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The aim of the study was to investigate the effect of histidine on the stability and physical properties of a fully human anti-IL8 monoclonal antibody (ABX-IL8) in aqueous and solid forms. Using a fractional factorial design, we tested many excipients, including histidine, sucrose, and other commonly used excipients, on the stability and physical properties of the antibody in both liquid and lyophilized forms. Antibody stability and physical properties were evaluated using size-exclusion high-performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a viscometer. Residual moisture content was determined by coulometric Karl Fischer titrator. Differential scanning calorimetry (DSC) was used to detect the glass transition temperatures (Tg) of the solid cakes and melting temperatures (Tm) of the antibody in liquid formulations. Fourier-transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure. Increasing the histidine concentration in the bulk solution inhibited the increases of high-molecular-weight (HMW) species and aggregates upon lyophilization and storage. In addition, histidine bulk enhanced solution stability of the antibody under freezing and thermal stress conditions, as evidenced by the lower levels of aggregates. Furthermore, histidine reduced viscosity of the antibody solution, which is desirable for the manufacture of the dosage form. However, high concentrations of histidine in liquid formulations led to coloration of the solution and high levels of aggregates on storage at elevated temperature (40 degrees C) after the formulations were exposed to stainless steel containers during bulk freezing-thawing. Histidine enhanced the stability of ABX-IL8 in both aqueous and lyophilized forms. Histidine also improved the physical properties such as reducing the solution viscosity. Liquid formulations containing high concentrations of histidine should not be stored in stainless steel tanks at elevated temperatures.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease. Thousands of recombinant antibodies enriched for specificity against defined targets are assembled in multivalent mixtures with enhanced therapeutic activity.

Edited by three pioneers in the field, each with longstanding experience in the biotech industry, and a skilled scientific writer, this is the first book to cover every step in the development and production of immunoglobulin Fc-fusion proteins as therapeutics for human disease: from choosing the right molecular design, to pre-clinical characterization of the purified product, through to batch optimization and quality control for large-scale cGMP production. The whole of the second part is devoted to case studies of Fc-fusion proteins that are now commercially successful products. In this section, the authors, several of whom were personally involved in clinical development of the products themselves, detail the product?s background and give insight into issues that were faced and how these issues were overcome during clinical development. This section also includes a chapter on promising new developments for the future. An invaluable resource for professionals already working on Fc-fusion proteins and an excellent and thorough introduction for physicians, researchers, and students entering the field.

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector $\\lambda $gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.

MOLECULAR fusions of CD4, the receptor for human immunodeficiency virus (HIV; refs 1—4), with immunoglobulin (termed CD4 immunoadhesins) possess both the gpl20-binding and HIV-blocking properties of recombinant soluble CD4, and certain properties of IgG, notably long plasma half-life and Fc receptor binding 5,6 . Here we show that a CD4 immunoadhesin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) towards HIV-infected cells, although, unlike natural anti-gpl20 antibodies, it does not allow ADCC towards uninfected CD4-expressing cells that have bound soluble gpl20 to the CD4 on their surface. In addition, CD4 immunoadhesin, like natural IgG molecules, is efficiently transferred across the placenta of a primate. These observations have implications for the therapeutic application of CD4 immunoadhesins, particularly in the area of perinatal transmission of HIV infection.

Tumor necrosis factors (TNF) α and β are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-α induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-α and TNF-β and exhibits 6- to 8-fold higher affinity for TNF-α than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-α or TNF-β on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF- α antibodies (4-fold) in inhibiting TNF-α. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-α in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.

A newly-constructed antibody-like molecule containing the gp120-binding domain of the receptor for human immunodeficiency virus blocks HIV-1 infection of T cells and monocytes. Its long plasma half-life, other antibody-like properties, and potential to block all HIV isolates, make it a good candidate for therapeutic use.