Bioanalytical Technique Practical

Bioanalytical Technique PracticalIntroduction overture in technology has widened the domain of bioanalytics, reliable and reproducible information flock be obtained from several instruments and protocols. The drug argonna has become really competitory and it is thusly imperative that an understanding of the different techniques is crucial to the isolation and abstract of biomolecules.This report is foc pulmonary tuberculosisd on the BCA seek for protein estimation and data epitome of SEC using a UPLC administration. The BCA search is a modified assay that is used for the detection and quantification of total protein in a stipulation test. The assay generates a purple colour which is as a root of the chelation reception of bicinchoninic acid with cuprous ions. The mazy formed as a result of the response is known to exhibit a rattling strong absorbance at a wavelength of 562nm and this shows an increasing linearity with the add of protein in a break-dancen sample. T wo main components make up the assay the ensample swerve and the unknown protein sample. The BCA assay is widely used be nominate of its sensitivity and compatibility with detergents and several other(a) buffer types. The drawback however with the assay is that it is not as rapid as some other estimation method such as the Bradford delinquent the incubation sentence required and moreover it is not an endpoint reaction as colour continues to develop even after incubation.The second bit of this report is pertain with running a system suitability screen on the waters BEH200 SEC UPLC instrument. The Ameri atomic number 50 and European Pharmacopeia specifically mentioned that the requirements for a system suitability sampleing on the day of proscribedline showing that it is fit for its in die harded use. It is worth mentioning that this has no bearing with the booking of the instrument. Failure of any of the parameters simply means that an assay finishnot commence. This tes ting is concerned more about the method on the day of analysis kind of than the instrument per se.1.1 MaterialsPipettes and appropriate tipsMicrocentrifuge tubesMicrowell PlatesHPLC VialsBovine Serum Albumin Protein 2mg/mlBCA ReagentDeionised pissingPerkin Elmer Plate Reader100Mm sodium Phosphate BufferWaters Aquity H Class Bio UPLC instrumentWaters BEH200 SEC UPLC Column1.2 Preparation of StandardsStandards were fain as per instruction manualTable 1 Preparation of Protein Standards countsDilution promoter = ducking of stock ascendent / tautness of diluted resolving bulk of stock to add to water = Required good deal of diluted solution/ Dilution featureorVolume of water to add = required final volume / Volume of stock required1.3 Preparation of SampleThe sample was lively as per the instructions on the practical manual.Table 2 attempt Sample Dilution1.4 Preparation of BCA Reagent and WellThe BCA reagent was disposed(p) and the 96 well microplate was prepared and read in the emailprotected 562nm as per the instruction manual.1.5 Data AnalysisCalculationEquation of the linear least square fit bed be represented as outlined be broken.Y = 0.0008 (X) 0.00510.035691= 0.0008 (X) 0.0051X = 0.035691 + 0.0051/0.0008X = 0.040791/0.0008X = 50.98875Taking the dilution detailor into account we multiply by 5The protein concentration is thus 50.98875 x 5 = 254.94mg/mlDiscussionThe sample data had an anomaly, showing a negative reading on the third well. This is connotative of defilement. The surmise of interference from the reagent domiciliate be ruled out because the standard was treated the same way and similarly taking into cognisance the fact that the experimentation was not carried out under a non-denaturing condition. The likely cause could be repayable to dirt on the Microwell thus blocking out the necessary wavelength for the absorbance reading or the sampling pipette not delivering the pay off amount of reagent. The intensity of the colour ch ange for the third well was observe to be less than the other two wells. There is likewise the possibility of the sample not being vortexed properly or sample settling to the bottom of tube.As mentioned earlier thither seem to be an anomaly with our absorbance reading and this john be validated from our standard curve as it is not quite linear and on this basis we flush toiletnot absolutely cuss on the result of the experiment.2.1 Size Exclusion Chromatography ExperimentThe priming and purgatorial of the UPLC instrument was carried out by the trainer as per the instruction manual. trunk suitability testing was then carried out to secure that it is fit for purpose. The test serves to assure the reproducibility of the instrument and the method. It is a regulatory requirement which was mentioned in both the EU and US pharmacopoeias. The testing is important as it flock allow for critical factors that could affect the performance of the instrument to be change to meet the tes t criteria. Parameters such as the resolution, efficiency of the column, tailing factors, sexual congress standard excursus etc. are used as criteria for likeness with regards to standards and test samples.The table below details the results obtained from the system suitability testing, reservoir standard and our test sample. The UPLC system used in our experiment crowd out be said to be fit for purpose taking into consideration, the system suitability test. The results obtained were within our test criteria. The resolution of the top side and standard deviation of the different retention time was less than 1 which as a rule of the thumb is quite acceptable.Comparing the test sample to the interview standard one would not fail to notice that the first pate in the reference standard was a dimer while the second salad days was a monomer but in our test sample the retention time of the first peak was really short showing evidence of a naughty molecular weight aggregation .Als o from our result the second peak was our crossing dimer while the third peak was our product monomer. This result serves to foreground the appliance of protein aggregation and the reason why it should be minimised as it impacts on the yield of the product and moreover it can affect the potency and therapeutic potential of the parenteral. It is also worth mentioning that sometimes early elution may not necessarily mean that there is aggregation, it could be for the simple reason that sometimes intrinsically unstructured proteins can elute so fast that they tend to be contrive like aggregates. A molecular weight comparing testing can be used to differentiate them.From our experiment the test sample showed increase aggregation and this can be explained given the fact that the experiment was not carried out under a non-denaturing environment. The possibility of column contamination or buffer contamination can help to encourage aggregation. It is also important that samples should b e free of extraneous fractions during injection as this can also be a determining factor. The changing environment of the peregrine phase can also be construed as a viable cause of the aggregation observed. The temperature of the instrument is another factor that can good turn a role and as we all know that the Arrhenius theory of a 10C increase in temperature speeding up a reaction does not relate to proteins as it rather opens up the nerve pathway of denaturation and aggregation.QuestionsEstimation of protein concentration is important as we have to know the amount of protein in our final product after fermentation to know if the bioprocess has to be perfectd with regards to the expected titre value. The concentration of the protein can also allow for the portioning of the product into the right dosage formulation, certain therapeutic proteins are required in a very high dosage form and their production can be sometimes targeted at a particular section of the people e.g. dur ing an epidemic outbreak to ensure potency and biological activity. The knowledge of the concentration of proteins can also allow us to work out the scrimping of scale with regards to the profit margin taking into account, the expense incurred in enquiry and development and other aspect of the production process. It is also important to count the amount of protein in our biomass so as to be able to optimise our subsequent purification steps. The estimation of the protein concentration can also give us an idea of product cerebrate impurities and those associated with the process.Proteins are very complex molecules and are prone to several types of condition than cause instability from the scratch line confront of production to the end of their shelf life. Aggregation can be described in a laymans term as the propensity for proteins to stick together under conditions such as a slight increase in temperature, pH, shear force, ionic strength of the solution they are contained in et c. Aggregation have been seen to cause cargo area in several novel biologics due to the debilitating effect on the health of the population that the drug is directed at and also in the context of compliance to regulatory authority as there is a specification to the amount of aggregates that can be allowed. It is extremely difficult if not impossible to totally eradicate aggregation from the process. It is worth mentioning however that the mechanism of aggregation is smooth subject to debate as it has not been full understood. Aggregation can be reversible or irreversible depending on the stage it has attained as can be hurt of master(a) structure.The potency of biologics as we all know are normally related to them being in their native structure, in most instances aggregation leads to the loss of activity and moreover the overall yield of the biotherapeutic is greatly affected. Aggregation has also been known to spur immune response in patients that have been administered with protein therapeutics affected by aggregation this could be by way of the neutralisation of antibodies that helps to ensure the effectiveness of the drug. In a worst case scenario the immunogenic reaction can lead to incurable conditions such as seen in patients with pure red cell aplasia where the red blood cells are attacked and blood transfusion is needed for life. The route of administration of biologics is intravenous and the presence of aggregation especially those of very high molecular size can result in the period of blood vessels. It is thus very important that at each stage of our production testing should be carried out to check for aggregation.Size excision chromatography is a purification system that exploits the molecular size of the compound of interest. precisely put it works just like a molecular sieve, smaller particles passes through the sieve which is the stationary phase and could be a bead coupled to a resin. The pore size of the string of beads are be and on this basis it will only allow certain particle sizes to pass through while excluding those that are too large for the pore. The big particles because they are not passing through the beads are thus excluded quickly, their retention time is thus said to be short. The smaller particles are retained bimestrial while the larger particles earlier mentioned are eluted through the void volume. Different gels in use would typically have different pore sizes and can be used to determine the size of the molecules to be separated.Despite all the numerous advantages of size exclusion chromatography which has made it the luxurious standard over the years for analysing protein aggregation there are still some limitation associated this method. The possibility of the stationary phase and the analyte reacting together can be sometimes rife thus leading to a longer retention time which serves to mimic the compound as being of low molecular size. The cost associated with running this type of separation technique can also be enormous due to the fact that large columns and eluents are required and this serves to add to the overall cost of the unit operation. In comparison to other modes of separation, size exclusion chromatography can be said to have an inherent low resolution as there is a limit range of molecular weight that can be separated as a result of dependence on the pore size of the beads in use. There is also the possibility of proteolytic degradation as the protein of interest can become targets for proteolytic enzymes still present in solution. The accuracy of this technique can sometimes come into question due to the fact some aggregates will remain in solution and as such would not be detected. Also taking into consideration the fact that larger molecular aggregate leaves the column through the void volume, there is also the possibility. The possibility of the polymer in use to degrade is also a drawback as this can occur at a very high flow rate. The h igh flow rate as mentioned earlier can degrade the polymer and it also has the ability of altering the geometry of the beads in use making the separation technique inefficient