Antibody charge heterogeneity formation in a mammalian cell culture fed-batch process

The charge heterogeneity of a monoclonal antibody (mAb) is as a sum factor of several post translational modifications and most of them are of high importance regarding product quality and efficacy. For this reason monitoring and controlling of this sum factor can be beneficial. The work presented here builds the basis for on-line monitoring and will help to achieve Quality by Control (Sommeregger et al., 2017). The aim of this work was to develop a method that allows fast and accurate determination of the charge profile of monoclonal antibodies directly from cell culture supernatants. We were able to circumvent a pre-purification step by adapting a cation exchange method (CEX) using a highly linear pH gradient (Lingg et al, 2013). The established method was then used to gain information about the formation of charge variants during a fed-batch process of an industrial relevant mAb produced by a Chinese Hamster Ovary (CHO) cell line. Please click Additional Files below to see the full abstract

Genotype of CHO host cell line has higher impact on mAb production and quality than process strategy or cell culture medium

Chinese hamster ovary (CHO) cells comprise a variety of lineages, including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite the fact that CHO cell lines share a common ancestor, extensive mutagenesis and clonal selection have resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are lacking from individual CHO cell lines and that each cell line harbors a unique set of mutations that are relevant to the bioprocess. However, literature outlining how the observed genetic differences affect CHO cell performance during bioprocess operations remains scarce. In this study, we examined 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 in all cell lines by using bacterial artificial chromosomes (BACs) as transfer vector. Cell-specific growth, product formation and heavy and light chain mRNA levels were studied in batch, fed-batch and perfusion cultures. Furthermore, two different cell culture media were investigated. Product quality was studied through glycoprofiling, and the thermal denaturation was analyzed using differential scanning calorimetry (DSC). We found CHO cell line-specific preferences for mAb production or biomass synthesis that were determined by the host cell line rather than product-specific mRNA levels. Additionally, quality attributes of the expressed mAb were influenced by the host cell line and medium used

Characterization of recombinant IgA producing CHO cells and product purification

Obwohl heutzutage mehrere Techniken zur Verfügung stehen, bleibt die Entwicklung gut produzierender rekombinanter Zelllinien schwierig. Die beiden IgA produzierenden Zelllinien 3D6-IgA/5C5 und 4B3-IgA/6H2, deren Produkte gegen das Hüllprotein gp41 des Humanen Immundefizienz-Virus 1 (HIV-1) gerichtet sind, wurden zuvor entwickelt und zeigten sehr unterschiedliche spezifische Produktionsraten. In der vorgelegten Arbeit wurden die Genkopienzahlen der schweren und leichte Ketten sowie der sogenannten Joining-Chain in beiden Produktionsklonen analysiert und auch die intrazellulären Mengen an Transkripten verglichen. Die Methode der Wahl für diesen Zweck war quantitative Real-time PCR, die für die isolierte genomische DNA und cDNA angewendet wurde. In verschiedenen Vorversuchen wurde die Methode optimiert und anschließend mehrere unabhängige Proben in der Real-time PCR analysiert und verglichen. Die erhaltenen Ergebnisse zeigten Unterschiede zwischen den Gen-Kopienzahlen und auch zwischen der Menge an Transkripten, allerdings konnte keine eindeutige Korrelation zwischen der Anzahl der Genkopien und der Transkriptionsrate sowie zwischen der Transkriptionsrate und den verschiedenen spezifischen Produktivitäten identifiziert werden. Die Ergebnisse legen nahe, dass in diesem Fall Gen-Kopien und Transkriptmengen keinen signifikanten Einfluss auf die Produktivität haben, aber Engpässe in anderen Teilen der Proteinexpressions-Maschinerie die Antikörper-Synthese beeinflussen. Neben der Bedeutung der Entwicklung gut produzierender Zelllinien für biotechnologische Prozesse besteht auch die Notwendigkeit funktionelle Methoden zur Reinigung der Produkte zu erarbeiten, die zu hoher Reinheit und akzeptablen Wiederfindungsraten führen. Im zweiten Teil dieser Arbeit wurden IgA Aufreinigungen mit Hilfe einer IgA Affinitätsmatrix erfolgreich durchgeführt.Although many techniques are available, the establishment of well-producing recombinant cell lines remains difficult. The two recombinant cell lines 3D6-IgA/5C5 and 4B3-IgA/6H2, that produce IgAs which are both directed against the gp41 envelope protein of the human immunodeficiency virus 1 (HIV-1), were previously generated and resulted in significantly different specific productivities. In this work, we compared the two cell lines according to their IgA heavy chain, light chain and joining chain gene copy numbers as well as the intracellular amount of transcripts. The method of choice for this purpose was quantitative real-time PCR and was performed using genomic DNA and cDNA. Method optimization was conducted and several quantification approaches of real-time PCR were analysed and compared. The obtained results showed differences among the gene copy numbers and also the amount of transcripts. However, no significant correlation between the number of product related gene copies and their transcription rates as well as between the transcription rates and the different specific productivities could be identified. The results suggested that gene copy and transcript numbers are not the only parameters influencing productivities, but bottlenecks elsewhere in the protein expression machinery may control antibody synthesis and secretion. In the second part of this work antibody purification using an IgA affinity matrix was successfully performed with camelid affinity ligands yielding highly pure IgAs with rather good recovery. This down-stream procedure enables IgA purification with high purity and acceptable recovery rates.submitted by Sommeregger WolfgangZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Masterarb., 2012(VLID)103612

Application of the Bradford Assay for Cell Lysis Quantification : Residual Protein Content in Cell Culture Supernatants

Österreichische Forschungsförderungsgesellschaft. Grant Number: 849725Bilfinger Industrietechnik Salzburg. Grant Number: 849725(VLID)384558

Ganglionic Local Opioid Analgesia at the Superior Cervical Ganglion: MRI-Verified Solution Spread

Abstract Introduction Ganglionic local opioid analgesia (GLOA) at the superior cervical ganglion (SCG) is performed for pain control and is known to be an effective procedure. In this study, we evaluated the spread of the injectate in the area of the SCG. Our expectation was that there would be a correlation between the area and volume of the injectate spread and post-procedural outcome measures. Methods This was a retrospective blinded review of magnetic resonance imaging (MRI) scans. Assessors evaluated the anatomical area of fluid spread, the furthermost spread from midline, any hampered spread and contact of contrast fluid with other structures. The efficacy of GLOA and complications were estimated. Results The main solution spread reached from the C1 to C3 vertebrae. The furthest spread in the lateral and sagittal planes was 21.2 and 15.2 mm, respectively. The furthest craniocaudal spread was 63.5 mm. In 53.3% and 33% of interventions, the solution was found in the parapharyngeal space and in its “medial compartment,” respectively. A correlation was found between pain relief and both solution spread and volume of solution spread. No hampered spread was recorded. A negative correlation between pain reduction and number of GLOA was observed. Higher pre-procedural pain intensity was correlated with higher pain reduction. We estimated pain relief in 93% of procedures correctly. No correlation between post-procedural Numerical Rating Scale (NRS) scores and different needle approaches was found. Conclusion For the transoral blocking technique, a strict laterodorsal needle direction is recommended to prevent possible block failures. A total volume of 2 ml injected into the parapharyngeal space and its “medial compartment” is recommended. Higher volumes may lead to uncontrolled distribution patterns. Trial registration Clinicaltrials.gov identifier NCT05257655; date of registration 2022-02-25; patient enrollment date from 2023-01-09 to 2023-08-31

Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.(VLID)458983