Analysis of regulatory guidance on antidrug antibody testing for therapeutic protein products.

Therapeutic proteins have the potential to induce unwanted immune responses. The potential impact of immunogenicity on pharmacokinetics, pharmacodynamics, safety and efficacy are well established. Here, we analyze key aspects of current US FDA and EMA guidelines on the development and validation of antidrug antibody assays. Although FDA and EMA guidance documents are in harmony on most points, EMA allows greater leeway for scientific judgement, while FDA recommends specific approaches that may not be appropriate in some situations. Many white papers suggest approaches different from the guidance documents, however, these can conflict with each other and are themselves only scientifically valid in certain situations. Here, we indicate when alternatives to guidance may be needed and what those approaches might be.

Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB).

We previously reported the development of a human monoclonal antibody (CS-D7, IgG(1)) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII(-/-) mice and FcγRIII(-/-) mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris.

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.

Impact of methionine oxidation on the binding of human IgG1 to Fc Rn and Fc gamma receptors.

Methionine oxidation commonly occurs in the Fc fragment of therapeutic monoclonal antibodies; however, its impact on antibody function has not been addressed. Using surface plasmon resonance and cell binding assays, we examined the impact of methionine oxidation on the binding of two humanized IgG1 antibodies to Fc gamma receptors (Fc gammaR) and to the neonatal Fc receptor (Fc Rn). A panel of Fc gammaRs, including Fc gammaRI, Fc gammaRIIa-131H, Fc gammaRIIa-131R, Fc gammaRIIb/c, Fc gammaRIII ALF, Fc gammaRIII ALV, and Fc gammaRIIIb was evaluated. The binding of oxidized IgG1 molecules to individual receptors remained the same with the exception of Fc gammaRIIa where a subtle decrease in binding to the 131H allele was observed. In contrast, but in agreement with recently reported structural changes associated with Met oxidation, binding to Fc Rn was significantly affected. An increase in K(D) values at pH 6.0 was observed with increasing degree of oxidation, reaching several-fold greater value in highly oxidized samples. To our knowledge this is the first report demonstrating that chemical degradations in the constant region of monoclonal antibodies can impact their function and it highlights the importance of avoiding oxidation in therapeutic antibodies.

Production of monoclonal antibodies by glycoengineered Pichia pastoris.

The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.

Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

Heterologous human immunodeficiency virus type 1 priming-boosting immunization strategies involving replication-defective adenovirus and poxvirus vaccine vectors.

We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag-expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.