INTEGRATED CHROMATOGRAPHY

Two or Three Steps. One Run. 2D/3D Integrated Batch.

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

One System. Two or Three Purification Steps.

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.

How It 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.

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

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.

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.

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

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

 

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.

One System. Two or Three Purification Steps.

Frequently Asked Questions

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