Estágio realizado na UNICER Bebidas, S.A. e orientado pelo Eng.ª Maria-Manuel Dantas / Tese de mestrado integrado. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 2009

Tese de mestrado integrado. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 2010

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One of most major concerns from both cost-effective and technical point of view in membrane process industry is membrane cleaning. The aim of the project was to investigate the variations in membrane surface properties and separation efficiency of reverse osmosis membrane. Compativtive analysis have to be performed on four RO membrane before and after exposing the virgin membrane into chemical cleaning to identify and analysis the impact of the chemical cleaning on the performance of RO membrane. Commerical chemical cleaning used in this project were caustic and acidic cleaning agent. The project’s aim is the investigation of simulation software’s precision for the four membranes performance projection at different conditions of the feed water. The assessment of the membranes performance was done in the Innovation Cluster at pilot plant that was industrial in size. The main commercial elements used were the thin-film composite membranes with a spiral-wound of 8-inch polyamide. Ultrafiltration (UF) and seawater RO membrane pretreatment process was done for the red sea sourced feed water. A pressure vessel dimensioned at 8-inch was operated in conjunction with an individual element at 8 -20 m3/hr feed flow rate, with an 8 to 12 % recovery and an average 35,000-42,000 mg/L of total dissolved solids (TDS) composition for the feed water. To achieve the project’s aim in assessing the membranes, three phase experimental stages were completed. The membranes performance was assessed in terms of their water flux, salt rejection, boron rejection, bicarbonate rejection and permeate quality. In addition, the membrane surfaces were characterized after exposing the fresh membranes with a chemical cleaning reagent. The experimental results showed an increase in both permeate flow and salt passage for all studied elements. The changes in the membranes performance were systematically explained based on the changes in the charge density and chemical structure of the membranes surface. The experimental results showed that both the physical and chemical surface properties of the membranes do not significantly alter under standard industrial conditions. These results shed some light on the effects of chemical cleaning in a pilot-scale RO plant and improve our understanding to provide a potential research direction for cleaning methods of membranes.

<p>Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.</p>

Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.

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<p>  </p> <p>Fouling is a severe problem for food processing equipment, including heat exchanger, filtration system, etc., which can decrease heat transfer efficiency, increase pressure drop, and affect food quality and safety. To ensure process efficiency and product quality, regular cleaning is necessary. On manufacturing lines in the food industry, cleaning is usually performed by cleaning-in-place (CIP) operations, which mainly comprise water rinse, alkaline wash and acid wash steps. Although CIP can ensure uniform cleaning of equipment, lower costs associated with labor and plant downtime, and improve personnel safety, it consumes large amounts of energy, chemicals and water and thus affects the environmental sustainability.</p> <p>Microbubbles (MBs) are fine gas bubbles with very different physicochemical properties from millimeter-sized bubbles, including longer residence time in liquid, higher mass transfer rate, larger surface-area-to-volume ratio, and generation of free radical when bubbles collapse. In addition, MBs feature hydrophobic liquid-gas interface, allowing hydrophobic and amphipathic substances to attach to and spread on bubble surfaces. MBs have been used in cleaning processes, such as oil flotation and fresh produce washing; however, their applications in CIP operations in food processing have not been explored.</p> <p>The objective of this dissertation is to develop a novel CIP operation with the incorporation of MBs for cleaning of food processing equipment. MBs were incorporated into rinsing water to clean milk deposit fouled on heat transfer surface. A computational fluid dynamics model was built to predict the contact frequency of MBs with deposit and further identify the flow conditions that provided maximum MB-deposit contact. Moreover, MBs were confirmed to be able to attach to milk deposit by microscopic imaging. Rinsing with MB-incorporated water noticeably enhanced the deposit removal at Re of 4392 and 5403, by 27−31%. For cleaning of microfiltration membrane reversibly fouled by palm oil-in-water emulsions as model wastewater, although adding MBs into alkaline wash increased the membrane flux recovery by 235%, increasing the crossflow velocity of MB-incorporated liquid did not guarantee the enhancement in cleaning performance. Lastly, a MB-assisted full CIP process was tested on an ultrafiltration system used for milk concentration. MB-assisted CIP showed an increased cleaning efficiency with up to 72% higher flux recovery than conventional CIP, and alkaline wash with MBs added was the major step accounting for enhanced protein removal.</p> <p>Overall, this dissertation proves the effectiveness of MBs in cleaning of different types of food deposits, and provides groundwork knowledge of MB incorporation into CIP operations for different food processing equipment. The results are expected to guide the scale-up of MB-assisted CIP processes that can reduce the water and chemical usage in food manufacturing sectors, ultimately improving both economic and environmental sustainability of the food industry.</p>

In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering. Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface ofStreptococcus. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix. Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue. The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region. In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein. <b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.