Chromatography Technique for Purification

Chromatography Technique for PurificationIntroductionColumn wadding is an integral embark on of the purification process in the manufacture of biologics. The goal is always to insure reproducibility with regard to the proficiency to be apply. Manual packing might sometimes involve several attempts to get an optimal packing as this would match the purification process. The rosin to be used in the packing process has to be well defined as it could impact on the eat rate which could lead to a trim down through put. The mobile phase is alike distinguished as the rationale behind the choice would be looking for a solvent that shtup pack the resin more tightly.In any chromatography proficiency that has to be utilised whether for the need to capture, purify or polish four integral parameters which includes, resolution, speed, capacity and recovery are always considered. Resolution is the most difficult to achieve especially during the polishing stage were impurities send away b e construed as having similar properties to the product. The efficiency of the chromatography chromatography column packing thus has a signifi poopt mapping to play on this basis as it is a good measure how consistently the column washbasin perform.1.1 Material As per SOP1.2 Key Instrument Comp peerlessntsBubble Trap BT1Filter Housing F1 inhalation / Outlet Valves V001 V101UV Sensor QIR4pH Probe QIR3Conductivity Meter QIR2Drain V102Column evanesce and bottom connections DN 1.3 Preparation of column for packing As per SOP TRG-DSP-052.1.4 Determination of % slurry Procedure was followed as per study manualResults Table 1Results Table 2r=5cmh= 15cm1.178Volume of gravity colonized resin for packing (Vgs)1.178 x 1.331.56674Slurry volume needed from container (SVc)(To give you the desired amount of gravity settled resin) (c)Vgs x 100% slurry in container= 1.55574 X 100/66% Slurry in container = 66%2.37384Adjusting the slurry to the desired % slow-wittedness for packingSlu rry volume required for packing (SVp)Vgs x 100% slurry for packing= 1.56674 X 100/ 70% Slurry for packing = 70%2.2382Volume packing buffer to addSVp SVc= 2.2382- 2.37384-0.13564Volume to be added is thus 0.13564 lCalculationsNumber of theoretical = NWhere VR = volume eluted from the start of sample application to the peak maximum= 8CMW h = peak width measured as the width of the recorded peak at half(a) of the peak height= 0.5CMN = 5.54 X Number of theoretical plates = 1418.24HETP = L/NWhere L = chouse height (cm)As we already know N (1418.24)HETP = 15/ 1418.24= 0.0105765Asymmetry factor (AS) = b/aWhere a = 1st half peak width at 10% of peak height (0.5cm)b = 2nd half peak width at 10% of peak height (0.5cm)= 0.5/0.= 1As rule of the thumb a good HETP grade should be at least two to trio times the average matrix bead size and normally in the range of 0.0018cm to 0.035cm. Looking at our column our HETP value was approximately 0.0106 and our bead has a pore size of around 40 mi crons which equates to 0.004cm and this is about 3 times our HETP. Our column can thus be confirmed to be within the welcome range.In the event that our column is not within the acceptable range several factors such as the following can be construed as being responsible.Uneven packing of the column or special the optimal packing flow rateThe possibility of channelling in the bedInadequate CIP can overly be a factor as this can result in a build-up of contamination in the column thus impacting on flow and other performance determinants of the column. Cleaning is also important to launder the matrix storage solution which is an unwanted entity during packing.Air entrapment prevalence of air bubbles can also affect the HETP values.The possibility of a void being present at the inlet can also be a contributing factor to the value of HETP not being within judicial admissionThe choice of resin is also very important as the possibility of the solute reacting with the resin can result in an uncertain HETP value.Peak asymmetry is an important measure in the determination of column efficiency and in lodge with the HETP value is always used in the calibration of a new or existing column. The atomic number 79 standard is the ability to achieve an asymmetry value of 1 although the acceptable range is normally amidst 0.8 and 1.2. An asymmetry value greater than 1 indicates the prevalence of extensive tailing while an asymmetry value less than 1 indicates extensive fronting.Taking our packed column into consideration, our asymmetry value from the chromatogram was 1 and one would generally thus expect a high efficiency and resolution.However, in the event of our column not being within the acceptable asymmetry value the following intellect are the possible develops. gigantic tailing which is characterised by an asymmetry value greater than 1 as mentioned earlier can be a reason. This factor is a result of column being packed too loosely and it can be observed from the chromatogram by the peak tailing gradually.Extensive fronting is also a possible cause and it is characterised by an asymmetry factor less than 1 which is normally as a result of the column being packed tightly and would be noticeable on the chromatogram by the peaks developing slowly.Possible causes of resin/column deterioration and their remediesTemperature the resins have a temperature range that is normally specified by the manufacturers and a commonly high temperature can cause irreversible damage due to loss of functional groups. It is thus important that operation should always within the optimal ranges and bearing in mind the fact that temperature maxima is only for indication. oxidisation The functional groups are also attached by oxidation and on this basis one has to ensure that oxidants such as hydrochloric acid , nitric acid are not utilised in the cleansing regime as they can accelerate oxidation which damages the polymer crosslinkFouling apart from impacting o n performance of the column can also cause irreversible damage to the resin. Fouling can result due to the presence of urge and silica for this reason special attention has to be paid to the type of resin to be used as prevention they say is better than cure.Drying out and cracking of the resin is also an important reason for column deterioration and this can be remedied by ensuring that the column is well equilibrated.High pressure -The build-up can also cause damage to the resin/column and it could be as a result of flow path restriction due to dirty or worn bed support. Manufacturers specification should always be adhered to in ensuring an optimal usage of the resin.The life span of the resin/column should also be taken into account and usage should always be as specified by the manufacturer. irate elution is another factor that is responsible for irreversible damages to resin/column. Every resin has a pH range that is optimal and this should be adhered to strictly.ConclusionThe practical experience was so interesting and brought the protein purification lectures acquire into perspective. A better understanding of the process was developed and the practical knowledge is quite adaptive to the twenty-four hours to day operation in a typical Biopharmaceutical plant.