and could be cultivated over a period of 8 months without loss in monoclonal antibody production. Immunoglobulin class, subtype, reactivity against HIV-1 proteins, Western blot patterns, immunofluorescence, and epitopes were characterized. The subtype of all antibodies was IgG1 or IgG3. The light chain was predominantly kappa. All antibodies showed reactivity against HIV-1 envelope or core protein. All hybridomas were stable and suited for mass production. Several Hu-MAbs are becoming an important tool in the field of diagnosis, research, and immunotherapy.

Recently we were able to show that the murine anti-idiotypic antibody (Ab2) 3H6 (Kunert et al., 2002; Antiidiotypic Antibody Inducing HIV-1 Neutralizing Antibodies Pending) significantly blocked the binding of the human mAb 2F5 to its synthetic epitope ELDKWA as well as to gp160. Furthermore, 3H6 was also capable of decreasing the in vitro neutralisation potency of 2F5 in a dose-related way. Finally, the murine 3H6 Fab fragments were capable to induce Ab1-like neutralising immune responses after applying the Ab2 to mice. Thus, Ab2/3H6 successfully mimics a neutralising epitope on gp41, which is either not or only poorly immunogenic on the native HIV-1 during natural infection. In the present study the murine Ab2/3H6 was partially humanized (the murine variable regions were fused with the corresponding human constant regions) and recombinantly expressed either as whole IgG1 (using two different expression strategies) or Fab fragment in CHO cells. Crude Ab2/3H6 IgG1 and Ab2/3H6 Fab expression supernatants were effectively purified using either protein A for Ab2/3H6 IgG1 (up to 90% recovery) or a combination of anion-exchange and size-exclusion chromatography in case of Ab2/3H6 Fab (up to 75% recovery). The purified Ab2/3H6 versions were further characterized by several specificity studies and competition experiments with neutralizing monoclonal antibody nMAb 2F5.

The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.

The human monoclonal antibody 2F5 (Buchacher et al., 1994) is a potent candidate for immunotherapy of HIV-1 (Katinger et al., 1995). The hybridoma derived humAb is of IgG3 kappa isotype. Since the IgGl isotype has a longer half-life in human than IgG3, we switched the subclass-type to IgGl by ligation of the 2F5 heavy chain variable region to an IgGl constant region and expressed the IgGl kappa molecule in CHO-cells. Stable recombinant 2F5-IgGl expressing CHO-cells were isolated and the recombinant protein compared to the hybridoma derived immunoglobulin. Here we confirm, that specificity and affinity of different isotypes has not changed. Stability assays of the recombinant 2F5 IgGl clone were performed with and without selection pressure.

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

Antibody engineering allows proper design of improved properties in antibody products such as a high binding affinity, stable biophysical properties and low immunogenicity. Besides these obvious features improved by elegant design the primary amino acid sequence also has tremendous effects on the performance of the expression host itself influencing cell culture parameters including specific productivities and growth rates therefore having major impact on the finally obtained antibody expression titers [1]. By understanding the underlying mechanisms how certain primary sequence combinations influence culture performance in conjunction with biophysical features of the antibody protein, we might rationally improve the production process of these expensive but essential biotherapeutic products. Many different antibody variants were expressed in our group in different projects based on various isotypes. For several antibody variable regions we could observe a direct correlation of advantageous cell performance and a high degree of similarity to the closest human germline sequence of the respective mature antibody leading to higher specific productivities. However, the fundamental principles of these consistent observations and the correlation of cell behavior and biophysical product properties remain quite elusive. Therefore, in this project we aim to set the proper foundations to investigate the generalization of a concept to improve cell performance and antibody expression based on the primary sequence at a high germinality degree (Figure ​(Figure1).1). To have proper control of transgene integration locus, gene copy numbers and mRNA transcript level a suitable host for recombinase-mediated cassette exchange (RMCE) was developed to compare different antibody variants under isogenic conditions. Open in a separate window Figure 1 Observed correlation of specific productivity and sequence identity to the closest human germline sequence (germinality) (A). Antibody expression levels are influenced by several factors. Recombinase-mediated cassette exchange was applied to control for gene copy number and mRNA transcript level. Low expressing antibody variants (2F5, 4B3 and 2G12) were compared to its closest human germline sequence together with a therapeutic antibody (Ustekinumab) with low degree of germinality (B). Respective germline antibody variants were constructed by combination of germline V-, D- and J-segments.

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

Experiments were carried out with five serum-free adapted subclones of a recombinant CHO DHFR - line producing a monoclonal antibody. The bioreactor cultivation was performed in the multiple fermenter system Sixfors (Infors HT) in repeated batch mode. In the course of inoculum preparation cells were cultivated in T-flasks and spinner flasks (WV 0.125l and WV 0.5l). The General Cell Screening System (GCSS) was used as a tool for comparing growth characteristics of subclones derived from different cultivation systems in the course of inoculum preparation and bioreactor cultivation. It enables non-invasive determination of growth kinetics in 96 well plates [Steindl, 1990]. In addition, part of the inocula was used to perform batch cultivations in spinner flasks. The obtained results were compared with those found in the bioreactor. Cell concentrations were determined measuring the cell nuclei with a Coulter Counter after cell desintegration. Viability

The large scale production of recombinant proteins is mainly routed by the stability and production potential of the master cell bank. Biological safety of the product is accessed to the history of the cell line as well as to production conditions, down stream procedures and quality control. The quality of a recombinant cell line is assessed predominantly at the lab scale fermentation but at that time the physiologic and genomic status is already defined. For that reason it is inevitable to control parameters influencing the productivity and growth properties already during clone development. Clonal changes in transcriptional and translational behavior according to stress during selection and amplification have to be identified and comprised into the screening process. In different examples we have analyzed production titers and growth rates as well as genotypic variables concerning house keeping genes, selection markers and target genes providing information on the further development of recombinant clones.

The expression of recombinant glycoproteins that bear a cytotoxic potential is a major challenge using animal cells. Product yields in a range of 0.1 g/l to 1.0g/l as commonly reached for proteins that do not adversely affect the cultured cells are out of reach. The permanent expression of a cytotoxic protein at low specific productivities requires a bioprocess that allows the accumulation of product at concentrations adequate for purification. We compared a continuous perfusion process based on an ultrasonic cell retention device with a discontinuous fed-batch using a balanced nutrient concentrate. Theproduct yield in batch mode was set to a relative concentration of 1.0. In fed-batch the product yield was increased by 40% to a relative value of 1.4 accompanied by a significant loss of culture viability indicating the detrimental effect. However, under perfused conditions not only the volumetric productivity was increased as expected, but also the relative product concentration reached a valueof 6.0 at viabilities of more than 90%. Furthermore the product was harvested almost cell free at perfusion rates of 2d−1. Combining the perfusion mode (cell accumulation) and the fed-batch mode (product accumulation) the product yield was significandy increased toa relative value of 10.0 at final viabilities of still 65%. We thus developed a process capable of accumulating a cytotoxic protein in concentrations sufficient for downstream processing requirements.

The ultimate goal of optimizing biological production is the establishment of simple, safe, cheap, consistent, and easily manageable production systems. Various complicated techniques have been used for animal cell culture. In practice, however, only the simple ones are used for production purposes. A high cell density continuous perfusion in vitro system simulates a situation that most closely exists in the in vivo maintenance of differentiated cells arrested in the G1/G0-phase of the cell cycle. In order to make this a technological reality, a macroporous open structured carrier has been designed using high pressure polyethylene as a primary matrix material. During long-standing continuous perfusion of cultured mammalian cells such as Chinese hamster ovary (CHO) cells with a protein-free medium, the cells are maintained at high cell density (> 10 8 per ml) in a fully productive but nonpropagating state. Using the fluidized bed porous bead technology we do not expect any limitation in the scale-up of any technology using continuous mammalian cell lines.

Als Alternativen zu Tierversuchen bieten sich fur viele Fragestellungen in vitro-Tests mit tierischen oder anderen Zellen an. 96 Well-Mikrotiterplatten erlauben eine grose Anzahl von Versuchen unter gleichen Bedingungen (Zeit, Temperatur, etc.) durchzufuhren. Die Quantifizierung solcher Tests erfolgt bisher anhand von indirekten Zellzahlbestimmungen oder direkter Zellzahlung nach dem Absaugen der Zellen aus den Wells. Diese Methoden sind alle Endpunktbestimmungen und konnen daher nur einmal pro Platte durchgefuhrt werden. Sei es die Quantifizierung uber den Einbau radioaktivmarkierter Nucleotide wie Thymidin (Hewlett G. et al., 1989), oder die Aktivitatsbestimmung von “Marker-Enzymen”, wie mitochondriale Dehydrogenasen mit MTT (Mosmann T., 1983), XTT (Paull K.D. et al., 1988), oder MTS (Baltrop J.A. et al., 1991) als chromogenem Substrat, wie Laktat-Dehydrogenase (Racher A.J. et al., 1990), wie Saure Phosphatase (Conolly D.T. et al., 1986) etc., erlaubt nur eine einzige punktuelle Momentaufnahme pro Platte, deren Ergebnis nicht nur von der vorhandenen Zellzahl, sondern auch vom Aktivierungsgrad der Zellen abhangig ist. Alle kinetischen Parameter und die gesamte Entwicklung des Versuches bis zum gemessenen Endpunkt bleiben bei diesen Methoden als “black box” verborgen.