Protein Sample Preparation and Analysis Application Manual - Purification Applications

Select a link below to access Protocols for Mustang Ion Exchange Membranes.

• Purification on an Acrodisc® Unit with Mustang Q Membrane (4.1.2)
• Purification on an Acrodisc Unit with Mustang S Membrane (4.1.3)

Introduction

In many areas, chromatography resins are the media of choice for chromatography applications. In some instances, where resin-based methods have limitations (e.g., purification of viruses or large molecules), membranes have proven to be a robust, scalable, and economical alternative to resins. Chromatography membranes offer faster flow rates as compared to their resin counterparts.

For ion exchange applications (see table below), Pall offers Mustang ion exchange membrane. This technology is available in several formats including a syringe filter column and a 96-well filter plate.

Ion Exchange Applications

Type of Ion Exchanger	Common Abbreviation	Functional Group	Product Name	Description
Strong Anion	Q	Quarternary Ammonium	Acrodisc Unit with Mustang Q AcroPrep™ Filter Plate with Mustang Q	Syringe filter column 96-well filter plate (350 µL or 1 mL)
Strong Cation	S	Sulfonic Acid	Acrodisc Unit with Mustang S AcroPrep Filter Plate with Mustang S	Syringe filter column 96-well filter plate (350 µL or 1 mL)

Select a link below to access Protocols for BioSepra Chromatography Resins.

• Protein Purification Using Ceramic HyperD® Ion Exchange Resin (4.2.2)
• Protein Purification Using Blue Trisacryl® M Resin (4.2.3)
• Using IMAC HyperCel™ Resin for Immobilized Metal Ion Affinity Chromatography (4.2.4)
• Protein Purification Using HA Ultrogel® Resin (4.2.5)
• Using MEP HyperCel Resin for Purification of Antibodies by Hydrophobic Charge Induction Chromatography (HCIC) (4.2.6)
• Protein Purification Using Heparin HyperD Resin (4.2.7)
• Protein Purification Using Lysine HyperD Resin (4.2.8)
• Protein Purification Using Ultrogel AcA Size Exclusion Chromatography Resin (4.2.9)

Introduction

Chromatography continues to be an essential technology for the purification of biomolecules. Pall offers a line of chromatography resin ideal for protein purification applications (see table below). This broad line of chromatography products exhibits superior performance and is useful for affinity, ion exchange, size exclusion, and hydrophobic interaction chromatography (HIC). Unique mixed-mode BioSepra products also exist to provide solutions to current sample preparation challenges.

The resins Pall offers for small-scale applications are the same ones offered to our customers currently manufacturing biopharmaceuticals. The ability to scale up is essential for those working in drug discovery, development, and manufacturing. These resins can be used in varying size chromatography columns, as well as in batch mode for single or high throughput mode. This is ideal for quick preps or in situations where optimizing purification conditions is required.

Chromatography Resins

Chromatography Type	Product Name	Description
Ion Exchange	Q Ceramic HyperD S Ceramic HyperD CM Ceramic HyperD DEAE Ceramic HyperD	Strong anionic exchanger, binds negatively-charged target Strong cationic exchanger, binds positively-charged target Weak cationic exchanger Weak anionic exchanger
Affinity	Protein A Ceramic HyperD   Blue Trisacryl Heparin HyperD Lysine HyperD IMAC HyperCel SDR HyperD	Binds IgG Binds albumin Direct binding to targets that have an affinity for heparin Direct binding to targets that have an affinity for lysine Binds tagged proteins using an immobilized metal compound Detergent removal
Mixed Mode	MEP HyperCel HA Ultrogel	Uses several binding mechanisms including hydrophobic interactions Hydroxyapatite
HCIC	MEP HyperCel	Uses several binding mechanisms including hydrophobic interactions
Size Exclusion	Ultrogel AcA	Separates targets by size

Available Separation Columns

Description	Column Volume	Available from Pall
Glass Chromatography Column	Varies	No
Disposable Chromatography Column	Varies	No
Spin Filter	< 1 mL	Yes
Deep Well Multi-Well Filter Plate	96 x < 1 mL	Yes
Multi-Well Filter Plate	96 x < 350 µL	Yes

Select a link below to access Protocols for Rapid Purification Development/Scouting (Process Proteomics).

• Screening of Chromatographic Resin in an AcroPrep™ Multi-Well Plate Format (4.3.2)
• Column Scale-Up to Pilot Process Applications (4.3.3)

Introduction

Chromatography continues to be an essential technology for the purification of biomolecules. Pall offers a line of chromatography resins ideal for protein purification applications. This broad line of chromatography products exhibits superior performance and is useful for affinity, ion exchange, size exclusion, and hydrophobic interaction chromatography (HIC). Unique mixed-mode BioSepra products also exist to provide solutions to current sample preparation challenges.

The resins Pall offers for small-scale applications are the same ones offered to our customers currently manufacturing biopharmaceuticals. The ability to scale up is essential for those working in drug discovery, development, and manufacturing. These resins can be used in varying size chromatography columns, as well as in batch mode for single or high throughput mode. This is ideal for quick preps or in situations where optimizing purification conditions is required.

Pall also offers recommendations and services for streamlining initial scouting of protein purification conditions through our Process Proteomic Service Centers. The identification and optimization of protein purification parameters can be a tedious task. Process proteomic methodologies can be used to streamline the initial scouting of protein purification conditions, as well as for some of the optimization steps. Process proteomics is performed using common chromatographic chemistries (e.g., anion exchange, cation exchange, IMAC) on a protein chip, in the wells of a multi-well filter plate, or using small columns. Using this approach, multiple binding, washing, and elution conditions can be tested on your sample simultaneously. Successful scale-up from these small-scale experiments to traditional chromatography columns has proven to be quite useful.

Select a link below to access Protocols for Buffer Exchange, Desalting, and Concentration.

• Buffer Exchange, Desalting, and Concentration Using Microsep™ UF Spin Filter for Samples (1-3.5 mL) (4.4.2)
• Buffer Exchange, Desalting, and Concentration Using Macrosep® UF Spin Filter for Samples (3.5-15 mL) (4.4.3)
• Buffer Exchange, Desalting, and Concentration Using Jumbosep™ UF Spin Filter for Samples (15-60 mL) (4.4.4)

Introduction

Biomolecule purification involves a complex series of steps where targets are selectively separated through a series of manipulations. The process by which separation is performed often creates a need for the sample to be desalted or concentrated to prepare the biomolecule sample for the next step in the purification process.

Pall offers several technologies to perform efficient sample concentration, desalting, and buffer exchange including ultrafiltration-based spin filters, ultrafiltration-based multi-well filtration plates, and bottled gel filtration media. Refer to the table below for a selection of ultrafiltration-based products available.

Ultrafiltration Products for Concentration and Desalting

Device	Sample Volume
AcroPrep 384 filter plate	< 100 µL
AcroPrep 96 filter plate	< 350 µL
Nanosep device	< 0.5 mL
Microsep device	0.5-3.5 mL
Macrosep device	3-15 mL
Jumbosep device	15-60 mL

Compared to other methods, ultrafiltration-based methods for concentration and desalting offer a number of advantages, including:

• Reduction of processing steps–desalting and concentration take place in a single step.
• Consistency–the use of ultrafiltration-based products eliminates problems with gel beds.
• Versatility–ultrafiltration is useful in both low and high throughput modes.

Pall also offers chromatography resins for desalting applications, including: Ultrogel AcA 202 and Trisacryl GF-05 resins. Refer to the below table for general properties.

Properties of Ultrogel AcA and Trisacryl GF-05 Resin Specifications

Specification	Ultrogel AcA 202 Resin	Trisacryl GF-05 M Resin
Particle Size	60-140 µm	40-80 µm
Monomer	20% (w/v) Acrylamide	N-acryloyl-2-amino-2-hydroxymethyl-1, 3-propanediol
Cross-linker	2% (w/v) Agarose	Hydroxylated acrylic bifunctional monomer
Exclusion Limit	22,000	3,000
Linear Fractionation Range	1,000-15,000	200-2,500
Resolving Power (plates/m)	3,000	2,500
Working pH Range	3-10	1-11

Centrifugal Spin Filters

Select a link below to access Protocols for Final Product Clarification.

• Clarification of Samples (< 1 mL) by MF Filtration in Nanosep® Spin Filters (4.5.2)
• Filter Sterilization of Samples (1-100 mL) in Sterile Acrodisc Syringe Filters (4.5.3)

Introduction
Although a basic filtration concept, the clarification and prefiltration of samples remains an important function within protein sample preparation applications. When filtration is used as a prefilter, matching the proper filter media and device to the application is critical. Large pore size filter materials are used to filter solutions prior to more detailed analysis. When selecting the best product for your application, several factors need to be considered. Sample viscosity, sample volume, and sample recovery are just some of the aspects that will drive the selection of the optimal device.

Pall offers a number of media and device options for fast, effective filtration with minimal sample hold-up for both single and high throughput sample processing. From sample volumes of a few microliters to multiple liters, Pall can supply the best product solution for your application.

Choose the Best Clarification Device for Your Needs

Acrodisc Syringe Filters for Analytical Sample Preparation