Lesson 26. UPSTREAM PROCESSING AND DOWNSTREAM PROCESSING
UPSTREAM PROCESSING AND DOWNSTREAM PROCESSING
26.1 Introduction
Industrial fermentation involves upstream and downstream processes (Figure 26.1).
Upstream processes, include selection of a microbial strain characterized by the ability to synthesize a specific product having the desired commercial value. This strain then is subjected to improvement protocols to maximize the ability of the strain to synthesize economical amounts of the product. Included in the upstream phase is the fermentation process itself which usually is carried out in large tanks known as fermenters or bioreactors. In addition to mechanical parts which provide proper conditions inside the tank such as aeration, cooling, agitation, etc., the tank is usually also equipped with complex sets of monitors and control devices in order to run the microbial growth and product synthesis under optimized conditions. The processing of the fermentation reactions inside the fermenter can be done using many modifications of engineering technologies. One of the most commonly used fermenter types is the stirred-tank fermenter which utilizes mechanical agitation principles, mainly using radial-flow impellers, during the fermentation process.
26.2 Overview of Upstream Processing
Upstream processing normally deals with three important points.
The first relates to fermentation media, especially the selection of suitable cost effective carbon and energy sources, along with other essential nutrients. The media optimization is a vital aspect of process development to ensure maximization of yield and profit.
The second aspect involves aspects associated with the producer microorganism. They include the strategy for initially obtaining a suitable microorganism, industrial stain improvement to enhance productivity and yield, maintenance of strain purity, preparation of a suitable inoculums and continuing development of selected strains to increase the economic efficiency of the process.
The third component relates to the fermentation which is usually performed under rigorously controlled conditions developed to optimize the growth of the organism or the production of a target microbial product.
26.2.1 Fermentation medium
The medium used for fermentation may be classified as defined, complex or technical medium. Defined medium consists only of precisely chemically defined substrates. Complex medium is composed of substrates with undefined composition, such as extracts or hydrolysates from waste products, which are cheap substrates commonly used in industrial production. Relatively expensive substrates, such as yeast extract, brain heart infusion, peptone, and tryptone are often used for complex medium. Technical media are used on an industrial scale and are cheaper. The substrate sources can also be derived from industrial waste, and are often highly impure mixtures, requiring pretreatment before they could be used for a fermentation process. Examples are soy meal, whey, fishmeal, malt extract, and sulfite waste liquor. Wastewater from monosodium glutamate production, which contains high levels of chemical oxygen demand (COD), sulphate, and ammoniacal nitrogen at a low pH, has been used as the nitrogen and water source, with sugar beet pulp as the carbon source, for the production of pectinase.
Media sterilization is necessary to ensure that only the desired microorganism is present to carry out the fermentation, that products are made of predicted quality, that the environment is protected from undesirable contamination, and that deterioration (microbial spoilage) of products is prevented. Sterilization by high temperature achieved by direct or indirect steam or electric heating, membrane filtration, microwave irradiation, high voltage pulses and photosemiconductor powders which involve the rupture of the cell membrane by increasing the transmembrane electric field strength beyond a certain threshold.
Inoculation is the transfer of seed material or inoculum into the fermentor. Inoculation of a laboratory fermentor is generally done using presterilized tubing and a peristaltic pump. However, on a larger scale, inoculum transfer is done by applying a positive pressure on the inoculum fermentor and connecting it aseptically to the production fermentor. The connecting lines are sterilized before being used for transfer of inoculum. Heat susceptible substances such as amino acids and some vitamins must be dissolved in small volumes of water, sterilized by filtration and added separately to the final medium aseptically.
26.2.2 Fermentation systems
A fermentation system is usually operated in one of the following modes: batch, fed batch, or continuous fermentation. The choice of the fermentation mode is dependent on the relation of consumption of substrate to biomass and products. The systems are batch, continuous and fed batch systems that were described earlier in lesson 21.
Today the most common type of upstream processing of proteins utilizes two tools: bioreactors a nd 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).
26.2.3 Innoculum
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.
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. 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 , 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. 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
26.3 Overview of Downstream Processing
Downstream processing encompasses all processes following the fermentation. It has the primary aim of efficiently, reproducibly and safely recovering the target product to the required specifications (biological activity, purity) while maximizing recovery yield and minimising costs. The target product may be recovered by processing the cells or the spent medium depending upon whether it is in intracellular or extracellular product. The level of purity that must be achieved is usually determined by the specific use of the product.
Each stage in the overall recovery procedure is strongly dependent on the protocol of the preceding fermentation. Fermentation factors affecting downstream processing include the properties of microorganisms, particularly morphology, flocculation characteristics, size and cell wall rigidity. These factors have major influences on the filterability, sedimentation and homogenization efficiency. The presence of fermentation by-products, media impurities and fermentations additives such as antifoams may interfere with downstream processing steps and accompanying product analysis.
The products of fermentation are usually found in complex mixtures of dilute solutions and must be concentrated and purified. The separation of the product of interest from the
fermentation broth depends on the accumulation of the product, which may be intracellular or extracellular.
The typical downstream operations and the unit operations involved in the processing of fermentation broth are:
- Cell disruption (high pressure homogenization, wet milling, and lysis)
- Clarification of extract (centrifugation, extraction, dead end filtration, and cross flow filtration)
- Enrichment (precipitation, batch adsorption, ultrafiltration, and partition)
- High resolution techniques (ion exchange, affinity, hydrophobic, gelfiltration, adsorption chromatography, and electrophoresis)
Fig. 26.3 Filtratin methods wherein separation of particles form liquid
occur by applying a pressure to the solution to force the solution through a filter
26.3.1 High resolution techniques like chromatography techniques
The molecules of interest are adsorbed or stuck to beads packed in the column. The higher the affinity of the molecule (protein) for the bead the more will be bound to the column at any given time. Proteins with a high affinity travel slowly through the column because they are stuck a significant portion of the time. Molecules with a lower affinity will not stick as often and will elute more quickly. We can change the relative affinity of the protein for the column (retention time) and mobile phase by changing the mobile phase (the buffer).
The most common type of adsorption chromatography is ion exchange chromatography. The others used in commercial biopharmaceutical production are affinity, hydrophobic interaction and gel filtration.
Column chromatography Separates molecules by their chemical and physical differences
Fig. 26.5 gelfiltration chromatography
26.3.2 Ion exchange chromatography
Ion Exchange Chromatography relies on charge-charge interactions between the protein of interest and charges on a resin (bead). Ion exchange chromatography can be subdivided into cation exchange chromatography, in which a positively charged protein of interest binds to a negatively charged resin; and anion exchange chromatography, in which a negatively charged protein of interest binds to a positively charged resin.
Fig. 26.6 Ion exchange chromatography (www.biomanufacturing.com)
26.3.3 Isoelectric focusing
Once the pI of your protein is known (or the pH at which your protein is neutral), you can place it in a buffer at a lower or higher pH to alter its charge. If the pH of the buffer is less than the pI, the protein of interest will become positively charged. If the pH of the buffer is greater than the pI, the protein of interest will become negatively charged.
26.3.4 Affinity chromatography
Affinity chromatography separates the protein of interest on the basis of a reversible interaction between it and its antibody coupled to a chromatography bead (here labeled antigen). With high selectivity, high resolution, and high capacity for the protein of interest, purification levels in the order of several thousand-fold are achievable.
Fig. 26.7 Affinity chromatography (www.biomanufacturing.com)
26.3.5 Hydrophobic interaction chromatography (HIC)
HIC is finding dramatically increased use in production chromatography. Antibodies are quite hydrophobic and therapeutic antibodies are the most important proteins in the biopharmaceutical pipeline. Usually HIC media have high capacity and are economical and stable. Adsorption takes place in high salt and elution in low salt concentrations.
