Plasmid Process Pack 1-1 mL

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CIM HiP² Plasmid Process Pack^TM

1. BEFORE YOU BEGIN

Before using the CIM HiP² Plasmid Process Pack^TM for the first time, read the instructions carefully.

WARNING: Use the product according to guidelines in this Instruction manual. Improper use may result in malfunction, personal injury, or damage of the product or material. Follow safety instructions, wear gloves, safety glasses, and a lab coat during operation.
WARNING: Adjust the pressure relief valve of the system (pump) to the maximum working system pressure of the column (see section 3.1., 3.2.).

2. INTRODUCTION

This high productivity CIM HiP² Plasmid Process Pack^TM is comprised of monolithic columns for fast, simple and robust purification of pharmaceutical grade supercoiled forms of plasmid DNA (pDNA). High productivity is enabled by special monolithic structure and design that ensure high dynamic binding capacity and flow independent properties. These characteristics of the CIM HiP² Plasmid Process Pack^TM assure simple scale up to pilot or production scale.
The chromatographic steps of the pDNA process start with removing impurities from the lysate by using the CIM^® DEAE-1 Tube Monolithic Column. It is followed by an efficient separation of supercoiled and open circular pDNA on the CIM^® C4 HLD-1 Tube Monolithic Column.

About the process (Scheme 1):

Purification process, which does not require ribonuclease treatment, starts with the alkaline lysate which is adjusted to 0.5 - 1.0 M CaCl₂ and, after clarification, loaded onto CIM^® DEAE-1 Tube Monolithic Column. RNA and proteins are separated from the pDNA. DEAE elution fraction is then (after hydrophobic conditions adjustment) further loaded onto the CIM^® C4 HLD-1 Tube Monolithic Column. Supercoiled pDNA is then separated from open circular form, genomic DNA and remaining endotoxins.
UV at 260 and 280 nm and conductivity should be monitored during the purification.

3. COLUMN CHARACTERISTICS

3.1. CIM^® DEAE-1 Tube Monolithic Column  specific information

BASIC CHARACTERISTICS

REGENERATION

To regenerate (to wash out from the monolithic column any ionically bound substances and to reintroduce the correct counter-ion) wash the column with at least 10 column volumes (CV) of a buffer containing 2 M NaCl at one-half of the working flow rate. For best results, this should be done at the end of each chromatographic run.

CLEANING IN PLACE (CIP)

In some cases, the simple regeneration of the monolithic column is not enough. Sample molecules may not completely elute from the column or may even precipitate on the column. This build up of contaminants on the monolithic column may cause loss of resolution and binding capacity, increased back pressure, or a complete blockage of the column. A specific CIP protocol should be designed according to the type of contaminants that are present in your sample. However, in most cases, the following procedures can be used:

1. Removal of precipitated proteins
• Wash with at least 10 CV (10 mL) of a solution containing 1.0 M NaOH/1.0 M NaCl.
Note: Use low enough flow rates to expose the column to the cleaning reagent for several minutes.
• Wash with at least 10 CV (10 mL) of deionized water at the working flow rate.
• Wash with at least 10 CV (10 mL) of a concentrated buffer (e.g. 0.1 to 0.5 M buffer) at the working flow rate in order to restore the appropriate pH.
• Re-equilibrate with at least 10 CV (10 mL) of the working mobile phase (buffer) at the working flow rate.
2. Removal of strongly bound hydrophobic proteins or lipids
• Wash with at least 10 CV (10 mL) of deionized water at one-half of the working flow rate.
• Wash with at least 5 CV (5 mL) of a 2-propanol (30 %) at one-half of the working flow rate.
• Wash with at least 10 CV (10 mL) of the working mobile phase (buffer) at the working flow rate.

SANITIZATION AND STORAGE

Sanitization of the monolithic column is achieved by thoroughly washing it with 1.0 M NaOH for at least 2 hours at room temperature.
CIM^® DEAE-1 Tube Monolithic Column should be stored at 4 °C (39 °F) to 8 °C (46 °F) in the presence of a suitable bacteriostatic agent, e.g. 20 % ethanol.

3.2. CIM^® C4 HLD-1 Tube Monolithic Column specific information

BASIC CHARACTERISTICS

CLEANING IN PLACE (CIP)

The build up of contaminants on the monolithic column may cause loss of  resolution and binding capacity, increased back pressure, or a complete blockage of the column. A specific CIP protocol should be designed according  to the type of contaminants that are present in your sample. However, in most cases, the following procedures can be used:

1. Removal of precipitated protein
• Wash with at least 10 CV (10 mL) of 1.0 M NaOH.
Note: Use low enough flow rates to expose the column to the cleaning reagent for several minutes.
• Wash with at least 10 CV (10 mL) of deionized water at the working flow rate.
• Wash with at least 10 CV (10 mL) of 20 % ethanol at the working flow rate.
• Re-equilibrate with at least 10 CV (10 mL) of the working mobile phase (buffer) at the working flow rate.
2. Removal of strongly bound hydrophobic proteins or lipids
• Wash with at least 10 CV (10 mL) of deionized water at one-half of the working flow rate.
• Wash with at least 10 CV (10 mL) of 2-propanol (30 % v/v) or ethanol (70 % v/v) at one-half of the working flow rate.
• Wash with at least 5 CV (5 mL) of 20 % ethanol at the working flow rate.
• Re-equilibrate with at least 10 CV (10 mL) of the working mobile phase (buffer) at the working flow rate.

SANITIZATION AND STORAGE

Sanitization of the monolithic column is achieved by thoroughly washing it with 1.0 M NaOH for at least 2 hour at room temperature. CIM^® C4 HLD-1 Tube Monolithic Columns should be stored at 4 °C (39 °F) to 8 °C (46 °F) in the presence of a suitable bacteriostatic agent, e.g. 20 % ethanol.

4. SUGGESTED PROTOCOL

4.1. Sample preparations

The plasmid DNA starting material is usually a bacterial lysate obtained by an alkaline lysis procedure. Bacterial cells suspended in 50 mM Tris-HCl, 10 mM EDTA, pH 8.0, are lysed by adding 0.2 M NaOH, 1 % SDS. After lysis, the suspension becomes viscous. Cellular debris and SDS complexes are precipitated by adding 3 M CH₃COOK, pH 5.5, and gently mixed while CaCl₂ is slowly being added to concentration 0.5 - 1 M. Incubate mixture for 15 minutes and clarify it with filtration or centrifugation. 
Note: Add CaCl₂ slowly during stirring to avoid local temperature increase. CaCl₂ can be added in the form of powder or concentrated solution (5 M).
When higher concentrations of RNA or/and genomic DNA are expected final CaCl₂ concentration can be increased up to 1 M. 
Just before applying to the column, lysate filtration through a 0.45 or 0.8 μm filter is strongly recommended. Avoiding this action can clog the columns.

4.2. Capture on AIEX CIM^® DEAE-1 Tube Monolithic Column

Buffers:
Equilibration buffer A1: 50 mM Tris, 10 mM EDTA, pH 7.2
Washing buffer A2: 50 mM Tris, 10 mM EDTA, 0.6 M NaCl, pH 7.2
Elution buffer A3: 50 mM Tris, 10 mM EDTA, 1 M NaCl, pH 7.2
Suggested flow rate: 8-16 mL/min

Method:
1. Dilute bacterial lysate with deionized water to conductivity of 35-40 mS/cm.
Note: Add 3 volumes (V) of deionized water to 1 V of bacterial lysate adjusted to 0.5 M CaCl₂. In case of 0.75 M CaCl₂ add 4 V of deionized water or 5 V in case of 1 M CaCl₂.  
2. Equilibrate CIM^® DEAE-1 Tube Monolithic Column by applying 20 CV of buffer A1.
3. Load cleared diluted bacterial lysate to the column.
Note: max. 6 mg of pDNA can be loaded on CIM^® DEAE-1 Tube Monolithic Column.
4. Wash the column with 20 CV of buffer A1.
5. Wash the column with 20 CV of buffer A2.
6. Elute and collect pDNA with 20 CV of buffer A3 (at half working flow rate).
Note: If pressure increases over specified limit for the columns (Section 2.1.) further reduce flow rate.
7. Perform sanitization after 10 purification runs.

4.3. Separations of supercoiled plasmid DNA on  HIC CIM^® C4 HLD-1 Tube Monolithic Column

Buffers:
Equilibration and washing buffer B1: 50 mM Tris, 10 mM EDTA, 1.7 M (NH₄)₂SO₄, pH 7.2
Elution buffer B2: 50 mM Tris, 10 mM EDTA, 0.4 M (NH₄)₂SO₄, pH 7.2
Regeneration buffer A1: 50 mM Tris, 10 mM EDTA, pH 7.2
Adjustment buffer: 4 M (NH₄)₂SO₄
Suggested flow rate: 8-16 mL/min

Method:
1. For further processing adjust eluate from CIM^® DEAE-1 Tube Monolithic Column containing pDNA by adding 3 V of 4 M (NH₄)₂SO₄ per 1 V of collected pDNA.
2. Equilibrate CIM^® C4 HLD-1 Tube Monolithic Column by applying 20 CV of buffer B1.
3. Load adjusted pDNA fraction eluted from CIM^® DEAE-1 Tube Monolithic Column.
Note: max. 2 mg of pDNA can be loaded on CIM^® C4 HLD -1 Tube Monolithic Column.
4. Wash the column with 20 CV of buffer B1.
5. Elute and collect pDNA with buffer B2.
6. Regenerate the column with 30 CV of buffer A1.
7. Repeat the method two more times.
8. Perform sanitization after 10 purification runs. 
Note: The number of CIM^® C4 steps can be reduced to one, if you need a lesser amount of plasmid. For some plasmids slight modification of (NH₄)₂SO₄ concentration in buffers might be needed.

4.4. Polishing & buffer exchange

Supercoiled pDNA fraction eluted from CIM^® C4 HLD-1 Tube Monolithic Column contains 0.4 M (NH₄)₂SO₄, which must be removed prior to biological application of the plasmid.
Buffer exchange can be performed with e.g. diafiltration or size exclusion chromatography.

5. SANITIZATION AND STORAGE

CIM^® DEAE-1 and CIM^® C4 HLD-1 Tube Monolithic Columns can be sanitized in 1 M NaOH for 2 hours. Sanitization can be performed after each purification run on the column or at least after 5 purifications .
CIM^® DEAE-1 and CIM^® C4 HLD-1 Tube Monolithic Columns should be stored at 4 °C (39 °F) to 8 °C (46 °F) in 20 % ethanol.
Note: Prior to storage in 20 % ethanol adjust the pH of the columns to neutral range.

6. ORDERING INFORMATION

CIM HiP² Plasmid Process Pack^TM 1-1             Catalog No.: 100.0001