Special report: Recombinant technology

Cultivating Factor VIII
from a human cell line

Mei Chuan Chiang Biopharmaceutical Production – Process Technician (Bio100 line 1 – Nuwiq®), Stockholm, Sweden

We receive batches of cultivated recombinant Factor VIII (rhFVIII) cells and our objective is to obtain the purified FVIII which has been produced inside the cells.

Purification removes unwanted proteins, cell residuals, DNA and potential viruses. This is done by several filtration and chromatography steps which give a solution of pure and concentrated FVIII protein.

Working on inventory raw material with OctaMES.

We first extract the FVIII from the cells and then remove all the cells by centrifugation and filtration. The FVIII is concentrated and purified by five different chromatography steps and two pathogen clearance steps, including virus inactivation by solvent detergent (SD) chemicals and DNA removal by nano-filtration, before formulation and polishing.

When we receive batches from cultivation, several steps have to be done by the purification group simultaneously. Luckily, our team helps one another and our great mix of competencies is used to approach all challenges. I have 30 years of experience working in pharmaceutical production but had, until 2014, only been working in “the last steps” of production. When packaging and visual inspection was moved to Germany in 2014, I joined biopharmaceutical production. I saw it as a great opportunity and challenge to move into recombinant production. It was especially interesting for me to learn about this new and innovative technology with a human cell line. While I do not have any academic background within biotechnology, luckily I work in a team which has a lot of this knowledge and experience. It feels great to work in an environment where everyone works together and contributes with their individual strengths.

There are new projects in the pipeline, and I look forward to learning new things. I enjoy being involved in an area that is always developing.


weeks to produce Nuwiq®


international units of Nuwiq® donated to 16 developing countries

In discussion with colleagues in front of the bioreactor equipment.

I arrive at work at 7am every weekday. I usually start my day by checking and calibrating all the instruments and equipment. Then I have to prepare different buffers, e.g. a buffer which we run through all the equipment to clean it, and a salt-buffer used to release cells from aggregation.

Recombinant production is a delicate and sensitive process. The cells must be carefully handled to avoid contamination and having to reject the batch. The cells are very sensitive to changes in temperature and pH and these parameters have to be monitored carefully. Recombinant production, compared with plasma fractionation, is on a much smaller scale, as we handle, at most, some hundreds of litres, whereas in fractionation they are working with several tons of plasma per day.

The process steps of purification are always the same since they are executed according to regulated and pre-defined standard operating procedures. It is rewarding when, time after time, we produce a product that fulfils all specifications and is approved.

I believe that Octapharma is a company that considers the long term aspects and puts the patients in focus. There are new projects in the pipeline, and I look forward to learning new things and I enjoy being involved in an area that is always developing. I am here to make sure patients receive quality-controlled products that really help them in everyday life.

Inserting the gene
to produce FVIII

Initial steps using recombinant DNA technology to create Nuwiq® production cells


Isolate human gene for FVIII


Insert gene into a production cell


Test and characterise the cell line


Freeze and store the permanent FVIII
production cell line

Ongoing steps to produce Nuwiq®


Thaw recombinant FVIII production cells


Introduce production cells intro a
bioreactor, where they grow and secrete
FVIII into the supporting medium


The factor VIII containing medium is
removed from the production cells.
Factor VIII is purified through
centrifugation, two filtration and five
chromatography steps


Solvent/detergent virus inactivation and
nanofiltration steps


With plasma-derived products, the desired protein, e.g. factor VIII (FVIII), is separated from other human plasma proteins by fractionation. In recombinant production, however, Octapharma produces FVIII by inserting the gene which produces FVIII into a human cell line and cultivating the cells. While the purification steps are similar, it is the cultivation steps and the technology behind the production of FVIII in a human cell line that really differentiates recombinant products from plasma-derived products. Our recombinant FVIII (rhFVIII) protein product is called Nuwiq®. From the thawing of a working cell bank to a final drug substance batch ready for filling and freeze-drying, Nuwiq® takes about eight weeks to produce.

Cell cultivation (seven weeks)

During cultivation, the recombinant FVIII cells are continuously supplied with nutrients (sugar, oxygen, salts, etc.) for growing and producing FVIII. Bioreactors (an apparatus in which a chemical process is carried out involving biochemically active substances) are used for inoculation and expansion of cells and the production of the FVIII protein. The cells are grown in progressively larger bioreactors, beginning with a 20-litre reactor. All bioreactors are operated in conjunction with systems which control the cultivation parameters, such as temperature, pH, dissolved oxygen and carbon dioxide. All steps in the cell cultivation process are performed in sterile conditions to avoid contamination.

Firstly, the cells are stored in a nitrogen freezer, in 1ml vials as a “working cell bank”. When the cells are thawed, they then have to be expanded in incremental steps. This is done in shaker flasks which are incubated in shaker incubators. The medium exchange and expansion is done manually and occurs every second and third day. It is important to keep the cells in their exponential growth phase during expansion, and the volume must be increased in proportion to the cell density. When a sufficient quantity of cells is reached, they can be transferred from the shaker flasks to the bioreactors.

The transfer from the shaker flasks to the bioreactors is done aseptically by connecting a sterile flask of concentrated cell suspension to the small inoculation 20-litre bioreactor. It takes approximately four weeks from the thawing and expansion in shaker flasks to inoculation and expansion in the 20-litre bioreactor. In this bioreactor, the cells are grown to inoculate the next bioreactor, of 100 litres capacity. The cells are cultivated in this larger bioreactor until they have reached a sufficient quantity to inoculate the 500-litre production bioreactor. This takes approximately a week.

The 500-litre bioreactor is the production bioreactor, in which the FVIII is produced. Production of one 500-litre batch takes two weeks. The cells are then harvested and transferred to continue with purification of the FVIII protein.

Purification (one week)

Purification removes unwanted proteins, cell residuals, DNA and potential viruses. This is done by several filtration and chromatography steps which give a solution of pure and concentrated FVIII protein.

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