A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development.

Glycosylation is a key quality attribute that must be closely monitored for protein therapeutics. Established assays such as HILIC-Fld of released glycans and LC-MS of glycopeptides work well for glycoproteins with a few glycosylation sites but are less amenable for those with multiple glycosylation sites, resulting in complex datasets that are time consuming to generate and difficult to analyze. As part of efforts to improve preparedness for future pandemics, researchers are currently assessing where time can be saved in the vaccine development and production process. In this context, we evaluated if neutral and acidic monosaccharides analysis via HPAEC-PAD could be used as a rapid and robust alternative to LC-MS and HILIC-Fld for monitoring glycosylation between protein production batches. Using glycoengineered spike proteins we show that the HPAEC-PAD monosaccharide assays could quickly and reproducibly detect both major and minor glycosylation differences between batches. Moreover, the monosaccharide results aligned well with those obtained by HILIC-Fld and LC-MS.

Bivalent non-human gal-α1-3-gal glycan epitopes in the Fc region of a monoclonal antibody model can be recognized by anti-Gal-α1-3-Gal IgE antibodies.

Monoclonal antibody (mAb) production using non-human cells can introduce non-human glycan epitopes including terminal galactosyl-α1-3-galactose (α1-3-Gal) moieties. Cetuximab is a commercial mAb associated with causing anaphylaxis in some patients due to the binding of endogenous anti-α1-3-Gal IgE to the Fab (containing bi-α1-3-galactosylated glycans) but not to the Fc region (containing mono-α1-3-galactosylated glycans). Despite being low in abundance in typical commercial mAbs, the inherent sensitivity of cell culture conditions on glycosylation profiles, and the development of novel glycoengineering strategies, novel antibody-based modalities, and biosimilars by various manufacturers with varying procedures, necessitates a better understanding of the structural requirements for anti-α1-3-Gal IgE binding to the Fc region. Herein, we synthesized mAb glycoforms with varying degrees and regioisomers of α1-3-galactosylation and tested their binding to two commercial anti-α1-3-Gal human IgE antibodies derived from a human patient with allergies to red meat (comprising α1-3-Gal epitopes), as well as to the FcγRIIIA receptor. Our results demonstrate that unexpectedly, anti-α1-3-Gal human IgE antibodies can bind to Fc glycans, with bi-α1-3-galactosylation being the most important factor, highlighting that their presence in the Fc region may be considered as a potential critical quality attribute, particularly when using novel platforms in mAb-based biotherapeutics.

A glyco-engineering approach for site-specific conjugation to Fab glycans.

Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab N-linked glycans in a narrow ratio range through functionalized sialic acids. Using a bacterial sialytransferase, we incorporated N-azidoacetylneuraminic acid (Neu5NAz) into the Fab glycan of cetuximab. Since only about 20% of human IgG1 have a Fab glycan, we extended the application of this approach by using molecular modeling to introduce N-glycosylation sites in the Fab constant region of other therapeutic monoclonal antibodies. We used trastuzumab as a model for the incorporation of Neu5NAz in the novel Fab glycans that we designed. ADCs were generated by clicking the incorporated Neu5NAz with monomethyl auristatin E (MMAE) attached to a self-immolative linker terminated with dibenzocyclooctyne (DBCO). Through this process, we obtained cetuximab-MMAE and trastuzumab-MMAE with drug/antibody ratios in the range of 1.3 to 2.5. We confirmed that these ADCs still bind their targets efficiently and are as potent in cytotoxicity assays as control ADCs obtained by standard conjugation protocols. The site-directed conjugation to Fab glycans has the additional benefit of avoiding potential interference with effector functions that depend on Fc glycan structure.

[Sharing information and respecting confidentiality: Standards for improving the quality of mental health services]. / Partage d'informations et respect de la confidentialité : repères pour améliorer la qualité des services en santé mentale.

Objectives Collaboration between family caregivers and professionals plays a critical role in the recovery of the person living with a mental health disorder. However, collaborative practices between family caregivers and professionals are impeded by issues relating to confidentiality, particularly in connection with bidirectional information sharing between the parties involved. In doing so, these issues affect the quality of mental health services. Method A qualitative study was conducted with 19 family caregivers and 19 mental health professionals from 2 Quebec regions[2] in order to identify issues related to information sharing and confidentiality from their combined perspective. The Photovoice method was used and individuals semi-directed interviews were conducted with the 38 participants. Results Confidentiality and the refusal of the person living with a mental health disorder to consent to share information remains important and current obstacles in mental health practises. The organization of mental health services should ensure better integration of family caregivers into care teams so that they can contribute to the person's recovery and thus receive all the support and information they need to exercise their role. This study shows that family caregivers have different information needs in order to carry out their role and accompany the person living with a mental health disorder, including the need for general and non-confidential information to better support the person. While respecting the fundamental rights and autonomy of the person, who is free to consent or not to sharing information concerning him or her, professionals and family caregivers can still interact and create an alliance that promotes collaboration and recovery. Conclusion This article offers benchmarks to facilitate dialogue among people living with a mental health disorder, family caregivers and professionals, and to support their actions around information-sharing and respect for confidentiality in mental health practises. Ultimately, the intention here is to foster collaborative practices that will help improve the quality of mental health services.

Personalized care planning and shared decision making in collaborative care programs for depression and anxiety disorders: A systematic review.

BACKGROUND: Collaborative care is an evidence-based approach to improving outcomes for common mental disorders in primary care. Efforts are underway to broadly implement the collaborative care model, yet the extent to which this model promotes person-centered mental health care has been little studied. The aim of this study was to describe practices related to two patient and family engagement strategies-personalized care planning and shared decision making-within collaborative care programs for depression and anxiety disorders in primary care. METHODS: We conducted an update of a 2012 Cochrane review, which involved searches in Cochrane CCDAN and CINAHL databases, complemented by additional database, trial registry, and cluster searches. We included programs evaluated in a clinical trials targeting adults or youth diagnosed with depressive or anxiety disorders, as well as sibling reports related to these trials. Pairs of reviewers working independently selected the studies and data extraction for engagement strategies was guided by a codebook. We used narrative synthesis to report on findings. RESULTS: In total, 150 collaborative care programs were analyzed. The synthesis showed that personalized care planning or shared decision making were practiced in fewer than half of programs. Practices related to personalized care planning, and to a lesser extent shared decision making, involved multiple members of the collaborative care team, with care managers playing a pivotal role in supporting patient and family engagement. Opportunities for quality improvement were identified, including fostering greater patient involvement in collaborative goal setting and integrating training and decision aids to promote shared decision making. CONCLUSION: This review suggests that personalized care planning and shared decision making could be more fully integrated within collaborative care programs for depression and anxiety disorders. Their absence in some programs is a missed opportunity to spread person-centered mental health practices in primary care.

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Chemoenzymatic synthesis of an α-1,6-glucan-based conjugate vaccine against Helicobacter pylori.

In this study, we investigated the utility of glycoconjugates based on a linear α-1,6-glucan chain synthesized using a recombinant α-1,6-glucosyltransferase from the 26695 strain of Helicobacter pylori. Capillary electrophoresis-mass spectrometry analysis confirmed the main product to contain 9-10 sequentially added α-1,6-linked glucose residues. This was consistent with a length of α-1,6-glucan structure present in the outer core region of H. pylori lipopolysaccharide (LPS) from strains 26695 and 26695 HP0826::Kan. The synthetic α-1,6-glucan was conjugated to either bovine serum albumin or tetanus toxoid and immunological properties of resultant glycoconjugates investigated. The conjugates were immunogenic in rabbits and mice and induced strong and specific IgG responses against purified LPS from typeable and nontypeable α-1,6-glucan-positive H. pylori strains. Furthermore, the post-immune sera from rabbits that received the conjugates were bactericidal and cross-reacted with selected clarithromycin-resistant and clarithromycin-susceptible clinical isolates of H. pylori. This technology offers a novel approach to the design of a synthetic carbohydrate-based vaccine against H. pylori.

In vitro Production and Immunogenicity of a Clostridium Difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine.

Abstract: The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of ß-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease.

Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors.

The effector functions of the IgGs are modulated by the N-glycosylation of their Fc region. Particularly, the absence of core fucosylation is known to increase the affinity of IgG1s for the Fcγ receptor IIIa expressed by immune cells, in turn translating in an improvement in the antibody-dependent cellular cytotoxicity. However, the impact of galactosylation and sialylation is still debated in the literature. In this study, we have investigated the influence of high and low levels of core fucosylation, terminal galactosylation and terminal α2,6-sialylation of the Fc N-glycans of trastuzumab on its affinity for the FcγRIIIa. A large panel of antibody glycoforms (i.e., highly α2,6-sialylated or galactosylated IgG1s, with high or low levels of core fucosylation) were generated and characterized, while their interactions with the FcγRs were analysed by a robust surface plasmon resonance-based assay as well as in a cell-based reporter bioassay. Overall, IgG1 glycoforms with reduced fucosylation display a stronger affinity for the FcγRIIIa. In addition, fucosylation, and the presence of terminal galactose and sialic acids are shown to increase the affinity for the FcγRIIIa as compared to the agalactosylated forms. These observations perfectly translate in the response observed in our reporter bioassay.

Strategies for engaging patients and families in collaborative care programs for depression and anxiety disorders: A systematic review.

BACKGROUND: Patients and families are often referred to as important partners in collaborative mental health care (CMHC). However, how to meaningfully engage them as partners remains unclear. We aimed to identify strategies for engaging patients and families in CMHC programs for depression and anxiety disorders. METHODS: We updated a Cochrane review of CMHC programs for depression and anxiety disorders. Searches were conducted in Cochrane CCDAN and CINAHL, complemented by additional database searches, trial registry searches, and cluster searches for 'sibling' articles. Coding and data extraction of engagement strategies was an iterative process guided by a conceptual framework. We used narrative synthesis and descriptive statistics to report on findings. FINDINGS: We found 148 unique CMCH programs, described in 578 articles. Most programs (96%) featured at least one strategy for engaging patients or families. Programs adopted 15 different strategies overall, with a median of two strategies per program (range 0-9 strategies). The most common strategies were patient education (87% of programs) and self-management supports (47% of programs). Personalized care planning, shared decision making, and family or peer supports were identified in fewer than one third of programs. LIMITATIONS: Our search strategy was designed to capture programs evaluated in clinical trials and so other innovative programs not studied in trials were likely missed. CONCLUSION: Most CMHC programs for depression and anxiety disorders adopted a limited number of strategies to engage patients and families in their care. However, this review identifies numerous strategies that can be used to strengthen the patient- and family-centeredness of collaborative care.

Production of α2,6-sialylated and non-fucosylated recombinant alpha-1-antitrypsin in CHO cells.

Alpha-1-antitrypsin (A1AT) is an abundant serum inhibitor of serine proteases. A1AT deficiency is a common genetic disorder which is currently treated with augmentation therapies. These treatments involve weekly injections of patients with purified plasma-derived A1AT. Such therapies can be extremely expensive and rely on plasma donors. Hence, large-scale production of recombinant A1AT (rA1AT) could greatly benefit these patients, as it could decrease the cost of treatments, reduce biosafety concerns and ensure quantitative and qualitative controls of the protein. In this report, we sought to produce α2,6-sialylated rA1AT with our cumate-inducible stable CHO pool expression system. Our different CHO pools could reach volumetric productivities of 1,2 g/L. The human α2,6-sialyltransferase was stably expressed in these cells in order to mimic elevated α2,6-sialylation levels of native A1AT protein. Sialylation of the recombinant protein was stable over the duration of the fed-batch production phase and was higher in a pool where cells were sorted and enriched by FACS based on cell-surface α2,6-sialylation. Addition of ManNAc to the cell culture media during production enhanced both α2,3 and α2,6 A1AT sialylation levels whereas addition of 2F-peracetylfucose potently inhibited fucosylation of the protein. Finally, we demonstrated that rA1AT proteins exhibited human neutrophil elastase inhibitory activities similar to the commercial human plasma-derived A1AT.

Role of the non-hypervariable FR3 D-E loop in single-domain antibody recognition of haptens and carbohydrates.

Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between ß-strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.

Neurostatin and other O-acetylated gangliosides show anti-neuroinflammatory activity involving the NFκB pathway.

In many neuropathologies activated microglia and macrophages cause neurotoxicity and prolong the inflammatory response. We have previously characterized the glycosphingolipid Neurostatin (Nst), which potentially reduces these detrimental mechanisms. Nst, isolated from mammalian brain, is the GD1b ganglioside with O-acetylation of the outer sialic acid residue. Using the enzyme sialate-O-acetyltransferase (SOAT), we obtained several O-acetylated gangliosides and O-propionylated GD1b (PrGD1b). In the present study we investigated the anti-inflammatory effects of these compounds. Nst and other O-acetylated gangliosides reduced nitrite production in microglial cells which were activated with lipopolysaccharide (LPS), but did not affect nitrite production after their stimulation with interferon gamma (IFNγ). Structure-activity relationship analysis showed that Nst was the most active ganglioside as inhibitor of nitrite production. Its ceramide moiety is essential for this, and both, the O-acetylation and the monosaccharide chain are important for the anti-inflammatory activity of the gangliosides. We also found that Nst reduced iNOS, IL-6 and IL-12 transcription in LPS-induced microglia, likely by inhibiting nuclear localization of NFκB. In co-cultures, Nst reduced neuronal cell death caused by LPS-activated microglia. In vivo, Nst diminished microglia activation in a mouse model of acute neuroinflammation. We propose that Nst and other O-acetylated gangliosides are neuroprotective regulators of microglia activity under both physiological and pathological conditions.

Selective Capture and Determination of Receptor-Binding Hemagglutinin in Influenza Vaccine Preparations Using a Coupled Receptor-Binding/RP-HPLC Assay.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy.

Immune targeting of (glyco)protein tumor markers has been useful to develop cancer and virus vaccines. However, the ganglioside family of tumor-associated glycolipids remains intractable to vaccine approaches. Here we show that synthetic antigens mimicking the carbohydrate moiety of GD2 or GD3 gangliosides can be used as vaccines to activate a selective humoral and cellular immunity that is therapeutic against several cancers expressing GD2 or GD3. Adoptive transfer of T cells generated after vaccination elicits tumor-infiltrating lymphocytes of the γδ T cell receptor and CD8+ phenotypes; and affords a high therapeutic index. The glycomimetic vaccine principles can be expanded to target the family of tumor gangliosides and other carbohydrates expressed primarily in pathological states.

Assessment of fed-batch cultivation strategies for an inducible CHO cell line.

In order to maximize cell growth and productivity for an inducible CHO cell line expressing rituximab, various fed-batch culture strategies were investigated. In each case, the performance was evaluated for cultures induced at moderate and high cell density conditions (4 × 106 and 10 × 106 cells/mL) to assess the impact of the timing of induction. We first demonstrate the importance of starting the feeding process during the growth phase, as this translated into significantly improved integral of viable cells and antibody concentration, when compared to post-induction feeding only. Secondly, we investigated the impact of the feed rate by maintaining different levels of glucose (25, 35 and 50 mM) via a dynamic feeding strategy. The highest antibody concentrations were achieved under a moderate feeding regime for both cell densities at induction, highlighting the risks of under- or over-feeding the cultures. We then evaluated the impact of performing a temperature shift at induction by testing different mild hypothermia conditions. At small-scale, the highest production yields (1.2 g/L) were achieved when the temperature was reduced from 37 to 30 °C during the production phase of a culture induced at high cell density. When the strategy was applied in bioreactor, the better controlled conditions led to even greater product concentrations (1.8 g/L). Furthermore, this production protocol was shown to promote a more galactosylated glycan profile than a bioreactor culture initiated at 34 °C during growth and downshifted to 30 °C during the production phase.

Genetics behind the Biosynthesis of Nonulosonic Acid-Containing Lipooligosaccharides in Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are the most common causes of bacterial gastroenteritis in the world. Ganglioside mimicry by C. jejuni lipooligosaccharide (LOS) is the triggering factor of Guillain-Barré syndrome (GBS), an acute polyneuropathy. Sialyltransferases from glycosyltransferase family 42 (GT-42) are essential for the expression of ganglioside mimics in C. jejuni Recently, two novel GT-42 genes, cstIV and cstV, have been identified in C. coli Despite being present in ∼11% of currently available C. coli genomes, the biological role of cstIV and cstV is unknown. In the present investigation, mutation studies with two strains expressing either cstIV or cstV were performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules using in vitro activity tests with recombinant GT-42 enzymes. Here we show that CstIV and CstV are involved in C. coli LOS biosynthesis. In particular, cstV is associated with LOS sialylation, while cstIV is linked to the addition of a diacetylated nonulosonic acid residue.IMPORTANCE Despite the fact that Campylobacter coli a major foodborne pathogen, its glycobiology has been largely neglected. The genetic makeup of the C. coli lipooligosaccharide biosynthesis locus was largely unknown until recently. C. coli harbors a large set of genes associated with lipooligosaccharide biosynthesis, including genes for several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide in Campylobacter jejuni In the present study, C. coli was found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact on our understanding of C. coli ecology, host-pathogen interaction, and pathogenesis.

Process intensification for the production of rituximab by an inducible CHO cell line.

Mammalian-inducible expression systems are increasingly available and offer an attractive platform for the production of recombinant proteins. In this work, we have conducted process development for a cumate-inducible GS-CHO cell-line-expressing rituximab. To cope with the limitations encountered in batch when inducing at high cell densities, we have explored the use of fed-batch, sequential medium replacements, and continuous perfusion strategies applied during the pre-induction (growth) phase to enhance process performance in terms of product yield and quality. In shake flask, a fed-batch mode and a complete medium exchange at the time of induction were shown to significantly increase the integral of viable cell concentration and antibody titer compared to batch culture. Further enhancement of product yield was achieved by combining bolus concentrated feed additions with sequential medium replacement, but product galactosylation was reduced compared to fed-batch mode, as a result of the extended culture duration. In bioreactor, combining continuous perfusion of the basal medium with bolus daily feeding during the pre-induction period and harvesting earlier during the production phase is shown to provide a good trade-off between antibody titer and product galactosylation. Overall, our results demonstrate the importance of selecting a suitable operating mode and harvest time when carrying out high-cell-density induction to balance between culture productivity and product quality.