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BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 48 of 151 Table 1(abstract P30) Relative potency (compared to biosimilar) of originators in ADCC and CDC assays Assay Originator Relative potency CV ADCC 2 140% 9.9% 3 141% 11.1% 4 135% 16.8% CDC 2 92% 15.1% 3 89% 10.2% 4 89% 16.7% A higher variability of the originators was found in ADCC assays (n = 6) besides the potency was higher than that of the Adalimumab biosimilar. Major differences between originators with regard to glycosylation were not found. The biosimilar showed a high galactose content and consequently a higher percentage of galactosylated glycan structures than the originators. Conclusions: In summary we show the suitability of an ADCC potency assay for investigation of functional comparability of Adalimumab and biosimilar candidate substances. Differences between biosimilar and originators in glycosylation might contribute to differences found in the ADCC potency assay but not with the CDC potency assay. References 1. Guideline in similar biological medicinal products containing monoclonal antibodies. , EMA/HCMP/BMWP/403543/2010. 2. Guideline on development, production, characterisation and specifications for mnoclonal antibodies and related products. , EMEA/ CHMP/BWP/157653/2007. 3. ICHQ6B Test procedures and acceptance criteria for biotechnological/ biological products. , CMP/ICH/365/96. P31 Cellular tools for biosimilar mAb analysis Carsten Lindemann * , Silke Mayer, Miriam Engel, Petra Schroeder EUFETS GmbH, 55743 Idar-Oberstein, Germany E-mail: Carsten.Lindemann@eufets.com BMC Proceedings 2013, 7(Suppl 6):P31 Background: For the development of biosimilar monoclonal antibodies (mAb) or related substances containing the IgG Fc part it is mandatory to fully compare immunological properties between originator and biosimilar in a “comparability exercise” [1]. The most complex Fc associated function to mediate antibody dependent cellular cytotoxicity (ADCC) needs to be characterized using the active substance of the biosimilar and the comparator. From a regulatory point of view potency assays should reflect the proposed mode of action but in vitro ADCC assays are considered difficult to validate due to the variability of the primary effector cells [2,3]. The requirement to test for ADCC with high precision and accuracy is challenging. Design of cell lines to replace primary cells for effector or target cells is a solution to provide tools for standardized and extensive biosimilar testing. Materials and methods: Retroviral vectors were used to generate cell lines with stable genetic modification. Vector particles were generated by transient transfection of 293T cells with plasmids encoding gag, pol/env and an expression plasmid containing the packaging region and the sequences of promotor and the transgenes, i.e. selection marker and gene of interest. Multiple gene expression was achieved either by using a bicistronic design enabling transcription from two promotor sequences, or by using an internal ribosomal entry site. Transduction of cells in log phase was followed by a selection of transduced cells and clonal selection by limiting dilution. Cell clones were expanded for primary and secondary cell banks and further characterised with regard to transgene expression and functional characteristics. We developed a human transgenic NK-cell line (YTE756.V#26, effector cell line) with stable expression of Fc gammareceptor IIIA (CD16, high affinity variant, valine at position 159) and stable functional characteristics. Target cell lines were generated similarly using different expression plasmid constructs. ADCC assays were developed by using design of experiments (DoE) to determine experimental factors of importance for assay suitability. To show assay suitability goodness of fit, the amplitude of sigmoid curve, slope and parallelism was determined for each sample compared to a standard. Hypoand hyperpotent samples (50%, 100%, 150% and 200% potency) of Rituximab, Trastuzumab, Adalimumab and Infliximab were analysed to determine accuracy and linearity of each method. Optimisation of each assay requires determining the relative importance of factors including E:T ratio, incubation time, target cell density and pre-assay schedules for target and effector cells. Analysis of critical factor interaction was performed using Minitab software. A list of established ADCC assays is shown in Table 1. CD16 expression was analyzed and quantified by flow cytometry. Cells were stained using anti-CD16 PE-conjugated antibodies. PE-fluorescence was correlated to number of PE-molecules per cell using BD Quantibrite beads. Primary NK-cells were isolated using Dynal beads (purity > 95%) from 3 healthy donors and used immediately after isolation. Results: In order to prove genetic stability of the transgenic NK cell line CD16 expression was analysed by flow cytometry for up to 22 passages. More than 95% of cells were CD16 positive, viability of cells was >90%. CD16 expression level was stable (19.000 - 28.000 CD16 molecules/cell). Functional stability of the effector cell line was shown for more than 30 passages. This was shown by a stable EC50 value obtained for a reference antibody in the Trastuzumab ADCC assay. The effector cell line was compared with primary NK-cells (purity > 95%) from 3 donors in a Trastuzumab ADCC assay. The data show high donor variability, mostly incomplete dose-response curves and a killing activity with a low dynamic range (baseline to top ratio: 3). For primary NK-cells the amplitude of the dose-response curve is dependent on both donor variability and the type of target cell. Using the effector cell line this is dependent on the target cell only. Assay variability was strongly reduced and sample throughput could strongly be increased by using the effector cell line in comparison to primary NK-cells. Optimization of each assay by DoE required determining the relative importance of various factors including effector to target cell ratio, incubation time, target cell density and pre-assay culture schedules for target and effector cells. Accuracy of these ADCC assays could be shown in between a range of 50% to 200% potency. Linearity was shown by a high coefficient of determination (>0.97) and other statistical methods. Inter-assay precision of all ADCC assays was
BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 49 of 151 Figure 1(abstract P31) Analysis of accuracy and linearity of the Infliximab ADCC assay. Data shown are sample dose response curves (left) determined by 4PL analysis and mean rel. potency +/- SD (dot, n = 3) compared to the standard (right). For precision analysis the relative potency of a sample was repeatedly analyzed on 4 days with 3 assays per day. Conclusions: Altogether these data show the feasibility of providing suitable tools for validation and routine testing of various mAbs in ADCC potency assays scalable to the analytical needs of biosimilar testing. References 1. Guideline in similar biological medicinal products containing monoclonal antibodies. EMA/HCMP/BMWP/403543/2010. 2. Guideline on development, production, characterisation and specifications for mnoclonal antibodies and related products. EMEA/CHMP/BWP/157653/2007. 3. ICHQ6B Test procedures and acceptance criteria for biotechnological/ biological products. CMP/ICH/365/96. P32 The successful transfer of a modern CHO fed-batch process to different single-use bioreactors Sebastian Ruhl * , Ute Husemann, Elke Jurkiewicz, Thomas Dreher, Gerhard Greller Sartorius Stedim Biotech GmbH, D-37079 Göttingen, Germany E-mail: sebastian.ruhl@sartorius-stedim.com BMC Proceedings 2013, 7(Suppl 6):P32 Introduction: Nowadays, single-use bioreactors are widely accepted in pharmaceutical industry. This is based on shorter batch to batch times, reduced cleaning effort and a significantly lower risk of cross contaminations [1,2]. One large field of the application of single-use bioreactors is the seed train cultivation of mammalian cells [1]. The focus is further extended to perform state of the art fed-batch production processes in such bioreactors. In this study an industrial proven CHO fed-batch process is established in different single-use and reusable bioreactors. Materials and methods: Cell line, medium and process strategy: For the fed-batch process the cell line CHO DG44 (Cellca, Germany) secreting human IgG1 was used. SMD5 medium (Cellca, Germany) was prepared for the seed train and PM5 medium (Cellca, Germany) as a basal medium for the fed-batch culture. The feeding procedure comprised the addition of three different feeds (feed medium A, feed medium B and concentrated glucose solution). After a 3 day batch phase, the 14 day fed-batch phase started. The automated discontinuous bolus feed of feed media A and B was supplemented by the glucose feed solution to keep the glucose concentration above 3 g/L. Bioreactors: The process was initially developed in a 5 L stirred glass bioreactor therefore the BIOSTAT® B with a UniVessel® 5 L was considered as a reference. Single-use bioreactors involved in this study were the stirred tank reactor BIOSTAT® STR 50 L with a CultiBag STR 50 L and the rocking motion bioreactor BIOSTAT® RM 50 optical with CultiBag RM 50 L. Process transfer: The used bioreactors were characterized in terms of process engineering [3]. Due to different agitation and gassing principles present in the BIOSTAT® STR and RM the k L a and mixing times were chosen as a scale-up criteria. The process conditions were specified to meet a k L a-value of > 7 h -1 [4] and a mixing time of < 60 s [5]. Sampling procedure: A daily sampling procedure was performed before the bolus feed. Metabolites like glucose and lactate were analyzed by the Radiometer ABL800 basic (Radiometer, Germany). Viable cell density (VCD) and viability were determined by the Cedex HiRes (Roche Diagnostics, Germany). Results: The process transfer is considered successful, if comparable cellular proliferation activities and product titers are obtained. The initial viable cell density in all systems was 0.3 - 0.4 × 10 6 cells/mL. At the start of the fed-batch phase a viable cell density of 4 - 5 × 10 6 cells/mL could be achieved. As seen in Figure 1A the viable cell density peak of 27 - 28 × 10 6 cells/mL was reached in all systems after 8 - 9 days. At the point of harvest after 17 days viable cell densities between 12 - 17 × 10 6 cells/mL and viabilities of 57 - 82% were reached. The cell broth was harvested for further downstream operations. A well-controlled pH value is essential for a reproducible cell proliferation. As seen in Figure 1B exemplarily shown for the BIOSTAT® STR 50 L small peaks occurred due to the daily addition of feed medium B (pH 11). The offline measured pCO 2 trend shows a constant decrease during the batch phase followed by an increase during the fed-batch phase with a maximum value of 135 mmHg.
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