Status: an intermediate only; not marketed; FDA approval only

Organizations involved:

Charles River Div. – Manuf.; Tech.

Immunomedics, Inc. – R&D; Tech.

Cross ref: See the entry for Arcitumomab (CEA-Scan) from Immunomedics, Inc., which is manufactured from this bulk monoclonal antibody concentrate (intermediate). See the entry for Monoclonal Antibodies (#300).

Description: IMMU-4 Bulk (For Further Manufacturing Use) is bulk IMMU-4 (originally called NP-4) murine monoclonal antibody with specificity for carcinoembryonic antigen (CEA) produced by the classic murine ascites method (cultured in the peritoneal cavity of mice). IMMU-4 is an IgG1 antibody with a lambda light chain derived from the murine IMMU-4 hybridoma cell line isolated from the fusion of murine myeloma cells with spleen cells from mice immunized with CEA.

CEA is a heavily glycosylated membrane-bound glycoprotein secreted by virtually all solid tumors. CEA exhibits marked heterogeneity due to quantitative and qualitative differences in its carbohydrate composition. The IMMU-4 antibody was selected for its binding to CEA, coupled with its lack of binding to meconium antigen and non-specific cross reacting antigen (NCA).

IMMU-4 is used by Immunomedics, Inc. for manu-facture of arcitumomab, a Fab fragment enzymatically derived from IMMU-4, which is chemically conjugated to a ligand complex containing Technetium-99m radioactive isotope, and administered to patients for radiodiagnostic imaging of CEA concentrations (indicating tumor tissue). The dating period for Immu-4 Bulk (For Further Manufacturing Use) is 12 months at -80˚C from the date of removal from mice, and 48 hours from the time of manufacture (initial thawing of frozen bulk) at 2-8 ˚C (refrigerated).

Nomenclature: CEA Mab, conc. [BIO]; Immu-4 Bulk (For Further Manufacturing Use) [FDA]; IMMU-4 Murine Ascitic Fluid (For Further Manufacturing Use) [FDA used at time of approval]; CEA monoclonal antibody [SY]; anti-carcino-embryonic antigen monoclonal antibody [SY]; carcinoembryonic antigen monoclonal antibody [SY]

Companies.: Immu-4 Bulk (For Further Manufacturing Use) is manufactured by Charles River Division, Wilmington Partners, L.P. (Wilmington, MA), CBER/FDA est. no. 1206, exclusively for further manufacturing of Arcitumomab (CEA-Scan) by Immunomedics, Inc.

Manufacture: The IMMU-4 hybridoma cell line was first isolated in 1981 from the fusion of myeloma cells with spleen cells (B-cells) from mice immunized with CEA. The IMMU-4 hybridoma cell line secreting a murine IgG1 CEA-specific monoclonal antibody was selected, cloned by serial dilution, and used to prepare the Master Cell Bank (MCB) and the manufacturer’s Working Cell Bank (MWCB). Frozen vials of the cell banks are stored by Charles River and Immunomedics. The MCB and MWCB (at time of approval) were shown to be free of adventitious agents including bacterial, mycoplasma, fungi, and exogenous viruses. No infectious murine viruses were detected. IMMU-4 Ascitic Fluid has been shown to contain murine retroviral particles (< 3.94 x 105/mL). Removal of 12.65-14.38 logs of Muloney leukemia virus (MuLV) by subsequent purification processes (by Immunomedics) has been demonstrated. These processes have also been shown to remove or inactivate 12.549-13.19 logs of pseudorabies virus and 6.5-8.6 logs of SV-40.

A vial of frozen IMMU-4 hybridoma cells is defrosted, passaged in DMEM +10 fetal bovine serum (FBS) for about 14 days in T-flasks and roller bottles. The expanded cells (5 x 106 to 1 x 107 IMMU-4 hybridoma cells) in 0.5 ml of saline are injected into the peritoneal sacs of CDF1 or BALB/c mice. The mice had previously been primed with pristane. When mice have produced sufficient ascitic fluid (about 15 days after inoculation), they are euthanized with carbon dioxide, the peritoneal cavity is aseptically exposed, and ascitic fluid is collected by single aspiration from each mouse. Generally, about 2,000 mice are injected to produce one lot of bulk product. The ascitic fluid harvests from each day’s collection are centrifuged, filtered through gauze, the pristane layer is aspirated, and the resulting bulk IMMU-4 is pooled and frozen at -80˚C. When fluid has been obtained from all mice, the frozen bottles from prior harvests are thawed, pooled, and dispensed into polypropylene bottles. Retention samples are removed and the ascitic fluid bottles are frozen. When Immunomedics is ready for another batch of bulk IMMU-4, the bottles are thawed, contents centrifuged, poured through sterilized glass wool, filled into polypropylene containers, and shipped overnight to Immunomedics, where purification begins within 48 hours.

The CDF1 and BALB/c breeds of mice used by Charles River Labs. are designated Viral Antigen Free. They are bred and housed in a barrier room maintained under positive pressure with the air supply HEPA filtered. Temperature, humidity, and entry are controlled. Cage bedding is autoclaved and food and water are ultrafiltered, ultraviolet light (UV) sanitized and 0.2 µm filtered. The rooms are cleaned and maintained. Serological testing and health monitoring are performed regularly. Sentinel mice are screened at 2 or 4 weeks or quarterly for rodent viruses and mycoplasma. Quality control is performed on raw materials and ingredients used in the preparation of the cell inoculum and in the harvesting and processing of ascitic fluid. The ascitic fluid is tested for bioburden (CFU/mL), IgG1 content (> 2.0 mg/mL), identity by IEF, MAP and mycoplasma.

FDA class: Biologic PLA

CBER class: Blood And Blood Derivatives

Approvals: Date = 19960628, first approval, PLA/ELA ref. no. 93-0678 and 93-0693

Indication: = approved exclusively for further manufacture of Arcitumomab (CEA-Scan) by Immunomedics