Production processes

Upstream (mAb)

According to the EMA’s European Public Assessment Reports on Herceptin and Kadcyla, trastuzumab is produced by a Chinese Hamster Ovary (CHO) cell line in suspension culture at Lonza’s Vacaville, California, USA site using a serum-free medium. Trastuzumab production begins with the thawing of recombinant CHO cells which are frozen at -80˚C in liquid nitrogen as part of the manufacturer’s Working Cell Bank (WCB). The cell number is expanded using a seed train which is a series of shake flasks and seed bioreactors used for inoculum expansion. The contents of the final seed bioreactor is used to inoculate a production bioreactor of 12,000 L volume which is operated in fed-batch mode.

Downstream (mAb)

The trastuzumab antibody is harvested from the cell culture using a centrifuge for cell removal and purified using several chromatographic and filtration methods. This downstream process is designed to remove process related impurities and clear adventitious viral agents.

Firstly, Protein A affinity chromatography is used to isolate the antibody and remove any unwanted protein or endotoxin contaminants. Cation exchange chromatography is used to remove antibody fragments and impurities from the CHO cell culture. Anion exchange chromatography is used to remove DNA, and any potential endotoxin or retrovirus. Finally, hydrophobic interaction chromatography is used to  remove any remaining antibody fragments, and CHO proteins (EMA, 2024).

The downstream process in trastuzumab manufacture also includes a viral inactivation step by pH and a viral filtration step. After viral filtration, trastuzumab is formulated in a L-histidine buffer with α,α-trehalose dehydrate and polysorbate 20 as excipients (Health Canada, 2024).

Conjugation (ADC)

The product pool is then filtered into bulk bioprocess containers and stored at 2-8˚C before transport to Lonza’s Visp, Switzerland facility where Kadcyla bulk drug substance manufacture occurs. It is here where the drug conjugation reaction takes place. The trastuzumab emtansine drug substance is then frozen and transported to the filling facility.

Formulation and Fill/Finish

Final drug product manufacture takes place at DSM, Inc., Greenville, North Carolina, USA. The frozen drug substance is thawed, and certain excipients are added to the drug substance such as succinic acid, sodium hydroxide, sucrose, and polysorbate 20.

This mixture is then filtered through a 0.22 μm sterile membrane filter into a filling vessel. Before filling, the mixture is once again passed through a 0.2 μm sterile filter. The drug product is filled into depyrogenated Type I 15 mL or 20 mL glass vials. The drug product is then lyophilised and stoppered.

After lyophilisation, the vials are sealed with aluminium seals and the outside of the vials are washed with WFI (water for injection). The vials are then dried using compressed air (EMA, 2023).