Chromatography Scaling Strategies
Goals of the Step
At the main capture phase of purification, the primary goals are to isolate the target product as quickly as possible from the crude or clarified feedstock to minimize its exposure to any destructive proteases present, to reduce the process volume to a manageable scale for subsequent steps, and to maximize yield and economy. Affinity chromatography using a highly selective ligand combined with a support optimized for maximal throughput and productivity can effectively achieve these goals.Subsequent purification and polishing steps further remove other impurities including DNA, viruses and endotoxins to achieve specified product purity and high recovery. Media selectivity and capacity are important at the intermediate stage moving to very high resolution, high recovery and low leakage characterizing the final polishing stage.
Methods and Protocols
- PROSEP - A High Capacity: the Throughput and Productivity Leader for Monoclonal Antibody Capture -- TB1010EN00
- PROSEP - A High Capacity and PROSEP-rA High Capacity Operating Instructions -- OIBP113118
- Determination of Dynamic Binding Capacity for ProSep-vA Media -- TB1175EN00
- PAB – An Enhanced Sanitization Option for ProSep® Chromatography Media -- TB0011EN00
- Cleaning ProSep-A Protein A Affinity Chromatography Media: Recommendations for minimizing or eliminating non-specific binding and for cleaning ProSep-A media -- TB1021EN00
- Increasing Purity on ProSep-vA Affinity Chromatography Media using an Intermediate Wash Step -- TB1026EN00
- Determination of Dynamic Binding Capacity for ProSep-vA Media -- TB1175EN00
- ProSep-vA Ultra Media 300 Cycle Lifetime Study -- TB1650EN00
Scale-Up Strategies
Once optimum conditions for product purity and yield have been determined through small-scale column experiments the results must now be related to the manufacture of larger columns for larger production processes. The product yield and quality must remain the same throughout scale-up. The sample loading volume should be directly related to the column size, so as to reduce sample wastage.The capture efficiency of a process column relates the quantity of sample applied to the yield of product produced. The capture efficiency, in turn, is directly related to the column residence time. Keeping the column residence time constant will maintain the same product yield when the same number of column volumes of sample is applied to a larger column. Also, a constant residence time ensures that the process cycle time is constant throughout scale-up.
The easiest way to achieve this is to maintain the same bed height, but increase the column diameter. The same linear flow rate will produce a constant residence time (See Table 1). However, this should not be the only method taken into consideration. Too large a column diameter can produce unstable column beds and will require good fluid distribution systems on the column inlet. Also, protein chromatography columns are produced to vary the column height per diameter. Changing only the column diameter during scale-up means the purchase of a significant number of different columns.
Table 1: Scaling up keeping the residence time constant, changing the column diameter and maintaining a constant linear flow rate
| Diameter (cm) | Height (cm) | Column Volume (ml) | Residence Time (min) | Flow Rate (cm/min) | Flow Rate (ml/min) |
2 | 10 | 31.4 | 5 | 2 | 6.28 |
5 | 10 | 196.25 | 5 | 2 | 3.92 x 101 |
10 | 10 | 785 | 5 | 2 | 1.57 x 102 |
Some process engineers recommend maintaining a constant aspect ratio (column diameter to column height). To retain the same residence time, the linear flow rate needs to be increased (see Table 2).
Table 2: Scaling up, keeping the residence time constant, changing the linear flow rate and keeping the aspect ratio constant
| Diameter (cm) | Height (cm) | Column Volume (ml) | Residence Time (min) | Flow Rate (cm/min) | Flow Rate (ml/min) |
2 | 10 | 31 | 5 | 2 | 6.28 |
5 | 25 | 490 | 5 | 2 | 9.81 x 101 |
10 | 50 | 3925 | 5 | 2 | 7.85 x 102 |
Scaling-up column sizes will necessitate using a combination of these methods as illustrated in Table 1 and Table 2. The scale-up of a column system from 30ml to 100 litres would probably proceed as illustrated in Table 3. PROSEP ® materials have a low backpressure in columns, even with low aspect ratios (long columns). This makes the scale-up process easier and more flexible.
Table 3: A summary of the column diameters and heights used to scale up from 30ml to 100 litres
| Diameter (cm) | Height (cm) | Column Volume (ml) | Residence Time (min) | Flow Rate (cm/min) | Flow Rate (ml/min) |
2 | 10 | 0.03 | 5 | 2 | 6.28 |
10 | 10 | 0.7 | 5 | 2 | 1.57 x 101 |
10 | 50 | 4 | 5 | 10 | 7.85 x 102 |
50 | 50 | 100 | 5 | 10 | 19.60 x 103 |
Scalable Chromatography Systems
The K-Prime systems are advanced, scalable, pre-engineered purification systems that make transferring separations from small to large scale fast, easy, reliable and economical.
These systems are highly suited to a wide range of biopharmaceutical applications using various chromatography media, including: Affinity Chromatography, Ion Exchange Chromatography, and Size Exclusion Chromatography.
The performance of Millipore’s Common Control Platform® (CCP®) operating system provides biopharmaceutical companies the ability to control all of their separation processes from a single robust software platform. Powered by iFIX® software, CCP is capable of controlling and linking Millipore’s biochromatography and TFF solutions.
K-Prime systems are designed to operate in cGMP facilities for the purification of licensed products, and are designed in accordance with GAMP and FDA 21 CFR Part 11 for electronic records and signatures. <
K-Prime 40-1
Smallest of the systems. Ideal for products undergoing process optimization prior to clinical trial evaluations, requiring flow rates from 20 to 500 mL/minute.K-Prime 40-II
Intermediate scale system enables rapid transfer of separation recipes from development to clinical trial evaluations. Ideal for products requiring flow rates from 0.1 to 3 liters/minute.K-Prime 40-III
Moderate scale system that facilitates regulatory approval of new drug products and are ready for small-scale production. Ideal for products requiring flow rates from 1 to 10 liters/minute.
