Identification of cell culture levers to lower trisulfide modifications in monoclonal antibodies produced in CHO cell culture

Trisulfide bond formations are a commonly observed modification of monoclonal antibodies produced in cell culture processes. Protein trisulfide bonds are post-translational modifications in which an extra sulfur atom is bound between two cysteines involved in disulfide bonding. This modification has been detected in both recombinant and endogenous plasma-derived IgGs. Inconsistent levels of trisulfides were identified as a source of variability observed during the conjugation process for antibody-drug conjugates (ADCs). To understand the root cause for variable trisulfide levels observed during process development for different molecule projects, cell culture studies aimed at identifying process levers to minimize trisulfide formation were conducted. Several cell culture factors, including different media components, were identified and tested to lower product trisulfides and minimize the variability at time of harvest. An assessment of concerns about product trisulfide levels will be presented. Finally, identified levers and potential mechanism(s) involved in product trisulfide formation during cell culture will be discussed in this presentation

Development towards a high-titer fed-batch CHO platform process yielding product titers \u3e 10 g/L

Roche’s current Global Cell Culture Platform (GCCP) using chemically-defined media was implemented in 2012 and has been successfully used in process development and clinical manufacturing for numerous molecules. Several minor version changes have been implemented since its inception mainly to further optimize product quality requirements. However, high lactate levels have been observed in several projects using our CHO-K1-M GS host cell line (Random Integration host), resulting in sub-optimal culture performance when not addressed by process modifications (e.g., off-platform pH changes, etc.). Understanding the “triggers” for undesirable lactate metabolism and identifying levers to control lactate metabolism are keys in improving process robustness and enabling further advances in platform process optimization towards higher titers required for high-demand products. Using a lactogenic model cell line, we examined numerous potential lactate levers including starting osmolality of production media and other factors that can mitigate the buildup of in-process osmolality (e.g., media components, media powder concentration, feed strategies, and process parameters). The results from these studies were then used to further optimize our existing platform media and process to develop a high titer proof-of-concept fed-batch process yielding \u3e 10 g/L. We also investigated the optimization of media solubility and stability of our proprietary liquid media, thus enabling the development of new highly concentrated liquid media which are required for high titer processes. Case studies that demonstrate the applicability of the newly developed high titer process with numerous mAb producing cell lines including our new Targeted Integration host will be discussed. The optimized “traditional” fed-batch process may ultimately lead to our next generation platform process, which still fits within our current manufacturing network, but will significantly reduce cost of goods and runs required to support clinical and commercial production of our biopharmaceutical proteins

Implementation activities for a chemically-defined media platform to minimize media variability impact to cell culture performance and product quality

Chemically-defined media have been developed for CHO cell culture platform processes. During implementation of the media at multiple scales and sites globally, additional activities were required to seamlessly balance practical considerations with desired process and product quality performance. Three main focus areas were further investigated and optimized to enable the platform implementation: 1) media preparation, filtration, and post-filtration stability; 2) media and supplement viral barrier compatibility; and 3) and raw material variability considerations. Overall, the effort provided an outlined approach for media implementation in the global network including scale and site-specific case studies where novel options were required. The data generated supported successful implementation of chemically-defined platform media at different global sites within our network. Manufacturing-scale media preparations met acceptance criteria and resulted in comparable cell culture performance and product quality to controls. Practical challenges were identified and risk mitigation efforts initiated. The challenges were primarily related to poorly-defined media preparation mixing environments as a result of variable equipment availability and use. Filtration capacity acceptance criteria were met in the scale-down model and large-scale filtration events were successful. HTST-compatibility studies showed that HTST-treated media performed comparably to control media in cell culture performance and product quality. Post-filtration media stability investigations led to the identification of key risk factors including temperature and agitation variation during storage, nucleation and media destabilization as a result of specific handling practices, and the impact of specific process-contacting materials. Options for mitigating the media stability risks and enabling successful implementation were developed and showed no negative impact for the application. Finally, platform media modifications were developed to mitigate raw material variation and product quality challenges. The overall evolution of the media platform to ensure flexibility and successful implementation will be summarized

Finiteness and children with specific language impairment: an exploratory study

Children with specific language impairment (SLI) are well known for their difficulties in mastering the inflectional paradigms; in the case of learning German they also have problems with the appropriate verb position, in particular with the verb in second position. This paper explores the possibilities of applying a broader concept of finiteness to data from children with SLI in order to put their deficits, or rather their skills, into a wider perspective. The concept, as developed by Klein (1998, 2000), suggests that finiteness is tied to the assertion that a certain state of affairs is valid with regard to some topic time; that is, finiteness relates the propositional content to the topic component. Its realization involves the interaction of various grammatical devices and, possibly, lexical means like temporal adverbs. Furthermore, in the acquisition of finiteness it has been found that scope particles play a major role in both first- and second-language learning. The purpose of this paper is to analyze to what extent three German-learning children with SLI have mastered these grammatical and lexical means and to pinpoint the phase in the development of finiteness they have reached. The data to be examined are mostly narrative and taken from conversations and experiments. It will be shown that each child chooses a different developmental path to come to grips with the interaction of these devices

Bridging the Gap between Glycosylation and Vesicle Traffic

Glycosylation is recognized as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae. Tethering of vesicles at the Golgi apparatus prior to fusion is regulated by Rab GTPases, coiled-coil tethers termed golgins and the multisubunit tethering complex known as the conserved oligomeric Golgi (COG) complex. In this review we discuss the mechanisms involved in vesicle tethering at the Golgi apparatus and highlight the importance of tethering in the context of glycan biosynthesis and a set of diseases known as congenital disorders of glycosylation

Linguistic foundations of heritage language development from the perspective of romance languages in Germany

This paper discusses the role of different factors determining the linguistic competence of heritage speakers (HSs) based on examples from speakers who speak a Romance language (French, Italian, Portuguese, or Spanish) as heritage language (HL) and German as the environmental language. Since the relative amount of contact with the HL and the environmental language may vary during the acquisition process, the role of language dominance (in terms of relative language proficiency) is of particular interest for HL development. In addition to dominance (and related to it), cross-linguistic influence (CLI) may have an influence on the outcome of HL acquisition. Finally, quality and quantity of input also determine HL acquisition and will be discussed in connection with heritage language education.info:eu-repo/semantics/publishedVersio