Manufacturing

Introduction

hI-con1™ is a new prototype targeting immunoprotein that has been designed with the intent of destroying pathologic, tissue factor (TF) expressing cells, without adverse effects on normal cells. Such cells include choroidal neovascularization (CNV) associated with wet age-related macular degeneration (wet AMD), TF-expressing cancer cells and tumor-associated pathologic blood vessels (PBV). Iconic Therapeutics, Inc. has successfully produced, using current good manufacturing practices (cGMP) over 1000 vials of hI-con1 ready for use in clinical studies. The purpose of this section is to provide a brief overview of the cGMP manufacturing process Iconic used to produce this hI-con1 protein.

The original hI-con1 protein is a fusion of an active site mutant of FactorVIIa with the hinge and Fc portions of a human IgG1, created by Drs Zhiwei Hu and Alan Garen at the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven (Hu et al, 2001). Shortly after licensing the composition of matter patent for this protein from Yale, Iconic Therapeutics engaged Avecia Limited of Billingham, UK as a contract manufacturer to devise a suitable procedure for production of adequate amounts of protein of acceptable quality for clinical evaluation. Avecia generated a cell line that produces increased levels of hI-con1 protein and designed an approach for the purification of the hI-con1 protein produced by these cells.

The pre-master cell bank stock produced by Avecia was transferred to BioReliance for GMP production of a Master Cell Bank (MCB). The purification approach was transferred to Laureate Pharma, Inc. of Princeton, NJ to produce clinical-grade hI-con1 protein under current good manufacturing practice (“GMP”) conditions. The upstream and downstream manufacturing processes were optimized by Iconic Therapeutics consultants resulting in adequate yield and purity of the hI-con1 protein for use in its wet AMD clinical studies. In parallel, a battery of tests to characterize the physical and biological properties of the protein have been developed at appropriate contract laboratories and qualified for their use in cGMP production.

Master Cell Bank

A “Cell Bank Certificate” was prepared by BioReliance containing the following test results: viability post-bank, thaw; viability post-bank, passaged; mycoplasma, sterility, number of vials produced, cell suspension volume, viable cell density and cryopreservative. Complete testing followed and individual reports were generated for the following tests: In Vitro Adventitious Viruses; in vivo adventitious viruses; reverse transcriptase assay for detection of retroviruses; isoenzyme assay; extended S+/K- test for detection of murine retroviruses; hamster antibody production in vivo test for the presence of viral contaminants; bovine viruses; and porcine viruses. BioReliance combined these results together into one Certificate of Analysis for the MCB. Vials of the MCB were transferred to Laureate Pharma for cGMP production of hI-con1.

Upstream Production

After MCB vial thawing, the upstream production process has a total of 6 passages to scale up for the 300L bioreactor. The first two passages are cultured in shaker flasks while the last four are cultured in Wave Cellbags. A commercial serum free and protein free medium is used throughout the process. At the end of the fermentation, the cells are removed by filtration and the clarified harvest is stored at 2-8 °C prior to purification. In-process tests include: mycoplasma (in the harvest), bioburden, protein concentration and virus safety (in the clarified harvest).

Downstream Processing

hI-con1 is purified from the clarified harvest by a series of standard chromatographic steps that include affinity chromatography, size exclusion chromatography and hydrophobic interaction chromatography. Potential virus contaminants are removed by nanofiltration. After formulation, the hI-con1 solution is filtered through 0.22µm filter and dispensed into appropriate sterile containers. In-process assays are conducted and validated assays are used to release the bulk drug substance and drug product.