Presentation on theme: "Design of RP-HPLC 1 for Insulin Production"— Presentation transcript:
1 Design of RP-HPLC 1 for Insulin Production Presented by:Patricia Yee, Nicholas Irianto, Thomas Fang
2 Overview Background Information Significance of RP-HPLC1 Design ParametersSelected Design and SpecificationsQuestions
3 Background Information What is HPLC?High Performance Liquid Chromatography- or -High-Pressure Liquid ChromatographyPat-used to separate components of a mixture based on chemical interactionsPhoto Credit:
4 How HPLC works… Pat -generally separate analytes by polarity “Like attracts Like”-column is packed with a stationary phase, which usually consists of small round particles with a selected surface chemistry-a liquid (the mobile phase) is pumped through the column under high pressure while the sample to be analyzed and separated is injected in smaller amounts-the velocity of the components in the analyte are slowed down by different degrees due to the chemical/physical interactions taking place between the analyte, the stationary and mobile phases, which have different chemical propertiesPicture References:
5 Reverse-Phase HPLC Pat -reversed phase chromatography -in comparison to normal phase chromatography (NP-HPLC), which uses polar stationary phase and non-polar mobile phase, RP-HPLC uses non-polar stationary and polar mobile phase-RP-HPLC gives better reproducibility of retention times-“like attracts like” therefore if the analyte is non-polar: the more non-polar the stationary phase or the more polar the mobile phase, the longer the retention time
6 Insulin Production Process: RP-HPLC 1 Pat-after reactor and before diafilter-components in mixture that are not desired in end product (e.g. enzymes, denature and contaminated proteins), therefore, take them out and keep what you want with RP-HPLC-mobile phase: acetonitrile/water/acid (water-miscible organic acid and other acids) used to control the retention times-the more water, the more polar the mobile phase, and the more acetonitrile, the less polar the mobile phasePicture References:
7 Insulin Production Process: RP-HPLC 1 What comes out…1) Contaminated protein, denatured insulin, enzyme and small amounts of insulin ester and WFI2) Majority of desired insulin ester and WFIPat-acetonitrile/acid/contaminated protein/denatured insulin/enzyme/and small amounts of insulin ester and WFI (water) get eluted first-then the majority of the desired insulin ester and WFI are eluted last
8 Insulin Production Process: RP-HPLC 1 Gradient Elution of mobile phase:Acetonitrile / Water / AcidPat-achieved through gradient elution: different ratios of acetonitrile/water/acid put through the column-stationary phase polarity basically fixed-but can change the percentage of acetonitrilemore acetonitrile: the more non-polar the wash, and non-polar components eluted earliermore water: the more polar the wash, and non-polar components eluted later-these ratios can be tweaked to optimize separation and processing time-the gradient concentrations and the times it changes can be automated
9 Main Design Parameters ColumnSize (Internal diameter)Material (Stainless Steels, Glass, etc)Column size - column that has a larger internal diameter has a larger loading capacity (the most common ID is 4-6mm these columns are called “analytical scale columns” for industrial scale or manufacturing purposes, the internal diameter is usually larger than mm)Column material - Usually made from stainless steels, due to the durability. We can also see glass columns for lab scale usage
10 Main Design Parameters ResinParticle size (10μm ~75μm )Pore size (300 Á)Material (Silica, styrene-divinylbenzene, etc.)Smaller particle size - more effective at accommodating separation of the insulin polypeptide (larger surface area for adsorption.) but can cause problems with the fluid flow through the column (need a bigger pressure)Larger particle size - Easier to pack, lower pressure drop through the column.Pore size - The pore size must be large enough for the polypeptide to properly adsorb to the resin, most polypeptide separations are performed on columns with particles with pores of about 300 A (Carr D., 2002)
11 Other Design Considerations Pump (pressure capacity)SolventAcetonitrilePolar solvent (increase retention time)Hydrophobic solvent (decrease retention time)Pump - pumps vary in pressure capacity, modern HPLC systems has been improved to work at higher pressure, Pressure may reach as high as atm. So therefore resin that has a smaller particle can be used in the column to get a better separation.Retention time - The retention time is longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily.The retention time can be increased by adding more polar solvent to the mobile phase, and decreased by addition of hydrophobic solvent.
12 Possible DesignThis shows the possible type of HPLC that can be used for insulin purification
13 Design Specifications 100% binding and 90% yield for insulin ester60% binding and 5% yield for denatured insulin precursorsPurification results in outlet stream consisting of 340 kg insulin ester/batchThe following specs were used when running the simulation in SuperPro. The expected insulin ester production rate is 340 kg insulin ester/batch. Designing for length and diameter in SuperPro, gives out the column volume and number of units required to do the purification step. We kept other parameters as default values since we were unable to find these parameters from vendors.Playing around with the column length and max diameter would affect the required column volume and number of units. Since SuperPro determined the column volume based on the number of cycles per batch and the specified column length and maximum diameter. If the calculated diameter goes above the maximum diameter, this means that an additional RP-HPLC working in parallel configuration would be required to do the separation.
14 Design Selections HPLC Columns Novasep Prochrom® DAC Columns LC1600 H x D: 450 x 1600 mmHPLC ResinsPRP-3 standard Poly(styrene-divinylbenzene) (PSDVB)Pore size 300 ÅDiameter 10 μmAfter contacting several vendors, we were able to obtain information from the hamilton company. This company specifically suggested that PRP-3 resins would be an ideal resin to be used for insulin purification primarily due to the molecular weight of insulin itself (5800 MW)In order to meet the process specification of producing 188 kg insulin/batch, 23 RP-HPLC units with maximum bed volume of 900 L are required. We were unable to find out the exact resin binding capacity from the Hamilton Company. Similar issues were faced when trying to determine the number of cycles per batch, resin replacement frequency and linear flowrate. Therefore, we decided to keep these values to SuperPro default values.Notice that assuming that the batch requires 60 hours of operation with 36 hours turnover time between two batches, if we are operating 7920 hours in a year, we would make roughly 82 batches which reflects to about kg insulin crystal/year. This exceeds the operational target of 4000 kg insulin crystal/year.According to the PRP-3 Resins manufacturing, 1 gram of the PRP-3 resin is applicable for 2.5 cm3 of the column volumes. Thus for a 900 L column volumes, we would require 360 kg of PRP-3 resins. Cost from the Hamilton Company is $24/g PRP-3 resin. This would translate to $ per column. For 21 columns, $10 microns were selected because it would provide a better separation a greater peak. PRP-3 are 300 Angstorms pores polymer materials.
15 Design Justifications Stable from pH 1-13Does not contain acidic silanol groups, which are present on silica supports.Highly cross-linked for exceptional durability and to prevent shrinking andConsistent performance10μm particle diameter is selected for better solution and peak shapeswelling.Compatible with 0-100% organic or aqueous solvents. Resins are highly cross-linked for exceptional durability and to prevent the resin from shrinking and swelling, which often occurs in other polymeric supportsThis eliminates irreversible, sample absorption on the support and thus greatly improves sample recovery and bioactivity. PRP columns are capable of providing sample recoveries of 95%Notice that assuming that the batch requires 60 hours of operation with 36 hours turnover time between two batches, if we are operating 7920 hours in a year, we would make roughly 82 batches which reflects to about kg insulin crystal/year. This exceeds the operational target of 4000 kg insulin crystal/year.
16 Design DetailsTwenty three LC 1600 HPLC column units with 0.91 hours process time would be required for the process according to SuperProFor one LC 1600 HPLC column with maximum bed volume of 900 L would require grams of PRP-3 resinsCOSTS$24/g of PRP-3 resinsThis translates to $ / LC 1600 HPLC columnFor 23 HPLC columns, it would cost $$ for a Novasep Prochrom DAC LC 1600 columnTotal Cost $Assuming that 1 cycle per batch with resin binding capacity of 20 mg/ml and 200 cycles resin replacement frequency (default values from SuperPro)From Hamilton vendor, 1 gram of resin is approximately equivalent to 2.5 cc of column volumesResins replacement frequency and beads replacement turnaround time cannot be found from vendor. Therefore we used SuperPro default valuesCost for column is obtained from linear interpolation based on volumeNotice that assuming that the batch requires 60 hours of operation with 36 hours turnover time between two batches, if we are operating 7920 hours in a year, we would make roughly 82 batches which reflects to about kg insulin crystal/year. This exceeds the operational target of 4000 kg insulin crystal/year.