Hi, I gave seminar on latest developments in downstream processing of biotechnology products in my III rd semester of Mpharm Pharmaceutical Biotechnology. It includes two parts 1. Use of Reverse Micelles in downstream processing of biotechnology products. 2. Use of Affinity Chromatography based upon uniquie single chain antibodies VHH ligands in downstream processing of biotechnology products. Reverse micelles in downstream processing, The selective separation and purification of target proteins in a mixture of both similar and dissimilar proteins in biological and chemical compounds and this separation is can be done by using reverse micelles. What are Reverse Micelles, Reverse micelles are nanometre sized 1-10 nm water droplets dispersed in organic phase obtained by action of surfactants. They consist of hydrophilic core and hydrophobic corona. Due to polar nature of hydrophilic core in RMs water is solubilized into these micelles solutions. This allows proteins and other hydrophilic molecules to be solubilized in aqueous microenvironment while organic reactants and products remain in bulk organic phase Structral Parmeters associated witn Reverse Micelles, There are two structural parameters associated with RMs which are the water content of the RMs Wo and the aggregation number Nag. Wowater/surfactant. Nag average molecular weight of RM/molecular weight of the surfactant. Nag enables to calculate number of micelles in system if surfactant concentration is known. Wo relates to size of RM Factors affecting protein solubilization by Reverse Micelles, Water content of RM, Wo and protein s Ex RM AOT, protein BSA Minimum Wo required for the solubilization of a fixed amount of protein. Aqueous phase pH and ionic strength Ex RM AOT, Protein BSA Salts Kcl, Nacl, Cacl2 and Mgcl2 Minimum Wo is independent of pH for Mgcl2 whereas increasing the concentration of Kcl and Cacl2 decreases minimum Wo. Surfactant concentration Ex RM DODMAC, Protein lysozyme The concentration of DODMAC has negligible influence on the amount of lysozyme extracted. Reverse Micellar Systems, Ionic surfactants based RMs are most commonly used due to their ability to solubilise a wide range of proteins . However electrostatic forces between proteins and ionic surfactants may denature the proteins and subsequently low yield of proteins. Non-ionic surfactants based RMs limited success due to difficulties in extraction process which produces low yields despite preventing denaturation. Mixed RMs are more efficient than ionic surfactants but limited success due to their complexities. Affinity based reverse micelle are surfactants ionic od non-ionic coupled with affinity ligands. The introduction of affinity ligands allows enhanced selectivity and extraction capacity. Ex ndash cibacron modified lecithin antibodies- ligand AOT Mechanism of purification of proteins by Rmrsquos. Forward extraction process ndash protein is transferred from a bulk aqueous phase to water pool of RMrsquos in an organic phase. Backward extraction process ndash proteins are recovered from the RMrsquos into a fresh aqueous phase Problems with back extraction process, Decrease in activity yields due to structural changes in proteins as a result of the strong interactions between proteins and micelles. Slow rate of back extraction due to the greater interfacial resistance towards protein release at the oil-water interface during the back extraction. Three notable techniques to improve the rate of back extraction process, 1. using aqueous stripping solution with high salt concentration or high pH, or varying the temperature of the system. 2. addition of appropriate alcohol . 3. addition of destabilizing solvent or dehydrating aqueous phase of RMs with silica gel or molecular sieves. 4. addition of suitable counter ionic surfactant. Illustration of Three notable techniques to improve rate of back extraction, 1. the first technique utilises the electrostatic repulsion that occurs between the surfactant and protein as a result of the pH difference the size exclusion when salt concentration is increased. 2. the second strategy involves the addition of suitable alcohol species during the back extraction process. 3. the last technique mentioned involves the use of a counterion surfactant such as TOMAC or DTAB which are oppositely charged to the surfactant . Counter-current chromatography using Reverse Micelles, Shen Yu 2007 investigated the possibility of protein separation and enrichment by CCC using RM solvent systems. Using two phase solvent systems that provides suitable partition coefficients k for target compounds. The major parameter for partition manipulation is electrostatic interaction between the protein and charged heads of the ionic surfactant. They investigated the effect on protein separation efficiency increased when pH and ionic strength gradient were applied simultaneously. The RM system involved was AOT in n-hexane which makes up stationary phase while the mobile phase was comprised of aqueous Kcl. Affinity based reverse micelles extraction and sepration ARMES, ARMES have the ability of to separate proteins with higher selectivity and higher purification levels as compared to ionic, non-ionic and mixed RMs The ARMES process involves four main steps 1. formation of a ligand-ligate complex . 2. selective removal of the complex by reverse micelles. 3. disassociation of complex into stripping solution. 4. separation of ligand and ligate, and regeneration of the ligate . Enzymatic reactions in Reverse Micelles, The Use of RMrsquos to solubilize enzymes in organic solution is another recent development due to three reasons 1. aqueous environment can be created in RMrsquos for hydrophilic enzymes which will be denatured in organic phase. 2. such a system allows the use of surface active enzymes. 3.the aqueous-organic interface of such systems is usually very large due to the small size of the RMrsquos. Microencapsulation Techniques of Enzymea, The solubilisation of enzymes into RMrsquos using these techniques depend on the pH, and ionic strength of the aqueous phase, the sizes of the enzyme and RM, as well as the surfactant. Three different methods are, 1. injection injecting concentrated aqueous enzyme solution into organic solution containing surfactant and mixing. 2. Phase transfer phase transfer between aqueous phase and organic solvent containing surfactant. 3. Dissolution adding lyophilised enzyme to two phase RM solution already containing aqueous phase. Conclusion, Traditional separation such as electrophoresis or chromatography expensive and hence economically unviable unless the product of interest is high value. Reverse micellar extraction is 1. cost effective. 2. easy scale up and offerrsquos continuous operation. 3. immense attention for isolation and purification of proteins and enzymes in recent times. 4 . liquid-liquid extraction technique. Use of CCC, which allows increases the ease of implementing a continuous system. Use of ARMES increases the selectivity and purification, which are highly valued in biotechnological products. ARMES potentially increases non- ionic surfactants where by preventing denaturation of proteins during extraction process. Enzymatic reaction in reverse micelles , which allows reactions between hydrophilic enzymes and both hydrophilic and hydrophobic reactants, and which provides a platform for surface active enzymes to create biopharmaceutical products. Latest developments in down stream processing of biotechnology products part II, Use of Affinity Chromatography based upon unique single chain antibodies VHH ligands for purification of biotechnology products, Structure of VHH ligand, Camelid antibody lacks light chains found in all classical antibodies as such it has only single variable domain VHH by which antigens are bound, and two constant domains CH2 and CH3. Because of this it has high affinity. VHH ligand is 12 K Da fragment derived from a fully functional immunoglobin. Consequently it improves affinity, solubility, stability when compared to conventional antibody fragments. It is smallest known functional antigen-antibody fragment that shows high affinity and stability. VHH domain exists as single polypeptide chain, it is extremely stable and easily produced fragment that retains the full binding activity of its parent heavy-chain antibody. Properties of VHH ligand needed for purification systems, 1. High affinity and High stability. 2. Specificity and multiple species specifity. 3. Elution profile. 4. Cleaning conditions Production of Custom affinity ligands, 1.library construction. 2.library screening. 3.Small scale chromatography testing. 4. industrial scale ligand manufacturing. Library construction, Immunization of llama with target molecule. Mrna encoding VHH fragments is isolated by PCR techniques . Mrna to DNA by reverse transcriptase enzyme DNA is cloned into saccharomyces cerevisiae creating VHH library Screening is done by ELISA technique If proportion of reactive clones that cross react with molecule of interest is high enough it is selected to screening stage. Conclusion 1 Lowering cost and improving quality and time. 2 A platform technology 2 A platform technology 3 New tools for purification of AAV, immunoglobulins, AAT etc. 4 Development of affinity matrices. Novel affinity ligands in bioprocessing. Search labels, Dong, X.-Y., Feng, X.-D., Sun, Y. 2009. A Metal-Chelate Affinity Reverse Micellar System for Protein Extraction. American Institute of Chemical Engineers , 150-158. Liu j, et al. Comparison of Camelot Antibody Ligand to Protein A for Monoclonal Antibody Purification. BioPharm Int, September 200935-43 Daliya, S. M., Juang, R.-S. 2007. Role of alcohols in the formation of inverse micro emulsions and back extraction of proteins/enzymes in a reverse micellar system. Separation and Purification Technology 53 , 199-215. Carvalho, C., Cabral, J. 2000. Reverse micelles as reaction media for lipases. Socieacuteteacute franccedilaise de biochimie et biologie moleacuteculaire , 1063-1085. Adachi, M., Harada, M., Katoh, S. 2000. Bio affinity separation of chymotrypsinogen using antigen-antibody reaction in reverse micellar system composed of a nonionic surfactant. Biochemical Engineering Journal 4 , 149-151.