PhyNexus has developed a unique technology for the micro scale purification of engineered proteins and antibodies which allow researchers to routinely purify and enrich sample volumes up to 1 mL. The exclusive design of the PhyTip™ columns allow for elution volumes as low as 10μL, thus producing enrichment factors as high as 50 x, with concentrations of purified protein of up to 5 mg/ml.

The process to purify and enrich is a simple three step technique where the protein of interest is first captured, then purified and finally enriched. The entire process can take less than 15 min to produce high concentrations of fully functional protein ready for further analysis.

Protein A PhyTip Columns demonstrate high binding affinity and high capacity affinity purification of functional antibodies (IgGs).

  • 70-90% IgG recovery
  • Obtain highly concentrated purified proteins
  • Protein A PhyTip columns are shown to extract IgG proteins with very high purity from varying levels of background proteins
Description

PhyTip columns with Protein A have been optimized for use with specific PhyNexus reagents and instrument flow rates/volumes as shown below. This information was collected using the PhyNexus Purification Systems. All PhyTip columns with Protein A are supplied with recommended PhyTip buffers including: Capture Buffer – provided for those situations where additional buffer needs to be added to supplement the volume of the sample and to ensure correct pH for capture Wash Buffer I – Phosphate Buffer solution pH 7.4 Wash Buffer II – Saline solution. NOTE: no buffering capacity so as to ensure effective elution Enrichment Buffer – for the final elution step – Phosphate Buffer solution pH 2.5 Neutralization Buffer. – Tris Buffer solution pH 9.0 Note: Enrichment buffer is supplied as 4 mL of a pH 2.5 Phosphate buffer solution, if protein to be purified requires less acidic elution conditions e.g. pH 2.8, the enrichment buffer pH can be changed as follows: Take 1 mL of standard Enrichment Buffer (pH 2.5) and add 30 μL of 1 M Tris Buffer standard Neutralization buffer to obtain 1 mL of pH 2.8 elution buffer (actual pH may vary depending upon volumetric accuracy) For a pH 3.0 enrichment buffer, take 1 mL of standard Enrichment Buffer (pH 2.5) and add 40 μL of 1 M Tris Buffer standard Neutralization Buffer to obtain 1 mL of pH 3.0 elution buffer (actual pH may vary depending upon volumetric accuracy) For the neutralization step add 25% v/v of the elution volume e.g. if the elution volume is 20 μL add 5 μL of 1 M Tris Neutralization Buffer.

200+ PhyTip columns with Protein A resin:

For a 200 μL sample with 5 μg IgG2a (anti-FITC MAb.) containing 1 mg BSA, processed using the conditions shown below, greater than 40% of the original IgG mass is recovered in the final sample volume. In addition, the recovered IgG is over 95% pure as determined by SDS-PAGE with Coomassie detection.

Capture: 200 μL sample captured by passing through the resin bed for four cycles at a flow rate of 250 μL per minute.

Purify: 200 μL of PhyNexus Protein A Wash Buffer I, passed over the resin bed for two cycles at a flow rate of 500 μL/min followed by a second wash with Wash Buffer II, passed over the resin bed for two cycles at a flow rate of 500 μL/min.

Enrich: elute the protein into solution with 10 μL of PhyNexus Protein A Enrichment Buffer, passed over the resin bed for five cycles at a flow rate of 500 μL/min. Neutralize with 3 μL of PhyNexus Protein A Neutralization Buffer.

Protein G Enrichment Buffer as shipped contains: 111mM NaH2PO4, 140mM NaCl in 14.8mM H3PO4, pH 3.0

1000+ PhyTip columns with Protein A resin:

For a 500 μL sample with 10μg IgG2a (anti-FITC MAb.) containing 5 mg BSA, processed using the conditions shown below, greater than 40% of the original IgG mass is recovered in the final sample volume. In addition, the recovered IgG is over 95% pure as determined by SDS-PAGE with Coomassie detection.

Capture: 500μL sample captured by passing through the resin bed for four cycles at a flow rate of 250 μL per minute.

Purify: 1000μL of PhyNexus Protein G Wash Buffer I, passed over the resin bed for two cycles at a flow rate 500 μL/min followed by a second wash with Wash Buffer II, passed over the resin bed for two cycles at a flow rate 500 μL/min.

Enrich: elute the protein into solution with 15mL of PhyNexus Protein A Enrichment Buffer, passed over the resin bed for five cycles at a flow rate of 500 μL/min. Neutralize with 5 μL of PhyNexus Protein A Neutralization Buffer.

Application

Performance Characteristics and Assay Procedures for Protein A PhyTip® Columns

Introduction

The PhyNexus PhyTip® columns are innovative purification tools that radically simplify the capture, purification and enrichment of proteins from a variety of sources. Key to the success of these purification tools is the design of the mechanism to retain the affinity resin bed, with minimum dead volume and maximum capture potential. One group of PhyTipcolumns incorporates Protein A to specifically bind antibody (IgG) from different sources under certain optimal conditions, thus allowing nucleic acids and other contaminants to be removed. Following a rapid wash step, purified antibodies are easily eluted with commonly used low pH buffer. This technique allows for exceptionally high yields of IgG, depending on various conditions and provides for highly selective purification in the presence of very high concentrations of carrier proteins like BSA. PhyTip columns have extremely high binding capacity that can bind greater than 100 μg IgG, and can efficiently recover as little as 200 ng of IgG – representing a range of nearly three orders of magnitude. PhyTip columns are ideal for antibody screening from hybridoma supernatants or for high throughput extraction and purification of affinity tagged proteins.

Performance and Assay Procedures

IgG binds protein A with high affinity and specificity. The strength and selectivity of this interaction enables protein A to effectively purify IgGs from complex protein mixtures. To examine the performance of PhyTip columns with Protein A resin, the percent recovery of purified IgG (an anti-FITC-MAb) using 1000+ Protein A PhyTip columns was measured.

Materials and Methods

Sample: 10 μg mFITC-MAb (IgG2a subclass) in 0.5 ml of
1. PBS containing 5 mg BSA, or
2. DMEM containing 10% FBS (Fetal Bovine Serum), or
3. SFM (Serum Free Medium) containing 5 mg BSA
Sample processing: All samples were processed on the semi-automated ME 1000 platform using the following protocols:

  1. (1)  Capture: Capture the specific protein by passing the sample over the resin bed with 2 in/out cycles (volume programmed for 0.6 ml @ 0.25 ml/min).
  2. (2)  Purify: Remove unbound proteins by washing the bound protein/affinity resin using 1 in/out cycle (volume programmed at 0.6 ml @ 0.5 ml/min) with 0.5 mL PBS (Wash Buffer I) followed by 1 in/out cycle (volume programmed at 0.6 ml @ 0.5 ml/min) with 0.5 mL of saline solution (Wash Buffer II).
  3. (3)  Enrich: Elute the IgG2a with 4 in/out cycles (volume programmed at 0.1 – 0.15 ml @ 1 ml/min) with 15 μL of pH 3.0 elution buffer. Once eluted, 5 μL of neutralization buffer was added. Note that current elution buffer supplied by PhyNexus with Protein A and Protein G PhyTip columns has a pH of 2.5 and that the current neutralization buffer is at pH 9.0.

Quantitation procedure

(1) To prepare reduced IgG2a, 15 μl of final elution volume was reacted with 5 μl of freshly prepared TCEP (15 μl of 10 mg/ml TCEP in water for a final volume of 30 μl).

(2) 20 μl of reduced IgG2a was injected into a non-porous polystyrene divinylbenzene reverse phase (C-18) column using an HP 1050 HPLC system. A gradient of 25% to 75% between solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN) was used for 5 minutes. Detection: UV at 214 and 280 nm.

  1. (3)  T wo major IgG2a peaks eluted around 3. 18 and 3. 34 min. Areas under these two peaks were integrated from (3. 13 – 3.5) min in each case and corresponding peak area was recorded at 214 nm.
  2. (4)  TCEP-treated IgG2a standard under identical reaction condition was loaded into the column and used as an input or standard for recovery calculation.

Results of IgG2a Recovery

Recovery in DMEM + 10% FBS = 77% Recovery in SFM + 5 mg BSA = 64%.

It is known that carrier proteins like albumin induce an immune response and help in the production of good antibodies, so it is often present in high quantities when antibodies are being raised. For that reason, any purification and enrichment procedure must be capable of tolerating high concentrations of albumin. In addition, the results shown above seem to indicate that the presence of high background protein concentrations (such as BSA) may have a beneficial effect on recoveries.

Reproducibility of IgG2a recoveries in presence of carrier protein

In order to verify the role of carrier protein for maximum IgG2a recovery, and to demonstrate reproducibility, IgG2a was purified from PBS (and also PBS containing 5 mg BSA) from multiple samples (n=4). Results suggest increased recovery (at least 20% or more) of purified IgG2a in the presence of 500-fold excess of BSA concentration and that the procedure is also highly reproducible.

Recovery in PBS = 42.25% (SD = 4.66%) Recovery in PBS + 5 mg BSA = 66.5% (SD = 7.5%)

Selectivity Performance and Assay Procedures

In order to characterize and verify the selectivity of the Protein A-IgG affinity interaction, 5 mg of BSA was added as a carrier protein to 15 μg IgG in a 0.5 ml sample volume (a 333-fold excess). The samples were processed using Protein A PhyTip columns.

Materials and Methods

Sample: 15 μg mFITC-MAb (IgG2a subclass) in 0.5 ml of PBS or PBS containing 5 mg BSA (10 mg/ml or 1% w/v BSA). Sample processing: As described above in Recovery Performance and Assay Procedures.

IgG quantitation and purity analysis procedures:

(1) 15 μl of final elution volume was divided into two parts: 13 μl was reacted with freshly prepared 13 μl of 10 mg/ml TCEP (final volume = 26 μl and [TCEP] = 17.5 mM) at room temperature for ~16 hours while 2 μl was loaded onto gel in each experiment.

(2) 20 μl out of above 26 μl reduced IgG2a was injected into a non-porous polystyrene divinylbenzene reverse phase (C-18) column using an HP 1050 HPLC system. A gradient of 25% to 75% between solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN) was used for 5 minutes. Detection: UV at 214 and 280 nm.

  1. (3)  Two major IgG2a peaks eluted around 3.17 and 3.3 min. The area under these two peaks was integrated from (3.13 – 3.5) min in each case and corresponding mAU was recorded at 214 nm.
  2. (4)  TCEP-treated IgG2a standards (injected amount 1. 08, 2. 16, 4. 32, 6. 48 and 8. 64 μg of FI TC-M Ab) under identical reaction condition were loaded into the column and used as a standard curve for recovery calculation.
Data

PhyNexus recommends the use of the PhyTip Starter Kit containing IgG as a standard to verify the use of PhyTip columns with all applications.

Samples for purification and enrichment must be clear and free from particulate matter. It is highly recommended to centrifuge samples and use the clear supernatant only, prior to use with PhyTip columns.

Shipping and Storage

Each pack of PhyTip columns has been manufactured and QC’d to the highest standards and shipped in retainer boxes that maintain the integrity of the specific affinity resin within each PhyTip column. This product is shipped at ambient temperatures, but on receipt should be stored in a standard laboratory refrigerator between 4 and 8◦C.

  • Do NOT freeze or store frozen.
  • When not in use, keep the lid of the box closed and sealed, store in the refrigerator.
  • Do not allow affinity resin to dry out by extended storage in a dry environment.

PhyTip columns with Protein A are stored in Glycerol when shipped from PhyNexus.

Documents
Robotics Systems Compatibility

Protein A PhyTip Columns are available for these robotic systems

Part Numbers

Protein A PhyTip Columns