Principles of Upstream Processing

Introduction

The manufacture of human proteins by the methods of modern biotechnology is separated into two stages: upstream processing during which proteins are produced by cells genetically engineered to contain the human gene which will express the protein of interest and downstream processing during which the produced proteins are isolated and purified. Following purification of the protein of interest, the final product is formulated (meaning excipients are added to the protein), filter sterilized, filled aseptically, lyophilized, sealed, inspected and labeled. Upstream processing, downstream processing, final drug production, and the general environment of the facility are monitored by the quality control division of the manufacturing facility. A master production batch record (MPBR) governs the behaviors of the people entrusted to carry out these various processes (upstream and downstream processing and quality control) according to validated protocols and standard operating procedures (SOPs).

Bioreactors and Cells

Today the most common type of upstream processing of proteins utilizes two tools: bioreactors {stainless steel are most common, but glass bioreactors (such as spinner flasks) and plastic bioreactors (such as hollow fiber bioreactors) are used as well} and suspension (or attached) cells transformed with expression vectors genetically engineered to contain one (or more) human genes that produce copious amounts of their protein(s). Upstream processing of proteins using bioreactors and cells usually begins with the preparation of the inoculum which proceeds in scale-up steps until enough inoculum is made to aseptically inoculate the final, sterile, media-filled bioreactor. Batch, fed-batch and continuous culture refer to whether or not the cells are fed after they are inoculated. In batch culture there is no additional feeding, while in fed-batch culture less media is used at the beginning of a run and feeding is done at intervals to a maximum volume during the run. In continuous culture feed is constantly added and media is constantly withdrawn. In fed-batch and especially continuous culture the cells can be maintained in a steady state, continuously producing human protein for up to several months at a time.

During the culture period samples are removed, aseptically, and various parameters are measured by fermentation technicians or operators including optical density (OD) and live cell count. Samples are also brought to quality control where other parameters may be measured such as the levels of glucose, lactate and ammonia, as well as the identity and concentration of the human protein that the cells are producing. Quality control would also collect environmental samples during the upstream processing run to ensure that the air quality and the quality of the water for injection (WFI) are maintained within acceptance criteria specified by the validation protocols, SOPs and PBRs.

Also part of upstream processing are the initial purification steps which could include centrifugation and/or filtration in order to separate cells from media. The cells or the media would be discarded to the kill tank, depending on where the protein was located. In this course we are using glass bioreactors and representative of three types of cells used in upstream processing of human protein pharmaceuticals: bacterial, animal, and fungal cells. In bacteria, such as biotechnology's workhorse, Escherichia coli , proteins remain inside the cell so the cells are separated from the media and the media is discarded to the kill tank. In animal cells, such as Chinese Hamster Ovary (CHO) cells, and in fungal cells, such as the yeast Pichia pastoris, proteins are secreted into the media so the media is saved for later isolation and purification of the protein of interest in downstream processing.

Plants and Animals as Bioreactors for the Manufacture of Human Proteins

Today, upstream processing can also take place in whole animals or plants, making the animal or plant a veritable bioreactor. This is a common occurrence in monoclonal antibody (Mab) production using mice. Other protein pharmaceuticals have been produced in the milk of goats and sheep and the leaves of tobacco to mention a few instances of this sort of upstream production of proteins.

A recent article on the production of therapeutic monoclonal antibodies using soybeans and corn stated that "transgenic soybeans and corn will prove competitive with bacteria, fungi and mammalian cell fermentation as production vessels for pharmaceuticals based on mammalian proteins. Purified products produced with existing technology can cost $100,000 to $10,000,000 per kg, compared to around $1,000 per kg for compounds produced in transgenic plants".

REFERENCES:

Holzmann, David 1994 Agracetus grows monoclonals in soybeans and corn plants. Genetic Engineering News, September 15: 1.


Sonia Wallman, NHCTC. 1997