A Bayesian Statistical Approach to Continuous Qualification of a Bioassay.

A validated bioassay is used to measure the potency of commercial lots, and as such, must be accurate, precise, and fit for its intended purpose. Regulatory expectations for a bioassay include a characterization of features, such as accuracy, precision, linearity, and range. The journey of a bioassay typically starts in a development lab, where it is initially qualified and used to support the release and stability testing of clinical lots.

Glycosylation of IgG and IgG-Like Recombinant Therapeutic Proteins: Why Is It Important and How Can We Control It?

Regulatory bodies worldwide consider glycosylation to be a critical quality attribute for immunoglobulin G (IgG) and IgG-like therapeutics. This consideration is due to the importance of posttranslational modifications in determining the efficacy, safety, and pharmacokinetic properties of biologics. Given its critical role in protein therapeutic production, we review glycosylation beginning with an overview of the myriad interactions of glycans with other biological factors.

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation.

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody.

Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies.

The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody.

Fc glycans of therapeutic antibodies as critical quality attributes.

Critical quality attributes (CQA) are physical, chemical, biological or microbiological properties or characteristics that must be within an appropriate limit, range or distribution to ensure the desired product quality, safety and efficacy. For monoclonal antibody therapeutics that rely on fraction crystalizable (Fc)-mediated effector function for their clinical activity, the terminal sugars of Fc glycans have been shown to be critical for safety or efficacy. Different glycosylation variants have also been shown to influence the pharmacodynamic and pharmacokinetic behavior while other Fc glycan structural elements may be involved in adverse immune reactions.

Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a relevant characteristic to measure for a number of therapeutic monoclonal antibodies (mAbs) under development. ADCC is a mechanism by which antibody-opsonized, infected, or cancerous cells are destroyed by FcγRIII (CD16)-expressing effector cells. Here we describe three methods that can be used to quantify the ADCC activity of mAbs by measuring distinct aspects of the ADCC mechanism.

Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity.

Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies. The most advanced uses of this approach, however, have been limited to E.

Development of an ELISA based bridging assay as a surrogate measure of ADCC.

Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) for many monoclonal antibody (mAb) therapeutics. As such, quantitative measurement of ADCC activity is key to drug development. Traditional cell lysis based ADCC assays using PBMCs or NK cell lines can be challenging to develop and implement for routine testing.

Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species.

Tumor-driven paracrine platelet-derived growth factor receptor alpha signaling is a key determinant of stromal cell recruitment in a model of human lung carcinoma.

Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRalpha ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass.

Bv8 and endocrine gland-derived vascular endothelial growth factor stimulate hematopoiesis and hematopoietic cell mobilization.

Bv8 and endocrine-gland-derived VEGF (EG-VEGF), or prokineticins, are two highly related, secreted proteins that we previously described as selective angiogenic mitogens. Here we describe the expression and functional characterization of Bv8 in peripheral blood cells, notably monocytes, neutrophils, and dendritic cells, and in the bone marrow. In human and mouse, the two Bv8 G protein-coupled receptors are expressed in hematopoietic stem cells and specific mature blood cells, including lymphocytes.

VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis.

We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth.

The endocrine-gland-derived VEGF homologue Bv8 promotes angiogenesis in the testis: Localization of Bv8 receptors to endothelial cells.

We recently identified an angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), with selective activity for endothelial cells of endocrine tissues. Here we describe the characterization of a highly related molecule, Bv8, also known as prokineticin-2. Human Bv8 shares 60% identity and 75% similarity with EG-VEGF.

The fission yeast ES2 homologue, Bis1, interacts with the Ish1 stress-responsive nuclear envelope protein.

In fission yeast, nutrient starvation induces physiological, biochemical, and morphological changes that enable survival. Collectively these changes are referred to as stationary phase. We have used a green fluorescent protein random insertional mutagenesis system to isolate two novel stress-response proteins required in stationary phase.