Bio-Rad Media Sampler Pack User Manual

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Macro-Prep®t-Butyl and Methyl Hydrophobic Interaction Chromatography Media

Instruction Manual

Please read these instructions prior to using Macro-Prep hydrophobic interaction chromatography media. If you have any questions or comments regarding these instructions, please contact your local Bio-Rad Laboratories representative.

Table of Contents

Section 1 Introduction ......................................................1

Section 2 Intended Use ....................................................1

Section 3 Media Screening...............................................1

Section 4 Technical Specifications

and Characteristics ..........................................2

Section 5 Preparation for Use ..........................................3

Section 6 Column Packing................................................3

Section 7 Column Packing Evaluation..............................5

Section 8 Operation and Maintenance .............................7

Section 9 Ordering Information ......................................10

Section 1 Introduction

All Bio-Rad products are manufactured under an externally approved ISO 9001 quality system. Bio-Rad Laboratories guarantees product quality and performance of unopened product for 1 year from date of shipment. Regulatory support files are available for Macro-Prep hydrophobic interaction chromatography (HIC) media. If you need assistance validating the use of Macro-Prep media in a production process, contact your local Bio-Rad Process Chromatography representative.

Section 2 Intended Use

Macro-Prep HIC media are designed specifically for intermediate purification steps that remove host-cell contaminants from partially purified targets. Due to their rigidity and unique surface chemistry, these media are particularly suited for HIC operations requiring high throughput and high recovery of target.

Both Macro-Prep t-butyl and Macro-Prep methyl HIC media operate on a mechanism of interaction that is based on hydrophobicity and charge.

The t-butyl and methyl groups are mildly hydrophobic. Depending on the chosen pH of loading and elution buffers, the carboxyl groups can be exploited to ionically repel target molecules while the hydrophobic groups retain contaminants. This is an ideal strategy to minimize product losses commonly experienced with HIC media due to denaturation of proteins when exposed to hydrophobic surfaces.

Section 3 Media Screening

For ease of screening of Bio-Rad HIC media, both forms of Macro-Prep HIC media are provided as prepacked 1 ml and 5 ml cartridges. In addition, these media are part of the media sampler pack, which also includes ion exchangers and ceramic hydroxyapatite media. See Ordering Information in Section 9 at the end of this instruction manual.

With Macro-Prep HIC media it is important to test both salt and pH conditions. Since HIC is usually used after an affinity or ion exchange step, it may be necessary to adjust the salt concentration and/or the pH of the sample load to obtain optimal purification and recovery with Macro-Prep HIC media.

Section 4 Technical Specifications and Characteristics

Macro-Prep t-butyl and Macro-Prep methyl media are based on rigid, epoxyactivated methacrylate beads derivatized with t-butyl or methyl groups, respectively, and carboxyl groups (Figure 1). These products are shipped as a slurry of 50% settled media in 20% ethanol. Table 1 lists the properties of the two media. The rigidity of these media allows high flow rates without bed compression (Figure 2). Testing in small laboratory-scale columns (10 mm in diameter x 100 mm in height) showed no bed compression at a pressure of 7 bar and a flow rate of 40 ml/min (3,000 cm/hr). This property of the media provides for high throughput during loading, column washing, equilibration, and cleaning.

37 75 114 159 195 233 271 313 359 409 458 487

Flow rate (cm/hr)

Pressure (psi)

Pressu re (bar)

Fig. 2. Pressure vs. flow for Macro-Prep media. Media bed dimensions were 140 mm diameter by 250 mm length.

Fig. 1. Macro-Prep HIC: Macro-Prep t-butyl contains t-butyl and carboxyl groups; Macro-Prep methyl contains methyl and carboxyl groups.

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