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- and or contact

PRODUCTION:

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orders@monoliths.com

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tech-support@monoliths.com

pDNA DOWNSTREAM PROCESSINGUSING CIM® MONOLITHS

CIM HiP2 Plasmid Process Pack™ is a perfect pDNA purifica-

and one C4 HLD column and is available in 1 mL or 8 mL sizes. Upon request, scale-up versions and cGMP Columns are available.

CIM HiP² Plasmid Process Pack™

Chemistry: weak anion exchanger; diethylamino Ligand density: 2.3±0.2 mmol/g dry support Support matrix: poly(glycidyl methacrylate -co- ethylene dimethacrylate)

Dynamic binding capacity:Working flow rates:

up to 9 mg pDNA/mL wet support (from crude bacterial lysate) • 1 mL column: max. 16 mL/min (625 cm/h)

• 8 mL column: max. 200 mL/min (670 cm/h) Maximum pressure:

• 1 mL column: max. 18 bar (1.8 MPa);• 8 mL column: max. 20 bar (2.0 MPa)

Temperature stability: 4 °C (39 °F) to 50 °C (122 °F)

Recommended pH: working range 3-9cleaning in place 2-14

Tube chemistry: butyl Ligand density: 3.2 mmol/g dry support Support matrix: poly(butyl methacrylate -co- ethylene dimethacrylate)

Dynamic binding capacity:Working flow rates:

up to 3 mg pDNA/mL wet support (from crude bacterial lysate) • 1 mL column: max. 16 mL/min (625 cm/h)

• 8 mL column: max. 200 mL/min (670 cm/h) Maximum pressure:

• 1 mL column: max. 18 bar (1.8 MPa);• 8 mL column: max. 20 bar (2.0 MPa)

Temperature stability: 4 °C (39 °F) to 50 °C (122 °F)

Recommended pH: working range 1-12cleaning in place 1-14

DEAE

C4 HLD

pDNA DOWNSTREAM PROCESSING USING MONOLITHS

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Monoliths’ convective interaction offer:- Extremely high binding capacity for pDNA

• DEAE up to 9 mg/mL;• C4 HLD up to 3 mg/mL)

- Accelerated process development - Increased manufacturing capacity- Preserved biological activity (low shear forces)

SPECIALLY DESIGNED FOR pDNA PURIFICATION.CIM® Monolithic Columns

CIM® 0.34 mL CIM® 1 mL CIM® 8 mL CIM® 80 mL CIM® 8000 mLCIM® 800 mL

Large Scale Production

Recommended flow rates:1 - 8 mL/min

CIMac™ pDNA 0.3 mLRecommended flow rates:0.2 - 2 mL/min

Recommended flow rates:1 - 16 mL/min

Recommended flow rates:10 - 200 mL/min

Recommended flow rates:40 - 250 mL/min

Recommended flow rates:400 - 2000 mL/min

Recommended flow rates:2000 - 10000 mL/min

Screening/Method DevelopmentAnalytical Scale

Preparative Scaleup to 48 g of pDNA

up to 4.8 g of pDNAup to 48 mg of pDNA

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Figure 1: pDNA Purification Process

E. coli culture with plasmid

Cell harvest

Alkaline lysis with adjustmentto 0.5 - 1.0 M CaCl2

Clarification

Adjustment to bindingconditions

CIM® DEAE

Adjustment with (NH4)2SO4

CIM® C4 HLD

Buffer exchange

Conditions: Column – 1 mL CIM® DEAE; Flow rate – 16 mL/min ; Buffer A1 – 10 mM EDTA, 50 mM Tris, pH 7.2; Buffer A2 – 0.6 M NaCl in buffer A1; Buffer A3 – 1 M NaCl in buffer A1; Buffer A4 –

2 M NaCl in buffer A1; UV detection – 260 nm

Conditions: Column - 1 mL CIM® C4 HLD; Flow rate – 16 mL/min; Buffer B1 – 1.7 M (NH4)2SO4 in buffer A1; Buffer B2 – 0.4 M

(NH4)2SO4 in buffer A1; Buffer A1 – 10 mM EDTA, 50 mM Tris, pH 7.2; UV detection – 260 nm

CAPTURE STEP ON CIM® DEAE-1 mL COLUMN POLISHING STEP ON CIM® C4 HLD-1 mL COLUMN

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New product timelines are getting constantly shorter creating pressure on all stages of new product development. To cope with these tight deadlines, researchers need a quick development tool to fully optimize their method in a fraction of the time ensuring a quick transition to full scale production. CIM® Monolithic columns are such a tool. The resolution of our columns enables researcher to separate the final product (sc)pDNA from (oc)pDNA and the rest of the impurities in just a fraction of the time, leaving more time for process optimization.

Research

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Developing a pDNA production process (Figure 1) is the simplest and quickest by using the CIM HiP² Plasmid Process Pack™ (see the back of the brochure). The process pack is equipped with a 2-step pDNA purification protocol with yields in excess of 80% (sc)pDNA. This flexible protocol can then be easily scaled carrying over the excellent yields, purity, and economics from the laboratory to production.

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Process yield > 80 %pDNA concentration < 400 µg/mLA260/280 1.93Time 33 min/mg pDNABuffer consumption 66 mL/mg pDNAIsolated pDNA 6 mgHomogeneity (SC pDNA) > 97 %Host cell DNA - removed > 99.5 %Host cell proteins - removed > 99%Endotoxin < 2 EU/mg pDNARNA - removed > 99 %Note: at the end buffer exchange is needed

Figure 5: Scale up options

Column size pDNA purified1 mL 6 mg8 mL 48 mg80 mL 480 mg800 mL 4.8 g8 L 48 g

Figure 4: Process results Figure 2 shows the complete purification process on CIM HiP2 Plasmid Process Pack™ with results that exceed FDA/EMEA require-ments (Figure 4). A single run on our 8 L units can produce 48 g of pharmaceutical grade (sc) pDNA (Figure 5). The protocol can be adjusted to meet your sample requirements or you can chose to implement either of the purification steps (HIC or AIEX) as a replacement for a parti-cle based purification or polishing step.

Figure 2: Downstream processing with the CIM HiP² Plasmid Process Pack™

Absorbance Conductivity

¹ F. Smrekar, A. Podgornik, M. Ciringer, S. Kontrec, P. Raspor, A. Štrancar, M. Peterka. 2010. Preparation of pharmaceutical-grade plasmid DNA using methacrylate monolithic columns.

Figure 3: Efficient separation of all three conformations of pDNA and gDNA on a CIM® C4 HLD Monolithic Column¹

pDNA DOWNSTREAM PROCESSING USING MONOLITHS

Closing the Loop on pDNA ProductionWould you like your entire pDNA production process to be based upon an easily scalable technology that will yield the highest capacities of supercoiled (sc) pDNA in the industry? Can you imag-ine getting your gene therapy or DNA Vaccine product to market years ahead of the competition? Using CIM® Monoliths in your Downstream Process (DSP) can give you that competitive edge.

CIM® Monoliths cover R&D, Analytical, and Production Scales for any pDNA production process. You are truly able to close the loop on your process. You can produce the highly pure supercoiled (sc) form of pDNA and ensure high batch to batch reproducibility by implementing real-time on-line monitoring of the different steps in

the production process. In addition, the high dynamic binding capacities (up to 9 mg/mL) and high flow rates of CIM® Monoliths yield high productivity that is unrivalled by traditional chromatographic methods. CIM® Monoliths provide high returns by being more productive at a lower cost than traditional methods and are better suited to meet the increasing

demands for improved purity of gene therapy products. Flexibility is the key element that we built into the process. The DEAE and HIC columns can be used from lab to production, but the DEAE column can be used alone on the lab scale or if the purity requirements are lower. For analysis, CIMac™ pDNA Analytical columns are available.

Below we explore how CIM® Monolithic Columns are already accel-erating pDNA production for gene therapy and the creation of lifesaving treatments.

POLISHING&

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E. coliWITH PLASMID

HARVEST & ADJUSTMENT STEPS

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FORMULATION & FILLINGDOWNSTREAM PROCESSING

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UPSTREAMPROCESSING

pDNA DOWNSTREAM PROCESSING USING MONOLITHS

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BIA Separations, an ISO-9000 certified company.

Purification Process DevelopmentBIA Separations’ Contract Research Laboratory has expertise in virus, pDNA, and monoclonal antibody downstream purifi-cation process development. Our team will deliver a robust and efficient purification process which will meet your company’s and local regulator’s requirements.Please feel free to contact us at sales@biaseparations.com should you wish to avail yourselves of our services. By taking advantage of CIM® monoliths, we are able to rapidly develop processes in our Biosafety Level 2 laboratories.

Meeting Regulatory DemandsTo meet the strictest regulatory demands, BIA Separations produces cGMP compliant Columns (stainless steel or dispos-able) that meet the strictest regulatory demands of agencies worldwide. It is very easy to move from method development to pilot and full scale cGMP production as CIM® monoliths have an identical performance profile regardless of scale. We currently have Drug Master Files for our QA, DEAE, and SO3 chemistries and will obtain others upon request.

To learn how CIM® Monoliths can help you purify your specific pDNA sample contact us at tech-support@monoliths.com.

Quality Control

Figure 7: The use of CIMac™ pDNA Analytical Column for in-process control

Column: CIMac™ pDNA Analytical Column (5.2 mm I.D. x 15.0 mm)

Injection volume: 20 µL Mobile phase A: Buffer A: 200 mM Tris, pH 8.0 Mobile phase B: Buffer B: 200 mM Tris, 1 M NaCl, pH 8.0

Detection: UV at 260 nm Flow rate: 1 mL/minHPLC system: A high pressure gradient HPLC system, Agilent 1200

Sample: Alkaline lysate of plasmid pEGFP-N1 (4.7 kbp) after adjust-ment to 0.5 - 1.0 M CaCl₂ (A.) was diluted 1:3 with water and filtered 0.45 µm prior analysis. Eluate of CIM® DEAE (B.) was diluted 1:3 with water, whereas eluate of CIM® C4 (C.) was directly injected onto CIMac™ pDNA Analytical Column

Each in-process control check is completed in less than 10 minutes (Figure 7) enabling the comparison of real-time data during the production of your gene therapy or vaccine product; saving time and expensive wastage. Additionally, the CIMac™ pDNA Analytical Column enables the separation of (oc) and (sc) pDNA conformations (Figure 7 A,B) and can monitor the removal of other impurities such as RNA.

The following chart demonstrates the advantages of monoliths over traditional particle based media for pDNA production. Monoliths shave off time, consume almost 30% less buffer, and consequently production costs 65% less per gram of pDNA produced. No matter how you look at it, mono-liths outperform particle supports.

CIMac™ pDNA Analytical Columns ensure that each production step (measured on-line and in real-time) meets your quality control standards. Each batch will have the highest level of supercoiled pDNA, the minimum amount of impurities, and an efficient, highly reproducible, and cost effective gene therapy/vaccine production run.

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Figure 6: Process economics

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Traditional kit Vs. Monolith based pDNA pack [%]