Chromatography Technique for Purification

Chromatography Technique for PurificationIntroductionColumn packing is an integral digress of the purification process in the manufacture of biologics. The goal is always to ensure reproducibility with regard to the technique to be utilize. Manual packing might sometimes involve several attempts to get an optimal packing as this would affect the purification process. The rosin to be used in the packing process has to be well defined as it could impact on the melt down rate which could lead to a reduced through put. The mobile phase is also all- pregnant(prenominal) as the rationale behind the choice would be looking for a solvent that send away pack the resin more tightly.In any chromatography technique 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 hard-fought to achieve especially during the polishing stage were impuritie s can be construed as having similar properties to the product. The efficiency of the pillar packing thus has a significant intent to play on this basis as it is a good measure how consistently the column can perform.1.1 Material As per SOP1.2 Key pawn Comp unityntsBubble Trap BT1Filter Housing F1Inlet / Outlet Valves V001 V101UV Sensor QIR4pH Probe QIR3Conductivity Meter QIR2Drain V102Column cabbage 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 instruction 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 % concentration for packingSlurry 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 cubic decimeterCalculationsNumber of theoretical = NWhere VR = volume eluted from the start of sample application to the peak maximum= 8CMW h = peak comprehensiveness measured as the width of the recorded peak at half of the peak height= 0.5CMN = 5.54 X Number of theoretical plates = 1418.24HETP = L/NWhere L = distinguish 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 look on should be at least two to three times the average matrix bead coat and normally in the browse 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 nearly 40 microns which equ ates to 0.004cm and this is about 3 times our HETP. Our column can thus be confirmed to be inside the acceptable 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 exceed the optimal packing flow rateThe possibility of channelling in the bedInadequate CIP can also 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 drizzle the matrix storage solution which is an unwanted entity during packing.Air entrapment preponderance of air bubbles can also affect the HETP values.The possibility of a void being set at the inlet can also be a contributing factor to the value of HETP not being within specificationThe choice of resin is also very important as the possibility of the solute reacting with the resin can result in an ambiguous HETP va lue.Peak asymmetry is an important measure in the determination of column efficiency and in conjunction with the HETP value is always used in the calibration of a new or existing column. The fortunate standard is the ability to achieve an asymmetry value of 1 although the acceptable range is normally between 0.8 and 1.2. An asymmetry value greater than 1 indicates the prevalence of extensive follow 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 reason are the possible causes.Extensive 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 tail ing 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 usually high temperature can cause irreversible defile 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.Oxidation 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 on performance of the column can also cause irreversible damage to the resin. Fouling can result due to the presence of iron 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 exercise 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. virulent 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 serviceable experience was so interesting and brought the protein purification lectures received into perspective. A better understanding of the process was developed and the practical knowledge is quite adaptive to the twenty-four hour period to day operation in a typical Biopharmaceutical plant.