Advances in Downstream Processing of Biopharmaceutical Proteins

Introduction

Downstream processing has to do with the attaining of targeted proteins from the biomass recovered from the upstream process. It generally refers to a purification process of Bio-Synthetic products that are the result of their crude preparations. The impurities may include tissue fluids and fermentation broth. It is basically a recovery of the targeted product from its basic form, for instance recovering antibiotics and vaccines from fermentation broths to be processed into tablets.

Traditionally, crude medicinal preparations were well known by indigenous practioners to cure certain ailments Examples of such medicines include silicylates that were used as pain killers and antimalarial compounds that were extracts of certain barks of trees. Moreover, animal organs such as the pancreas and the placenta have been traditionally used as a source of hormone considered adequate for therapeutic use (Jozala, Geraldes, Tundisi, Feitosa, Breyer, Cardoso, & Oliveira, 2016).However, the levels of impurities in these crude forms limit the efficiency of the desired medicine.

In today’s technologically advanced world, biotechnology helps us to produce drugs like vaccines and antibodies. Most biotechnological processes can be divided into three major steps. The first step is cloning. It involves a process of inserting a gene in a microorganism, and in turn the cells produce a protein which can be used as a major component of the final medicine.

The second process is to control and closely monitor the microorganism in a bio reactor to produce the protein in high concentrations. This is what is called the upstream processing.  It is the formative step which involves biomolecules which are grown, most often by cell lines from bacteria or mammals, inside bioreactors (Fujiwara, Nagy, Chew, & Braatz, 2005). Whenever they achieve the required density, they are collected and transferred to the downstream are of the bioprocess.

Nevertheless, what is gotten from the upstream processing is a mixture of different molecules proteins and cells. The process of getting the final product, which is ready for use, from this mixture is what we call downward processing ( Khanra, Mondal, Halder, Tiwari, Gayen, & Bhowmick, 2018 ). It is defined as the recovering and purifying of biosynthetic products, mostly pharmaceutical products, which are from natural sources such plant or animal tissue.

After the fermentation process, and the bacteria or the yeast has produced the protein of interest, we get an impure mixture. The goal of purification is to get a pure and concentrated product. High levels of drug purity and safety are strictly demanded by regulatory authorities all over Europe, The United States of America among other countries (Demian, 2009).

As mentioned above, downward processing involves purification of a specific molecule. The molecule could be a protein or a nucleic acid emanating from the wet biomass which is a result of the upward processing ( Khanra et al., 2018 ). The ultimate goal is to clean the robust pharmaceutical component to make it into a tablet, vaccine or a final product fit for consumption.

Downstream processing employs a variety of fractionation and purification methods which include: filtration, chromatography, distillation, centrifugation and precipitation. It is worthy to note that the most significant principle of the down streaming processing is that there is no universally acceptable arrangement of operational units which can dictate the most best downstream processing process (Fujiwara et al., 2005). Rather, a combination of more than one technique for purification need to be employed for successfully separating the pure product.

The cheapest and simplest separation methods should be done early in the process. While the complex and expensive separation techniques may be required, should be carried out towards the end of the process in order to capitalize on an economic friendly production in the long run.

Body

Downstream process steps include: Disruption and harvesting of the cell, cell lysis, cleaning and brightening, clarification, capture, and crafting the fill and finish.

Harvesting and cell disruption is the first process of the downstream processing. Clarification follows closely to remove all the materials that will be insoluble in eth main medium. The initial stage in the whole process is getting the cells by delinking the cellular biomass away from the fermentation broth ( Khanra et al., 2018 ). It is usually arrived at through centrifugation which entails using separators or tangential flow filtration on micro porous membrane.

Cell Lysis comes through a mechanical activity which disrupts the cell, under pressure in order to release the targeted outcome into the lysate. The process is succeeded by a new mode of tangential flow filtration by employing much finer micro porous membrane to get rid of the subcellular impurities ( Khanra et al., 2018 ).

Clarification relies upon the nature of the product protein. The determining factor is whether it can dissolve. If it is soluble it finds itself in the clarified permeate and can move on to the next processing phase that entails product capture. If on the other hand the product gets attached on an inclusion body, repeated centrifugation can aid in separating the concentrated addition of bodies from other particulate matter ( Khanra et al., 2018 ). Furthermore, additional solubilization phase is needed which involves using robust denaturing and deactivating buffers prior to protein refolding and recovery by using refined chromatography with high resolution.

The next stage is the capture stage. The beginning place for material capture is a clear filtrate that comprises the targeted element and many other contaminants. As expected, capturing is normally achieved through the initial chromatography stage aimed at eliminating as many contaminants as possible ( Khanra et al., 2018 ). The capture technique is presently employed where the capturing is believed to be the route to good purification the product in a single phase and then decreases the input volume through making dense the protein product at the outflow section.

The next stage is the Purification and polishing stage. Purification is removing all the dirty molecules and subsequent display of the final outcome in a uniform and homogenous quality. It is just a battery of chromatography and filtration process ( Khanra et al., 2018 ). Because polishing comes at the tail-end phases of purification, it poses the greatest challenge in the stage because it entails separation of the desired product from the contaminants. At this point, different types of chromatography can be employed to achieve the desired results.

Chromatography is a construct of two basic terms, Chromo- which means color and Graphy- which means making an impression of an idea on a surface ( Khanra et al., 2018 ). Chromatography is a manual segregation technique where various elements of a mixture get layered due to variations in their distribution between two phases. One of the phases is the stationary phase while the other is the mobile phase.

The first type of chromatography is the Affinity Chromatography. It uses the specifics between one type of solute molecule and a second one that is immobilized in a phase that is stationary. The principle behind the method is that the stationary stage is usually a pulp mixture (mostly agarose) ( Khanra et al., 2018 ). The most valuable molecule has a well-known and articulated attribute. The system that is designed to entrap in where the desired molecule is mined on the stationery phase. Stationary stage can then be captured, alienated form the process and washed then the target molecules can be freed from the entrapment. The process works by use of ligand which is a molecule that specifically binds to the protein of interest. An affinity material is prepared using inert support and the lingad. The affinity material is combined with the mixture of proteins from which we have the desired product. Once the targeted molecules combines with the inert support, the unwanted proteins are disposed of and a solute with competitive lingad is introduced to the combination of the inert support and the desired product. The elute frees the desired molecules from the inert support. Finally we remove the competitive lingad by dialysis and the end product is the desired molecules in pure form.  

Figure 2

Affinity chromatography was discovered by Pedro Cuatrecasas and Meir Wilcheck in the year 1968. The method can be applied to cleanse and make dense the enzyme solution. It can also be used in purification of recombinant proteins or in the purification of antibodies.

The second type of chromatography is the ion-exchange chromatography. This process is a mechanism that facilitates the delinking of ions and polar molecules in line with the ionoc charges that they have. The principle is that this type of chromatography remains with model molecules on the column based on the interactions of the ions (Fujiwara et al., 2005). The exterior of the static stage reveals ionic functional groups (R-X) that react with analyte ions carrying an opposite charge. Eventually the negatively charged analyte (anion) is drawn to the positive surface. However, the positively charged analyte is attracted to the negative exterior.

There are two types of ion exchange chromatography. The cation exchange chromatography maintains positively charged cations since the static stage displays a negatively charged active faction. The second type is the Anion exchange chromatography. The process maintains anions using positively charged active groups.

The applications of ion exchange chromatography includes: use in some types of of charged molecules including very big proteins amino acids or slightly minute nucleotides and amino acids, cleasnsing of proteins and water analysis.

Figure 2

The third type of chromatography is the hydrophobic interaction chromatography. This type of chromatography is a layering methods that utilizes the characteristics of hydrophobicity to separate proteins from other similar elements. Hydrophobic interaction chromatography is a unique because the proteins coagulate at high salt concentration and break at low salt concentration (Hong et al, 2018). This situation is evident in a reverse salt gradient. HIC forms a versatile liquid chromatography technique. The principle behind the technique is that the proteins are split by hydrophobic i on columns with hydrophobic groups attached. For instance (phenyl-, and octyl- groups) ( Khanra et al., 2018 ). In this type of chromatography, hydrophobic groups such as the phenyl, octyl and butyl are attached to the stationary columns. HIC separations are mostly designed using antagonistic situations from those in ion exchange chromatography. A buffer with an enormous ionic strength normally ammonium sulphate is in the first stages evident in the column. The more hydrophobic the molecule, the less salt needed to promote the binding. Under high salt conditions there are enforced hydrophobic interactions. The hydrophobic regions have a higher tendancy to aggregate.

The fourth type of chromatography is the Reversed phase chromatography which works on the same rule as HIC although it uses resins with greater hydrophobicity such as the C4 to C18 alkyl groups ( Khanra et al., 2018 ). A high resolution possible with a fast speed reversed- phase liquid chromatography makes it suitable for the separation of small proteins and peptides as small as the size of insulin and generally used in the last stages of the downstream process.

Conclusion

The role of biotechnological downstream process is to offer markets the most promising outcome which is pure and within a short period. The process saves on the overall cost of production. However, traditional processes (not the ancient ones) and regular quality control systems do not aid the efficacy required to keep track of the current upstream production. The process of mitigating present challenges have led to emergence of some general trends, the most relevant including continuous production, the single use modules, process analytical technology, and quality by design. 

The expanding biotechnology market has resulted in the emergence of noble and perfected alternatives to chromatography to aid improve yields and lower costs of production, at the same time maintaining excellent product purity. A number of valuable substitutes have been proposed in academic literature. The proposals include filtration methods relying on thiophilic and affinity interactions, affinity precipitation, two-phase aqueous processes high-purpose tangential flow filtration, and affinity interactions, , high-gradient magnetic fishing among others.

References

Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology advances , 27 (3), 297-306.

Fujiwara, M., Nagy, Z. K., Chew, J. W., & Braatz, R. D. (2005). First-principles and direct design approaches for the control of pharmaceutical crystallization. Journal of Process Control , 15 (5), 493-504.

Hong, M. S., Severson, K. A., Jiang, M., Lu, A. E., Love, J. C., & Braatz, R. D. (2018). Challenges and opportunities in biopharmaceutical manufacturing control. Computers & Chemical Engineering , 110 , 106-114.

Jozala, A. F., Geraldes, D. C., Tundisi, L. L., Feitosa, V. D. A., Breyer, C. A., Cardoso, S. L., ... & Oliveira, M. A. D. (2016). Biopharmaceuticals from microorganisms: from production to purification. brazilian journal of microbiology , 47 , 51-63.

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