PDA Biosimilars Workshop Report (September 27-28, 2018)-Getting It Right the First Time for Biosimilar Marketing Applications.

This workshop report summarizes the presentations, the breakout session outcomes, and the speaker panel discussions from the PDA Biosimilars Workshop held September 27-28, 2018, in Washington, DC. This format was deliberately selected for the workshop with the expectation of delivering a post-workshop paper on current best practices and existing challenges for sponsors. The event, co-chaired by Dr.

Maintaining consistent quality and clinical performance of biopharmaceuticals.

Introduction: Biopharmaceuticals are large protein based drugs which are heterogeneous by nature due to post translational modifications resulting from cellular production, processing and storage. Changes in the abundance of different variants over time are inherent to biopharmaceuticals due to their sensitivity to subtle process differences and the necessity for regular manufacturing changes. Product variability must thus be carefully controlled to ensure that it does not result in changes in safety or efficacy.

Target-directed development and preclinical characterization of the proposed biosimilar rituximab GP2013.

Biosimilar development involves a target-directed iterative process to ensure a similar product to the originator. Here we report the preclinical development of the proposed biosimilar rituximab (GP2013). Post-translational modifications and bioactivities of GP2013 versus originator rituximab were engineered and monitored to ensure similar pharmacological profiles.

Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab.

Background: Regulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis.

Hydrophilic-interaction liquid chromatography (HILIC), reversed-phase chromatography (RPC) and porous graphitic carbon (PGC) chromatography are typically applied for liquid chromatographic separations of protein N-glycans. Hence the performances of these chromatography modes for the separation of fluorescently labeled standard glycan samples (monoclonal antibody, fetuin, ribonuclease-B) covering high-mannose and a broad range of complex type glycans were investigated. In RPC the retention of sialylated glycans was enhanced by adding an ion-pairing agent to the mobile phase, resulting in improved peak shapes for sialylated glycans compared to methods recently reported in literature.

Solvent effects on the retention of oligosaccharides in porous graphitic carbon liquid chromatography.

Porous graphitic carbon (PGC) is known as well suited adsorbent for liquid chromatography of carbohydrates. In this work we report on systematic investigations of solvent effects on the retention mechanism of fluorescence labeled malto-oligosaccharides on PGC. The adsorption mechanism was found to depend on the type of organic modifier used in the mobile phase.

Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides.

Retention of hydrophilic compounds on porous graphitic carbon (PGC) is afforded by polar interactions with induced dipoles within this polarizable stationary phase. These interactions depend on the redox state of PGC, which can be influenced by application of an electrical field or by chemical means. We explored the impact of oxidizing and reducing agents on the retention of fluorescence labeled neutral oligosaccharides.

HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals.

In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry.

Nix directly binds to GABARAP: a possible crosstalk between apoptosis and autophagy.

Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail.

Ligand binding mode of GABAA receptor-associated protein.

The gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA(A) receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 A resolution.

An indole-binding site is a major determinant of the ligand specificity of the GABA type A receptor-associated protein GABARAP.

The role of tryptophan as a key residue for ligand binding to the ubiquitin-like modifier GABA(A) receptor associated protein (GABARAP) was investigated. Two tryptophan-binding hydrophobic patches were identified on the conserved face of the GABARAP structure by NMR spectroscopy and molecular docking. GABARAP binding of indole and indole derivatives, including the free amino acid tryptophan was quantified.

Identification of clathrin heavy chain as a direct interaction partner for the gamma-aminobutyric acid type A receptor associated protein.

Gamma-aminobutyric acid type A receptors (GABAA receptors) are the major sites of GABA-mediated fast synaptic inhibition in the central nervous system. Variation of the cell surface receptor count is postulated to be of importance in modulating inhibitory synaptic transmission. The GABAA receptor associated protein (GABARAP) is a ubiquitin-like modifier, implicated in GABAA receptor clustering, trafficking, and turnover.

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library.

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions.

Competitive displacement of full-length HIV-1 Nef from the Hck SH3 domain by a high-affinity artificial peptide.

We studied the interaction of the artificial 12-aa proline-rich peptide PD1 with the SH3 domain of the hematopoietic cell kinase Hck and the peptide's potency in competitively displacing HIV-1 Nef from the Hck SH3 domain. PD1 was obtained from a phage display screen and exhibits exceptional affinity for the Hck SH3 domain (K(d)=0.23 microM).

Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes.

We studied the interaction of hematopoietic cell kinase SH3 domain (HckSH3) with an artificial 12-residue proline-rich peptide PD1 (HSKYPLPPLPSL) identified as high affinity ligand (K(D)=0.2 muM). PD1 shows an unusual ligand sequence for SH3 binding in type I orientation because it lacks the typical basic anchor residue at position P(-3), but instead has a tyrosine residue at this position.

Solution structure of the X4 protein coded by the SARS related coronavirus reveals an immunoglobulin like fold and suggests a binding activity to integrin I domains.

The SARS related Coronavirus genome contains a variety of novel accessory genes. One of these, called ORF7a or ORF8, code for a protein, known as 7a, U122 or X4. We set out to determine the three-dimensional structure of the soluble ectodomain of this type-I transmembrane protein by nuclear magnetic resonance spectroscopy.

Structure of human immunodeficiency virus type 1 Vpr(34-51) peptide in micelle containing aqueous solution.

Human immunodeficiency virus type 1 protein R (HIV-1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G(2) cell cycle arrest and is involved in cellular differentiation and cell death. Vpr subcellular localization is as variable as its functions. It is known, that consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells.

Solution structure of human GABA(A) receptor-associated protein GABARAP: implications for biolgoical funcrion and its regulation.

Control of neurotransmitter receptor expression and delivery to the postsynaptic membrane is of critical importance for neural signal transduction at synapses. The gamma-aminobutyric acid, type A (GABA(A)) receptor-associated protein GABARAP was reported to have an important role for movement and sorting of GABA(A) receptor molecules to the postsynaptic membrane. GABARAP not only binds to GABA(A) receptor gamma2-subunit but also to tubulin, gephyrin, and ULK1.