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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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In a SARS-CoV-2 seroprevalence study conducted with 1,655 working adults in spring of 2020, 12 of the subjects presented with positive neutralization test (NT) titers (>1:10). They were here followed up for 1 year to assess their Ab persistence. We report that 7/12 individuals (58%) had NT_50 titers ≥1:50 and S1-specific IgG ≥50 BAU/ml 1 year after mild COVID-19 infection. S1-specific IgG were retained until a year when these levels were at least >60 BAU/ml at 3 months post-infection. For both the initial fast and subsequent slow decline phase of Abs, we observed a significant correlation between NT_50 titers and S1-specific IgG and thus propose S1-IgG of 60 BAU/ml 3 months post-infection as a potential threshold to predict neutralizing Ab persistence for 1 year. NT_50 titers and S1-specific IgG also correlated with circulating S1-specific memory B-cells. SARS-CoV-2-specific Ab levels after primary mRNA vaccination in healthy controls were higher (Geometric Mean Concentration [GMC] 3158 BAU/ml [CI 2592 to 3848]) than after mild COVID-19 infection (GMC 82 BAU/ml [CI 48 to 139]), but showed a stronger fold-decline within 5–6 months (0.20–fold, to GMC 619 BAU/ml [CI 479 to 801] vs. 0.56–fold, to GMC 46 BAU/ml [CI 26 to 82]). Of particular interest, the decline of both infection- and vaccine-induced Abs correlated with body mass index. Our data contribute to describe decline and persistence of SARS-CoV-2-specific Abs after infection and vaccination, yet the relevance of the maintained Ab levels for protection against infection and/or disease depends on the so far undefined correlate of protection.

The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs but due to their high oligomeric complexity,in vitroproduction, biochemical and biophysical characterizations are challenging. In the present study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (AUC, SEC-MALLS, Mass Photometry and Transmission Electron Microscopy). Each IgM oligomer has individual specific expression pattern and yield with different protein quality likely due to intrinsic IgM properties and patterning. Nevertheless, the purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. And more importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma.

Twelve subjects with positive SARS-CoV-2 neutralization test (NT) titers (>1:10) identified in a seroprevalence study with 1655 working adults were followed up for one year. Here we report that 7 of these 12 individuals (58%) still had NT titers ≥1:50, S1-specific IgG concentrations ≥50 BAU/ml and ≥26% ACE2 receptor binding inhibition, measured with surrogate virus NT one year after mild COVID infection. Furthermore, NT_50 titers >1:10 and S1-specific IgG levels >60 BAU/ml present at three months post-infection persisted at detectable levels for 1 year and correlated with circulating S1-specific memory B-cells. Vaccine-induced SARS-CoV2 immune responses decline at similar rates as those after infection; thus the describes threshold of 60 BAU/ml at three months post infection might also be relevant for assessment of Ab persistence after vaccination.

IgM antibodies are arousing considerable interest as biopharmaceuticals. Despite their immunotherapeutic potential, little is known about the impact of environmental conditions on product quantity and quality of these complex molecules. Process conditions influence the critical quality attributes (CQAs) of therapeutic proteins and thus are important parameters for biological safety and efficacy. Here, the results of a systematic study are presented that characterized the influence of temperature and pH on cell-specific productivity and IgM quality attributes. Biphasic temperature and pH shift experiments were performed as batch cultures in DASGIP® bioreactors under controlled conditions and defined by a specific design of experiment (DOE) approach. An internally-developed recombinant IgM producing CHO cell line was used. With respect to product quality, after an initial purification step efforts were focused on pentamer content, nucleic acid (NA) impurities and the glycosylation profile after an initial purification step. All quality attributes were evaluated by densitometric and chromatographic methods. The reduction of cultivation temperature severely reduced IgM titers, while pH variation had no impact. In contrast, IgM quality was not significantly influenced by bioprocessing parameters. Data revealed that an additional purification step is required to reduce the presence of NAs for in vivo applications. In conclusion, the results showed that for the chosen IgM model, IgM012_GL, variation in quality attributes is not caused by the environmental conditions of temperature and pH.

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.

Monoclonal antibodies (mAbs) represent the largest group of biopharmaceuticals used for human therapy, diagnostics, and imaging. Full-length mAbs with human-like posttranslational modifications are almost exclusively expressed in mammalian cell cultures. Progress in genetic engineering techniques and process development enables the development of optimized cell lines and manufacturing processes. The main factors that influence the yield of a production process are the time to accumulate a target amount of biomass, the process duration, and the specific productivity of the employed cell line. In this chapter, the contribution of the cell line, culture medium, cultivation parameters, and mode of process operation to product yield and quality are discussed.

Nomenclature of monoclonal antibodies traditionally followed a strict scheme indicating target and species information. Because of the rapid advances in this field, emphasized by approval of four humanized and six human antibodies in 2017, the International Nonproprietary Name of new antibodies was updated profoundly by removing the species substem completely. In this review we give an overview about what developments led to the preference of the scientific community towards human-like antibodies. We summarize the major updates in naming schemes that tried to classify antibodies according to their humanization technique or to the final primary sequence and how this led to the erroneous perception to indicate expected immunogenicity. Following the new 2017 nomenclature update, there will not be any information available about the species origin in the names of new antibodies, which emphasizes the need for providing additional supplemental information to the scientific community and develop tools to accurately estimate and control the safety of new monoclonal antibody molecules.

Immunoglobulin M (IgM) antibodies are reckoned as promising tools for therapy and diagnostic approaches. Nevertheless, the commercial success of IgMs is hampered due to bottlenecks in recombinant production and downstream processing. IgMs are large, complex and highly glycosylated proteins that are only stable in a limited range of conditions. To investigate these sensitive IgM antibodies we optimized the elution conditions for a commercially available IgM affinity matrix (CaptureSelect™). Applying a small-scale screening system, we optimized our single step purification strategy for high purity, high yield and retained antigen binding capacity. Here we show that IgMs are sensitive to aggregation at very acidic conditions (pH ≤ 3.0) despite often being used for affinity chromatography. We combined pH 3.5 with a high salt concentration to prevent aggregation during elution. The elution strategy presented in this paper will improve IgM processes for further applications. The herein used IgMs were produced in Chinese hamster ovary (CHO) cells. We present the first detailed glycan analysis of IgM produced in CHO cells with predominantly complex type structures at Asn171, Asn332 and Asn395 and oligomannosidic structures at Asn402 and Asn563 similar to human serum-IgM.

Perfusion is considered as the preferable unit operation mode for fully integrated continuous bioprocessing. However, the inherent complex process control, long process development times, and lack of suitable scale-down models for high-throughput screening are reasons why perfusion processes are still not routinely applied in cell culture technology. Advantages of perfusion are maintenance of a consistent cellular environment, a constant high-quality product flow, enhanced volumetric bioreactor productivity, and small lab footprint. Here, we provide guidelines for screening different proprietary but commercially available HyClone™ Cell Boost™ supplements in a Design of Experiment (DoE) approach to spike the HyClone™ CDM4NS0 basal media for enhanced product titers in small-scale TubeSpin models. These surrogate semi-perfusion cultures were successfully realized by a daily complete media exchange routine resulting in high viable cell densities for extended time periods at minimal media consumption. This technique was leveraged to define the potential of different perfusion media formulations.

Immunoglobulin M (IgM) antibodies are considered as promising biopharmaceutical drugs in the future despite recombinant production is quite challenging as incomplete polymer formation or nucleic acid adherence can decrease the quality of the IgM preparation. Therefore, we defined densitometric and chromatographic methods as suitable tools to analyze the polymer distribution and the remaining nucleic acid content after initial IgM purification. Additionally, the quality of the glycosylation pattern is an important parameter for biological safety and efficacy.

Perfusion is considered as the preferable unit operation mode for fully integrated continuous bioprocessing. However, the inherent complex process control, long process development times, and lack of suitable scale-down models for high-throughput screening are reasons why perfusion processes are still not routinely applied in cell culture technology. Advantages of perfusion are maintenance of a consistent cellular environment, a constant high-quality product flow, enhanced volumetric bioreactor productivity, and small lab footprint. Here, we provide guidelines for screening different proprietary but commercially available HyClone™ Cell Boost™ supplements in a Design of Experiment (DoE) approach to spike the HyClone™ CDM4NS0 basal media for enhanced product titers in small-scale TubeSpin models. These surrogate semi-perfusion cultures were successfully realized by a daily complete media exchange routine resulting in high viable cell densities for extended time periods at minimal media consumption. This technique was leveraged to define the potential of different perfusion media formulations.

Immunoglobulin M (IgM) antibodies are considered as promising biopharmaceutical drugs in the future despite recombinant production is quite challenging as incomplete polymer formation or nucleic acid adherence can decrease the quality of the IgM preparation. Therefore, we defined densitometric and chromatographic methods as suitable tools to analyze the polymer distribution and the remaining nucleic acid content after initial IgM purification. Additionally, the quality of the glycosylation pattern is an important parameter for biological safety and efficacy.

Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry. This is due to increased space–time‐yields (STYs) and a short residence time of the recombinant protein in the bioreactor. Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone‐specific, high‐performing perfusion media we present a straightforward and rapid two‐step approach combining commercially available basal media and feed supplements using design‐of‐experiment. First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small‐scale semicontinuous perfusion cultures. The final media formulation is supported by statistical response surface modeling of a set of cultivation experiments with blended media formulations. Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 106 c/ml within 2 weeks at minimum cell‐specific perfusion rates as low as 10–30 pL/c/d. Obtained STYs of 0.4–1.2 g/L/d represent a 10‐fold increase compared to batch cultures. This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium, and established feed solutions.

The transmembrane protein CD19 is exclusively expressed on normal and malignant B cells and therefore constitutes the target of approved CAR-T cell-based cancer immunotherapies. Current efforts to assess CAR-T cell functionality in a quantitative fashion both in vitro and in vivo are hampered by the limited availability of the properly folded recombinant extracellular domain of CD19 (CD19-ECD) considered as “difficult-to-express” (DTE) protein. Here, we successfully expressed a novel fusion construct consisting of the full-length extracellular domain of CD19 and domain 2 of human serum albumin (CD19-AD2), which was integrated into the Rosa26 bacterial artificial chromosome vector backbone for generation of a recombinant CHO-K1 production cell line. Product titers could be further boosted using valproic acid as a chemical chaperone. Purified monomeric CD19-AD2 proved stable as shown by non-reduced SDS-PAGE and SEC-MALS measurements. Moreover, flow cytometric analysis revealed specific binding of CD19-AD2 to CD19-CAR-T cells. Finally, we demonstrate biological activity of our CD19-AD2 fusion construct as we succeeded in stimulating CD19-CAR-T cells effectively with the use of CD19-AD2-decorated planar supported lipid bilayers.

Rational design of chimeric antigen receptors (CARs) with optimized anticancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative, single-molecule, live-cell imaging and found the sensitivity of CAR T cells toward antigen approximately 1,000-times reduced as compared to T cell antigen-receptor-mediated recognition of nominal peptide-major histocompatibility complexes. While CARs outperformed T cell antigen receptors with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-protein kinase ZAP-70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR designs, which presently limit the efficacy of CAR T cell therapies to target tumors with diminished antigen expression.

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.

Cell culture media are highly complex solutions of multiple nutrients for mammalian cells and lay the foundation for enhanced quality of the expressed protein and the performance of the bioprocess. Especially for long term, large-volume continuous production processes the constant quality of media without compositional variation is desirable, despite exposure to various stress conditions like (UV)-light or (high)-temperature cannot be represented by a measurable parameter yet. In this study, chemometric analysis of non-enhanced RAMAN spectra was used to identify different cell culture media and to track accelerated degradation conditions by light or temperature in aqueous solutions of glucose and chemically defined media. To link the changes in the RAMAN spectra with cell culture parameters we used the untreated and stressed media for biophysical and biochemical measurements as well as cell culture experiments. The two tested media were exceptionally stable against heat, but showed diminished biological performance after light exposure accompanied by reduced concentrations of the key amino acids Met, His, Trp and Lys. RAMAN spectroscopy provides a rapid and non-destructive method to distinguish individual cell culture media and to find even minor differences between otherwise comparable media lots. In this study we demonstrate the suitability of RAMAN spectroscopy for identity- and quality testing of various process media as an exciting tool for implementation in quality control of bioprocessing.

Feed supplements are concentrated cell culture media that contain a variety of nutrients, which can be added during a bioprocess. During fed-batch cultivation, feed media are typically added to a growing cell culture to maximize cell and product concentrations. In this study, only a single shot of feed medium was added on day 0 to a basal cell culture medium and compared to non-supplemented basal medium (feed-spiked at day 0 versus control experiments) by cultivation of a recombinant mAb expressing CHO cell line in batch mode under controlled conditions in a bioreactor. Since the feed-spike at day 0 was based on existing medium components without introducing additional supplements, a desirable process with decreased complexity was generated. Unlike cells in basal medium, feed-spiked cultures reached almost 2× higher peak cell concentrations (10 × 106 c/mL vs. 18 × 106 c/mL) and 3× higher antibody concentrations (0.8 g/L vs. 2.4 g/L). Batch process time and the integral over the viable cell count were similar for both process types. Constantly high cell-specific production rates in feed-spiked cultures (70 pg/cell/day) compared to continuously declining rates in basal medium (from 70 to 10 pg/cell/day) were responsible for an overall 70% higher cell-specific production rate and the higher product concentrations. To associate gene expression patterns to different process proceedings, transcriptome analysis was performed using microarrays. Several transcripts that are involved with glutamine de novo synthesis and citric acid cycle were significantly upregulated on several days in feed-spiked cultures. The top identified gene ontology (GO) terms related well to cell cycle and primary metabolism, cellular division as well as nucleobase formation or regulation, which indicated a more active proliferative state for feed-spiked cultures. KEGG biochemical pathway analysis and Gene set enrichment analysis (GSEA) further confirmed these findings from a complementary perspective. Moreover, several interesting gene targets, which have not yet been associated with recombinant protein expression, were identified that related to a higher proliferative state, growth, protein synthesis, cell-size control, metabolism, cell survival as well as genes that are associated with the control of the mammalian target of rapamycin (mTOR) in feed-spiked cultures. Analysis of critical product quality attributes (i.e. glycosylation, charge variants and size distribution) showed that feed-spiking did not change antibody quality.

Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44, and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis, and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing show that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations. In this study, the host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S, and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format by using bacterial artificial chromosomes (BACs) as transfer vector in all cell lines is examined. Cell-specific growth and product formation are studied in batch, fed-batch, and semi-continuous perfusion cultures. Further, two different cell culture media are used to investigate their effects. The authors find CHO cell line-specific preferences for mAb production or biomass synthesis that are determined by the host cell line. Additionally, quality attributes of the expressed mAb are influenced by the host cell line and media.

Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called ‘tyrosine cage’. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5., Protein Engineering, Design & Selection, 31 (7-8), ISSN:1741-0126, ISSN:1741-0134, ISSN:0269-2139, ISSN:1460-213X

The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MSE was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 106 dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control.

Single-chain fragment variable-fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.

Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The ever-increasing knowledge about the potential of IgAs has renewed interest in this antibody class for therapeutic use against a variety of infectious and malignant diseases, and as a preventive agent for mucosal pathogens. Despite the considerable therapeutic potential of IgA the exploration thereof has often been hampered due to difficulties in producing and purifying desired quantities. Large amounts of pure IgA will be required for in vivo studies. This work reviews current achievements and bottlenecks in upstream and downstream processing of recombinant IgA from a biotechnological point of view. We also highlight recent accomplishments with diverse expression systems and presents different affinity techniques for the capture of recombinant IgA to compare their purification potential.

Human host cell lines for the production of biopharmaceutical proteins are of interest due to differences in the glycosylation patterns of human and animal cell lines. Specifically, sialylation, which has a major impact on half-life and immunogenicity of recombinant biopharmaceuticals, differs markedly. Here, we established and characterized an immortalized well documented and serum-free host cell line, RS, from primary human renal proximal tubular epithelial cells (RPTEC). In order to test its capacity to produce complex glycosylated proteins, stable recombinant human erythropoietin (rhEpo) producing clones were generated. The clone with highest productivity, RS-1C9 was further characterized and showed stable productivity. Biological activity was observed in in vitro assays and 28% of rhEpo glyco-isoforms produced by RS-1C9 were in range and distribution of the biological reference standard (BRP) isoform, as compared to 11.5% of a CHO based rhEpo. Additionally, cellular α-2,6 sialylation, Galactose-alpha-1,3-galactose (alpha-Gal) and N-glycolylneuraminic acid (NeuGc) patterns compare favourably to CHO cells. While productivity of RS still needs optimization, its amenability to upscaling in bioreactors, its production of glyco-isoforms that will increase yields after down-stream processing of about 2.5 fold, presence of sialylation and lack of Neu5Gc recommend RS as alternative human host cell line for production of biopharmaceuticals.

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells' quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell's productivity.

Designing appropriate expression vectors is one of the critical steps in the generation of stable cell lines for recombinant protein production. Conventional expression vectors are severely affected by the chromatin environment surrounding their integration site into the host genome, resulting in low expression levels and transgene silencing. In the past, a new generation of expression vectors and different strategies was developed to overcome the chromatin effects. Bacterial artificial chromosomes (BACs) are cloning vectors capable of accommodating up to 350 Kb. Thus, BACs can carry a whole eukaryotic locus with all the elements controlling the expression of a gene; therefore, BACs harbor their own chromatin environment. Expression vectors based on BACs containing open/permissive chromatin loci are not affected by the chromatin surrounding their integration site in the host cell genome. Consequently, BAC-based expression vectors containing the appropriate loci confer predictable and high levels of expression over time. These properties make BAC-based expression vectors a very attractive tool applied to the recombinant protein production field.

Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using amplicon- or plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool.

Single-chain fragment variable-fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.

For many years, the potential of immunoglobulin M (IgM) antibodies was not fully understood because of characteristics different to the well-known immunoglobulin G type like low target affinity, cross reactivity and complex protein structure. In the meanwhile IgMs have been positively evaluated for their use as therapeutic agent in the mucosal environment but also in serum to eradicate upcoming tumor cells and invading antigens. Therefore IgM class of antibodies will play a significant role in clinical applications but also diagnosis in the future. To evaluate the full potential of this kind of antibody molecules large amounts of high quality product will be needed. In this review the focus is set on the biotechnological aspect of producing IgM class antibodies recombinantly in mammalian cells. Current achievements in expression and purification of this molecule are highlighted and compared.

Vector engineering approaches are commonly used to increase recombinant protein production in mammalian cells, and among various concepts, bacterial artificial chromosomes (BAC) have been proposed to serve as open chromatin regions to omit chromosome positional effects. For proof of concept, we developed stable recombinant Chinese hamster ovary (CHO) cell lines using different expression vector systems: the plasmid vectors contained the identical expression cassette as the BAC constructs. Two anti-HIV1 antibody derivates served as model proteins (3D6scFc and 2F5scFc) for generation of four stable recombinant CHO cell lines. The BAC-derived clones showed three to four times higher specific productivity, and therefore, gene copy numbers and transcript level were quantified. The active chromatin region provided with the BAC environment significantly improved transcription evidenced with both model proteins. Specific transcription was approximately six times higher from BAC-based vectors compared to the corresponding plasmid vectors for both single-chain fragment crystallizable (scFc) proteins. Our accurate investigations elucidated also differences between translational activities related to the protein of choice. 3D6scFc expressed specifically three to four times more product than 2F5scFc indicating that the product by itself also contributes to enhanced productivity. This study indicated comparable increase of transcription level for both scFc proteins when using the BAC system, but translation, maturation, and secretion of individual proteins seem to be protein specific.

Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB , gyrB , and ycfQ . Simultaneous gene scanning of fljB , gyrB , and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S . Dublin isolates, an additional single PCR assay was developed for specific identification of S . Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S . Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.

MicroRNAs (miRNAs) are a novel class of short non-coding RNAs, which negatively regulate target gene expression at post-transcriptional level. They mediate an important layer of control in the global regulation of gene networks, controlling a broad range of physiological as well as patho-physiological pathways including development, cancer, metabolism, proliferation, and stress resistance. So far, more than 365 miRNA genes have been identified in CHO cells. The functional analysis of the physiological effect of such large numbers of miRNAs, however, requires an efficient functional screening method. In the current study, we therefore established and evaluated a protocol to perform miRNA overexpression and to screen their effect on bio-industrially relevant phenotypes, such as growth, viability and productivity, using a recombinant, Epo-Fc producing CHO cell line. For protocol optimization, four CHO miRNAs (cgr-miR-17, cgr-miR-221, cgr-miR-21, and cgr-miR-210) were cloned into small hairpin vectors including a GFP cassette and transfected. After transfection cells were analyzed for growth and productivity over a 4-day period. Even from this small set of four miRNAs, the overexpression of miR-17, one of the members of the oncogenic miR-17-92 cluster, gave proof of principle that this method enables the identification of miRNA engineering candidates as its overexpression increased the speed of cell proliferation without negatively impacting specific productivity. The here presented method is applicable for medium-throughput screening for microRNA, miR-sponge, siRNA, or mRNA overexpression along with detailed functional characterization using the same experimental set up. As the same procedure can be applied to different production cell lines, the protocol can also be used to test for individual, cell line specific responses to microRNAs. Thus our system represents a general platform to functionally screen candidates for rational cell factory design. Biotechnol. Bioeng. 2012; 109:1376–1385. © 2012 Wiley Periodicals, Inc.

Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions.

Immunoglobulins of isotype A (IgA) mediate a key role in mucosal immunity and are promising new immunotherapeutic candidates, but difficulties in obtaining enough material often hamper their in vivo exploration. We established recombinant Chinese hamster ovary (CHO) cells which stably expressed two IgA1 antibodies under serum-free conditions. The two cell lines achieved significantly different specific productivities of 16 pg per cell and day and 100 times less, a common phenomenon in recombinant antibody expression which challenges the production and purification process. Polymeric IgA in crude culture supernatants was assembled with J chain and showed expected specificity. We employed an immobilized camelid anti-human alpha-chain VHH ligand and isolated both recombinant IgAs at high purity and yield in a single chromatographic step. The described method was irrespective of the light chain and specificity and may be used as a generic capture step for the isolation of any IgA. Results were compared with a multistep purification process consisting of an affinity step based on immobilized jacalin followed by anion exchange and hydrophobic interaction chromatography.