INTEGRATED CHROMATOGRAPHY

2D/3D Chromatography — Two or Three Steps. One Run

Your batch method is already step one. ChromaCraft™ adds the next — orchestrating multi-column purification (2D/3D) on the Contichrom® CUBE in a single integrated run, with automated transfer, in-line dilution, and full method control across every dimension.

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Two or Three Purification Steps in One Integrated Run.

The moment you run your first batch method on the Contichrom® CUBE, you have the foundation for a 2D purification process. The same hardware, columns, and ChromIQ® software that ran your Protein A capture can route the eluate directly onto a second column — IEX polishing, HIC, desalting — without breaking the run.

The CUBE handles the handoff automatically:

In-line dilution between steps
No hold tank
No manual intervention
No separate method file per column

That’s 2D integrated batch chromatography. Add a third column step and it becomes 3D. The CUBE’s twin-column valve architecture supports both natively, and ChromaCraft™ designs the complete method in the same visual Gantt chart editor you already use for single-column batch.

The result is an automated, end-to-end purification train — capture through polishing to desalting — completed in a single unattended run. Collect one product pool or fractionate as desired. Time from load to final purified fraction drops from days to hours.

What Is 2D Chromatography in Preparative Purification?

2D chromatography combines two chromatographic purification steps in sequence. A selected product-containing portion of the eluate from the first column is transferred directly to a second column, typically using a different and complementary separation mode.

On the Contichrom® CUBE, this is implemented as an integrated batch process. Each dimension follows its own batch chromatography method, while ChromaCraft™ and ChromIQ® control the Product Transfer Window, column-to-column routing, and any required in-line dilution. This makes it possible to connect workflows such as Protein A capture followed by ion exchange polishing or anion exchange followed by reversed-phase purification without an intermediate hold step or manual reloading.

Batch & Multi-Column Processes on the Contichrom® CUBE:

Process format	Number of purification steps	Transfer between columns	Example workflow	Method control
Single-column batch	1	No inter-column transfer	Protein A capture	One single-column batch method
2D integrated batch	2	Selected product window transferred automatically from Dimension 1 to Dimension 2	Protein A → IEX or AEX → RP	One integrated ChromIQ® method
3D integrated batch	3	Automated sequential transfer across three column steps	Capture → polishing → desalting	One multi-step ChromIQ® method

The 2D and 3D configurations use the same ChromaCraft™ Batch Editor as single-column batch methods. A third dimension requires the additional column position enabled by the Column Selection Valve Kit.

Integrated 2D Chromatography vs Separate Batch Steps

Process factor	Separate batch purification steps	Integrated 2D chromatography on the CUBE
Product transfer	The first product pool is collected and subsequently loaded onto another column	The selected product window is transferred automatically
Intermediate handling	Operator intervention and an intermediate container are normally needed	No manual transfer or intermediate hold tank
Stream conditioning	Salt concentration, pH, or solvent composition may require offline adjustment	The pump network applies the defined in-line dilution
Method execution	Each column is commonly run as a separate method	Both dimensions run within one ChromIQ® method
Data review	Each purification step produces a separate run record	Both dimensions appear in one continuous Evaluation Center record
Fraction collection	Intermediate and final pools may need to be collected separately	Product collection can take place after the final dimension

Unlike separate batch steps, integrated 2D chromatography connects both purification dimensions within one automated workflow. On the Contichrom® CUBE, the selected product window from Dimension 1 can be transferred, conditioned through in-line dilution, and processed in Dimension 2 without manual reloading or an intermediate hold step. This reduces handling between stages while keeping method execution and data evaluation within one continuous process record.

How an Integrated 2D/3D Workflow Works.

The CUBE’s valve network connects the outlet of the first column directly to the inlet of the second. When the Dimension 1 UV trace meets the Product Transfer Window you set in ChromaCraft™, ChromIQ® diverts the eluate to Dimension 2 and applies in-line dilution at the ratio you specify.

The transfer window can be triggered two ways:

Absolute mAU threshold — fires at a fixed UV value (peak front marker)
Relative percentage of peak maximum — fires at a fraction of the observed peak height (peak tail marker)

The pumps adjust automatically. No manual action between steps.

Dimension 2 then runs its own equilibration, bind, wash, and elution sequence, governed by the method parameters defined for that column. Both steps run within the same ChromIQ® method.

For 3D methods, add a third column position using the Column Selection Valve Kit — a full capture → polishing → desalting train in one automated run.

Method Design with ChromaCraft™

Step 1 — Same editor, extended for two or three dimensions

2D and 3D methods are built in the same ChromaCraft™ Batch Editor used for single-column batch — no new software, no new interface to learn. The Gantt chart extends to show all dimensions in sequence. If both dimensions share a pump at any point, ChromaCraft™ immediately flags the conflict as a red zone — before you generate the method, not after the first failed run.

Step 2 — Choose process type and import your existing method

Select 2D or 3D mode and assign each dimension’s buffers to the appropriate CUBE pump inlets. Any previously saved single-column ChromaCraft™ method can be uploaded directly into Dimension 1, 2 or 3 — all column parameters and step timings are imported. Your existing batch protocol becomes a new dimension instantly.

Step 3 — Configure the Product Transfer Window

Define UV triggers to start and stop column-to-column product transfer. ChromaCraft™ automatically selects a trigger for absolute mAU (peak front marker) or relative % of peak maximum (peak tail marker) depending on product transfer start and end locations. Next – set the in-line dilution factor — ChromIQ® automatically applies it during product transfer to condition the eluate for re-binding to the column downstream. An optional delay time is set to prevent early-eluting species from triggering the transfer prematurely.

Step 4 — Set fractionation triggers for the final dimension

Define product collection criteria for Dimension 2 (or 3) — UV threshold, volume/time window, or a combination. Deactivating “Continue after End” cuts the Dimension 1 elution gradient the moment the transfer window closes, skipping the unnecessary remainder and going straight to regeneration — a meaningful cycle time saving on every run.

Step 5 — Handle special cases and auto-generate

For immiscible solvent combinations (e.g., RP organic → IEX aqueous), ChromaCraft™ automatically inserts a water purge step at the pump — no manual intervention needed. Hit Save: the complete multi-step ChromIQ® method is generated as a single ready-to-run file, with all dimensions, all transfer parameters, and fractionation logic compiled in.

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Analysing Results: The Evaluation Center

Both dimensions of a 2D or 3D run are captured in a single, continuous run record in the Evaluation Center. The UV trace spans the complete method — you see the Dimension 1 elution, the transfer point, and the Dimension 2 (and Dimension 3) chromatogram as a single timeline.

Peak integration — automatic or manual, applied independently to each dimension’s elution profile
Fraction analysis — final collected fractions mapped against the full run trace; purity and yield assessed in one view
Run comparison — overlay multiple 2D/3D runs to confirm reproducibility of both the transfer timing and the final product peak
PDF report export — full structured report spanning all dimensions, transfer parameters included
Excel / CSV export — complete signal data and integration results across the entire multi-step run

The ChromIQ® logbook records every system event — including the Product Transfer Window trigger — in an unalterable audit trail with data integrity checksums and multi-level user rights management.

Multi-Column Applications

Molecule class	Typical 2D/3D configuration
Monoclonal antibodies (mAbs)	2D: Protein A capture → CIEX polishing; 3D: Protein A → CIEX → desalting — full downstream train, single run
ADCs	2D: Protein A capture → HIC (DAR fractionation); or HIC → IEX polishing
Recombinant proteins / hormones	2D: His-Tag IMAC → IEX polishing; 3D: Protein L → IEX → SEC
Peptides	2D: RP polishing (Dim 1) → AIEX polishing (Dim 2) — orthogonal gradient-gradient; or RP → desalting
Oligonucleotides	2D: AEX gradient → RP polishing; orthogonal selectivity, single automated run
AAV / gene therapy	2D: Affinity capture → IEX polishing
mRNA / LNP	2D: Affinity → IEX at appropriate scale
Nutraceuticals / small molecules	2D: RP → mixed-mode or normal-phase polishing

Supported step combinations on the Contichrom® CUBE:

Format	Step 1	Step 2	Step 3	Required accessories
2D	Isocratic (e.g. Protein A, IMAC)	Isocratic (e.g. SEC, desalting)	—	Column Bypass Valve Kit
2D	Isocratic (e.g. Protein A)	Gradient (e.g. CIEX, AEX, HIC)	—	Column Bypass Valve Kit
2D	Gradient (e.g. RP)	Gradient (e.g. AIEX)	—	Column Bypass Valve Kit + Buffer Selection Valve Kit
3D	Isocratic	Gradient	Isocratic (e.g. desalting)	Column Bypass + Column Selection Valve Kits
3D	Isocratic	Gradient	Gradient	Column Bypass + Buffer Selection + Column Selection Valve Kits

Contichrom® Systems for 2D Integrated Batch and Continuous Chromatography

All Contichrom® Systems Perform 2D Integrated Batch Chromatography in Addition to Continuous Chromatography, With Predictable Scalability from CUBE to PILOT and TWIN System Lines. 3D capabilities are exclusive to the CUBE.

System Overview

$Contichrom CUBE multi-column FPLC chromatography system with UV detectors and FOXY fraction collector$

Contichrom® CUBE

The essential platform for chromatography process development. This versatile 100-bar benchtop FPLC system runs batch, integrated batch, and continuous chromatography — including MCSGP and CaptureSMB — out of the box. Develop purification processes for mAbs, peptides, oligonucleotides, ADCs, and viral vectors, with verified scale-up at over 100× to pilot and production systems.

Contichrom® PILOT

Scale peptide and oligonucleotide purification to pilot and clinical GMP manufacturing. This 100-bar preparative HPLC system features MCSGP with AutoPeak® to eliminate the yield-purity trade-off — delivering +10–50% absolute yield increase at target purity and significantly less solvent than batch HPLC. ATEX Zone 2 / Class I Div 2 certified for organic solvents. Flow rates up to 300 mL/min.

Contichrom TWIN preparative HPLC chromatography system

Contichrom® TWIN HPLC

Produce peptides and oligonucleotides at commercial GMP scale. This 80-bar industrial HPLC system uses MCSGP with AutoPeak® to eliminate the yield-purity trade-off — increasing recovery to >90% while reducing solvent consumption compared to batch. ATEX Zone 2 / Class I Div 2 certified for organic solvents, and flow rates up to 36 L/min.

Contichrom TWIN LPLC continuous chromatography system for capture and polishing

Contichrom® TWIN LPLC – Capture

Accelerate biopharmaceutical capture chromatography at production scale. This sanitary GMP system uses CaptureSMB® with AutomAb® to maximize resin utilization — achieving up to 3× faster processing and 50% less Protein A resin and buffer consumption compared to batch. CIP-ready design for mAbs, viral vectors, ADCs, and recombinant proteins.

Contichrom® TWIN LPLC – Polishing

Bring continuous polishing to biopharmaceutical manufacturing at production scale. This sanitary GMP system uses MCSGP with AutoPeak® to eliminate the yield-purity trade-off achieving +10–50% absolute yield increase at target purity while reducing buffer consumption compared to batch. CIP-ready design for mAbs, viral vectors, ADCs, and recombinant proteins.

System Overview

Platform Advantages at a Glance

Advantage	What it means in practice
No hold tanks between steps	Eluate transfers column-to-column automatically — no intermediate container, no manual handling, no wait
In-line dilution built in	Ionic strength or pH adjustment between dimensions is handled by the pump network — no separate dilution step
Single product pool	Fraction collection happens once, at the end of the final dimension — not after every step
Reduced QC burden	One run record, one chromatogram, one report — instead of three per downstream step
Same hardware as batch	No additional system purchase — 2D/3D runs on the CUBE you already have, with accessory valve kits
Direct scale-up path	2D methods transfer directly to the Contichrom® PILOT 300X and TWIN systems

How Does Preparative 2D Chromatography Work?

In preparative 2D chromatography, the outlet of the first column is connected directly to a second purification column. On the Contichrom® CUBE, ChromIQ® transfers the selected eluate when the Dimension 1 UV signal meets the Product Transfer Window defined in ChromaCraft™. The system can apply the specified in-line dilution during transfer before Dimension 2 performs its own binding, washing and elution steps within the same automated method.

Is preparative 2D chromatography the same as comprehensive 2D-LC?

No. Comprehensive analytical 2D-LC normally transfers or repeatedly samples the first-dimension effluent into a rapid second-dimension separation to increase analytical peak capacity.

The Contichrom® process is an integrated preparative workflow. It transfers a defined product window from the first purification column to a second preparative column to continue product purification without an intermediate hold or manual reload.

What hardware accessories are needed to run 2D or 3D on the Contichrom® CUBE?

All 2D configurations require the Column Bypass Valve Kit, which allows the system to flush or sanitise between dimensions without dismounting columns. Gradient-to-gradient 2D methods additionally require the Buffer Selection Valve Kit for Gradient Pump 1A & 1B, which enables independent eluent selection for each dimension’s gradient pump. 3D methods require all of the above plus the Column Selection Valve Kit, which enables the third column position. All accessories are fully integrated into ChromaCraft™ and ChromIQ® as standard — no additional configuration required.

Does the CUBE need new columns or resins for 2D/3D?

No. The columns and resins used in your existing batch method are the starting point. The same Protein A column you use for batch capture becomes Dimension 1 in a 2D process. Your existing IEX or RP polishing column becomes Dimension 2. No new column procurement is required to configure a 2D method.

What is the Product Transfer Window and how does it work?

The Product Transfer Window is a ChromaCraft™ step configuration that defines the criteria for transferring the eluate from Dimension 1 to Dimension 2. You set a UV absorbance threshold — when the Dimension 1 UV trace crosses that threshold during elution, ChromIQ® diverts the eluate to Dimension 2. In-line dilution is applied simultaneously at the defined ratio. The transfer is fully automated — no manual action is required at the switchover point.

Does the eluate from Dimension 1 need to be diluted before loading onto Dimension 2?

Often yes — particularly for isocratic-to-gradient transfers where the Dimension 1 elution buffer (e.g., low-pH Protein A elution buffer) is not compatible with optimal binding conditions on Dimension 2 (e.g., IEX). The CUBE’s pump network handles this in-line, applying the dilution you define in the Product Transfer Window. You set the ratio; the system does the rest.

Can I still run single-column batch and continuous processes on the same CUBE after adding 2D/3D accessory valve kits?

Yes. The accessory valve kits expand capability without removing any existing functionality. The CUBE with a Column Bypass Valve Kit runs all standard 1D batch modes (isocratic, gradient, alternating batch), all 2D integrated batch configurations, and all continuous processes (CaptureSMB, MCSGP, N-Rich) on the same hardware. Process mode is selected in ChromaCraft™ or the relevant wizard — the hardware configuration handles the rest.

How does 2D/3D integrated batch relate to MCSGP or CaptureSMB?

They are complementary but operate differently, and importantly, a single Contichrom® CUBE can run either a twin-column continuous process (CaptureSMB, MCSGP, or N-Rich) or a 2D/3D integrated batch process — not both simultaneously. Full downstream integration combining, for example, a CaptureSMB capture step with a 2D polishing-polishing train, or a CaptureSMB capture step with MCSGP polishing, is achieved using two CUBE systems operating in series — with viral inactivation or another required hold step between them. This architecture delivers the benefits of both continuous capture and multi-step automated polishing across a complete downstream train.

Can the 2D/3D method be transferred to larger systems?

Yes for 2D, no for 3D. 2D methods developed on the Contichrom® CUBE transfer directly to the Contichrom® PILOT 300X (up to 300 mL/min) and TWIN LPLC and HPLC production systems. The same ChromIQ® method logic, Product Transfer Window parameters, and in-line dilution ratios apply at larger column scale — no method redevelopment required. 3D methods are not natively supported on the larger scale systems, though customized solutions are possible – please enquire.

One System. Two or Three Purification Steps.

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