Amino acid degradation pathway inhibitory by-products trigger apoptosis in CHO cells.

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. Improving their productivity is necessary to fulfill the growing demand for such products. One way to enhance productivity is by cultivating cells at high densities, but inhibitory by-products, such as metabolite derivatives from amino acid degradation, can hinder achieving high cell densities. This research examines the impact of these inhibitory by-products on high-density cultures. We cultured X1 and X2 CHO cell lines in a small-scale semi-perfusion system and introduced a mix of inhibitory by-products on day 10. The X1 and X2 cell lines were chosen for their varied responses to the by-products; X2 was susceptible, while X1 survived. Proteomics revealed that the X2 cell line presented changes in the proteins linked to apoptosis regulation, cell building block synthesis, cell growth, DNA repair, and energy metabolism. We later used the AB cell line, an apoptosis-resistant cell line, to validate the results. AB behaved similar to X1 under stress. We confirmed the activation of apoptosis in X2 using a caspase assay. This research provides insights into the mechanisms of cell death triggered by inhibitory by-products and can guide the optimization of CHO cell culture for biopharmaceutical manufacturing.

Long-term safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2 in adults aged 18-55 years or ≥56 years: 12-month results from a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years. METHODS: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 µg, 15 µg, or 45 µg vaccine, or one 45 µg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. FINDINGS: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. INTERPRETATION: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. FUNDING: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Strategies for developing a recombinant butyrylcholinesterase medical countermeasure for Organophosphorus poisoning.

Organophosphorus nerve agents represent a serious chemical threat due to their ease of production and scale of impact. The recent use of the nerve agent Novichok has re-emphasised the need for broad-spectrum medical countermeasures (MCMs) to these agents. However, current MCMs are limited. Plasma derived human butyrylcholinesterase (huBChE) is a promising novel bioscavenger MCM strategy, but is prohibitively expensive to isolate from human plasma at scale. Efforts to produce recombinant huBChE (rBChE) in various protein expression platforms have failed to achieve key critical attributes of huBChE such as circulatory half-life. These proteins often lack critical features such as tetrameric structure and requisite post-translational modifications. This review evaluates previous attempts to generate rBChE and assesses recent advances in mammalian cell expression and protein engineering strategies that could be deployed to achieve the required half-life and yield for a viable rBChE MCM. This includes the addition of a proline-rich attachment domain, fusion proteins, post translational modifications, expression system selection and optimised downstream processes. Whilst challenges remain, a combinatorial application of these strategies demonstrates potential as a technically feasible approach to achieving a bioactive and cost effective bioscavenger MCM.

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 µg dose (7492, 4959-11 319), and the two 45 µg dose cohorts (8770, 5526-13 920 in the two-dose 45 µg cohort; 8793, 5570-13 881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102 400, 64 857-161 676), with two 15 µg doses (74 725, 51 300-108 847), with two 45 µg doses (79 586, 55 430-114 268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.

Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Working Hard or Hardly Working? Regulatory Bottlenecks in Developing a COVID-19 Vaccine.

Vaccine solutions rarely reach the public until after an outbreak abates; an Ebola vaccine was approved 5 years after peak outbreak and SARS, MERS, and Zika vaccines are still in clinical development. Despite massive leaps forward in rapid science, other regulatory bottlenecks are hamstringing the global effort for pandemic vaccines.

RNA-Seq Highlights High Clonal Variation in Monoclonal Antibody Producing CHO Cells.

The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster drugs produced in CHO. However, engineering superior CHO cells with improved production features has had limited success to date and cell lines are still developed through the generation and screening of large strain pools. Here, we applied RNA sequencing to contrast a high and a low monoclonal antibody producing cell line. Rigorous experimental design achieved high reproducibility between biological replicates, remarkably reducing variation to less than 10%. More than 14 000 gene-transcripts are identified and surprisingly 58% are classified as differentially expressed, including 2900 with a fold change higher than 1.5. The largest differences are found for gene-transcripts belonging to regulation of apoptosis, cell death, and protein intracellular transport GO term classifications, which are found to be significantly enriched in the high producing cell line. RNA sequencing is also performed on subclones, where down-regulation of genes encoding secreted glycoproteins is found to be the most significant change. The large number of significant differences even between subclones challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines.

Accelerating patient access to novel biologics using stable pool-derived product for non-clinical studies and single clone-derived product for clinical studies.

Cell cloning and subsequent process development activities are on the critical path directly impacting the timeline for advancement of next generation therapies to patients with unmet medical needs. The use of stable cell pools for early stage material generation and process development activities is an enabling technology to reduce timelines. To successfully use stable pools during development, it is important that bioprocess performance and requisite product quality attributes be comparable to those observed from clonally derived cell lines. To better understand the relationship between pool and clone derived cell lines, we compared data across recent first in human (FIH) programs at Amgen including both mAb and Fc-fusion modalities. We compared expression and phenotypic stability, bioprocess performance, and product quality attributes between material derived from stable pools and clonally derived cells. Overall, our results indicated the feasibility of matching bioprocess performance and product quality attributes between stable pools and subsequently derived clones. These findings support the use of stable pools to accelerate the advancement of novel biologics to the clinic. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1476-1482, 2017.

Nanocell targeting using engineered bispecific antibodies.

There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDV(TM)nanocell) to the epidermal growth factor receptor (EGFR). EDV(TM)nanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV(TM)nanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDV(TM)nanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDV(TM)nanocells. BsAbs therefore provide a functional means to deliver EDV(TM)nanocells to target cells.

Intracellular trafficking pathways for nuclear delivery of plasmid DNA complexed with highly efficient endosome escape polymers.

Understanding the pathways for nuclear entry could see vast improvements in polymer design for the delivery of genetic materials to cells. Here, we use a novel diblock copolymer complexed with plasmid DNA (pDNA) to determine both its cellular entry and nuclear pathways. The diblock copolymer (A-C3) is specifically designed to bind and protect pDNA, release it at a specific time, but more importantly, rapidly escape the endosome. The copolymer was taken up by HEK293 cells preferentially via the clathrin-mediated endocytosis (CME) pathway, and the pDNA entered the nucleus to produce high gene expression levels in all cells after 48 h, a similar observation to the commercially available polymer transfection agent, PEI Max. This demonstrates that the polymers must first escape the endosome and then mediate transport of pDNA to the nucleus for occurrence of gene expression. The amount of pDNA within the nucleus was found to be higher for our A-C3 polymer than PEI Max, with our polymer delivering 7 times more pDNA than PEI Max after 24 h. We further found that entry into the nucleus was primarily through the small nuclear pores and did not occur during mitosis when the nuclear envelope becomes compromised. The observation that the polymers are also found in the nucleus supports the hypothesis that the large pDNA/polymer complex (size ~200 nm) must dissociate prior to nucleus entry and that cationic and hydrophobic monomer units on the polymer may facilitate active transport of the pDNA through the nuclear pore.

Thermoresponsive worms for expansion and release of human embryonic stem cells.

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC.

High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system.

Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

Bridging the gap: facilities and technologies for development of early stage therapeutic mAb candidates.

Therapeutic monoclonal antibodies (mAbs) currently dominate the biologics marketplace. Development of a new therapeutic mAb candidate is a complex, multistep process and early stages of development typically begin in an academic research environment. Recently, a number of facilities and initiatives have been launched to aid researchers along this difficult path and facilitate progression of the next mAb blockbuster. Complementing this, there has been a renewed interest from the pharmaceutical industry to reconnect with academia in order to boost dwindling pipelines and encourage innovation. In this review, we examine the steps required to take a therapeutic mAb from discovery through early stage preclinical development and toward becoming a feasible clinical candidate. Discussion of the technologies used for mAb discovery, production in mammalian cells and innovations in single-use bioprocessing is included. We also examine regulatory requirements for product quality and characterization that should be considered at the earliest stages of mAb development. We provide details on the facilities available to help researchers and small-biotech build value into early stage product development, and include examples from within our own facility of how technologies are utilized and an analysis of our client base.

Stem cell integrins: implications for ex-vivo culture and cellular therapies.

Use of stem cells, whether adult or embryonic for clinical applications to treat diseases such as Parkinson's, macular degeneration or Type I diabetes will require a homogenous population of mature, terminally differentiated cells. A current area of intense interest is the development of defined surfaces for stem cell derivation, maintenance, proliferation and subsequent differentiation, which are capable of replicating the complex cellular environment existing in vivo. During development many cellular cues result from integrin signalling induced by the local extracellular matrix. There are 24 known integrin heterodimers comprised of one of 18 α subunits and one of 8 ß subunits and these have a diverse range of functions mediating cell-cell adhesion, growth factor receptor responses and intracellular signalling cascades for cell migration, differentiation, survival and proliferation. We discuss here a brief summary of defined conditions for human embryonic stem cell culture together with a description of integrin function and signalling pathways. The importance of integrin expression during development is highlighted as critical for lineage specific cell function and how consideration of the integrin expression profile should be made while differentiating stem cells for use in therapy. In addition this review summarises the known integrin expression profiles for human embryonic stem cells and 3 common adult stem cell types: mesenchymal, haematopoietic and neural. We then outline some of the possible technologies available for investigating cell-extracellular matrix interactions and subsequent integrin mediated cell responses.

Enhanced CHO cell-based transient gene expression with the epi-CHO expression system.

Transient gene expression systems in mammalian cells continue to grow in popularity due to their capacity to produce significant amounts of recombinant protein in a rapid and scalable manner, without the lengthy time periods and resources required for stable cell line development. Traditionally, production of recombinant monoclonal antibodies for pre-clinical assessment by transient expression in CHO cells has been hampered by low titers. In this report, we demonstrate transient monoclonal antibody titers of 140 mg/l with CHO cells using the episomal-based transient expression system, Epi-CHO. Such titers were achieved by implementing an optimized transfection protocol incorporating mild-hypothermia and through screening of a variety of chemically defined and serum-free media for their ability to support elevated and prolonged viable cell densities post-transfection, and in turn, improve recombinant protein yields. Further evidence supporting Epi-CHO's capacity to enhance transgene expression is provided, where we demonstrate higher transgene mRNA and protein levels of two monoclonal antibodies and a destabilized enhanced green fluorescent protein with Epi-CHO compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection. The results demonstrate the Epi-CHO system's capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions.