This section describes purification of monoclonal antibodies with HiTrap® MabSelect SuRe. The protocol includes a CIP procedure to minimize cross-contamination from different antibodies between purification runs, as well as for general column cleaning after a purification run.
Figure 1 shows capture of a human monoclonal antibody by affinity chromatography using HiTrap® MabSelect SuRe. SDS-PAGE of pooled, eluted fractions shows the high purity of the human MAb obtained in a single-step purification (SDS gel, lane 3).
Figure 1.(A) One-step puriﬁcation of a human monoclonal antibody on HiTrap® MabSelect SuRe 1 mL on AKTAexplorer 100. (B) SDS-PAGE analysis on ExcelGel SDS Gradient 8-18, reducing conditions.
Refer to Desalting and Buffer Exchange for general considerations.
Centrifuge samples (10 000 × g for 10 min) to remove cells and debris. Filter through a 0.45 µM ﬁlter.
IgG from many species has a medium to strong afﬁnity for protein A at physiological pH. Sample pH should be between 6 and 9 before applying to the column. If required, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column or dilution and pH adjustment.
Binding buffer: 0.02 M sodium phosphate, 0.15 M sodium chloride, pH 7.2
Elution buffer: 0.1 M sodium citrate, pH 3.0 to 3.6
Neutralizing buffer: 1 M Tris-HCl, pH 9.0
Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 µM ﬁlter before use.
General instructions for puriﬁcation using HiTrap® columns
To preserve the activity of acid-labile IgG, we recommend adding 60 to 200 µL of 1 M Tris-HCl pH 9.0 to collection tubes, which ensures that the ﬁnal pH of the sample will be approximately neutral.
* 1 mL/min corresponds to approximately 30 drops/min when using a syringe with a 1 mL HiTrap® column; 5 mL/min corresponds to approximately 120 drops/min when using a 5 mL HiTrap® column.
When optimizing elution conditions, determine the highest pH that allows efﬁcient elution of antibody from the column. This will prevent denaturing sensitive antibodies due to exposure to low pH.
Stepwise elution allows the target antibody to be eluted in a more concentrated form, thus decreasing buffer consumption and shortening cycle times. It might be necessary to decrease the ﬂow rate due to the high concentrations of protein in the eluted pool.
Desalt and/or transfer puriﬁed IgG fractions to a suitable buffer using a desalting column (Desalting and buffer exchange).
Reuse of HiTrap® columns depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.
CIP can be performed to remove very tightly bound, precipitated, or denatured substances from the medium. If such contaminants are allowed to accumulate, they can affect the chromatographic properties of the column, reducing capacity and potentially contaminating subsequent puriﬁcation runs. If the fouling is severe, it can block the column, increase back pressure, and reduce ﬂow rate.
Regular CIP prevents the build-up of contaminants and helps to maintain the capacity, ﬂow properties, and general performance of HiTrap® MabSelect SuRe. When an increase in backpressure is seen, the column should be cleaned. We recommend performing a blank run, including CIP, before the ﬁrst puriﬁcation is started to wash out possible trace amounts of leached protein A.
Store in 20% ethanol at 2 °C to 8 °C.