TWIN-COLUMN ENRICHMENT

Continuous Enrichment Chromatography with N-Rich®

Automated Trace Impurity Isolation. Accumulate what’s barely there — cyclic enrichment for reference standard preparation, charge variant isolation, and ICH Q3A/Q6B characterization: 100–1,000× Enrichment factor | 80–95% Purity overnight | fully automated via ChromIQ® N-Rich® Wizard.

Sources: Bigelow et al. (BMS), J. Chromatogr. A 2021; Weldon & Müller-Späth, J. Chromatogr. A 2022

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N-Rich Tutorial Paper

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N-Rich Tutorial Paper

The Isolation Bottleneck

Regulatory agencies require pure, isolated impurity reference standards — not just detected peaks. ICH Q3A and Q6B mandate structural identification and toxicological qualification of trace impurities and product-related variants. The problem is that these compounds exist at 0.1–5% of the feed, co-eluting with a dominant main peak that is 10–1000× more abundant.

Conventional batch preparative HPLC has no mechanism to address this ratio. Hundreds of injections, progressive pooling of dilute fractions, weeks of processing time — and at the end of it, material that may still be too impure to use as a reference standard. Custom synthesis costs tens of thousands of dollars per compound and is simply impossible for biologic impurities such as charge variants, glycoforms, or partially filled capsids that only exist as product-related species.

N-Rich® Changes What’s Possible

N-Rich® does not try to isolate a trace compound in a single pass. Instead, it accumulates it on-column across many cycles — progressively building concentration in the system until enough pure material is available to collect in a single, fine-fractionated elution.

Each enrichment cycle adds more of the target to the column while simultaneously removing the dominant main compound to waste. After enough cycles, the target has been concentrated to the point where it can be eluted and fractionated with purity that would be impossible at the original abundance. The process runs fully automated and unattended — overnight, or over multiple days for very low-abundance targets.

The results across validated applications are consistent:

95% purity

mAb charge variants in 10h vs. 300h by HPLC (BMS)

3 days

vs. 30+ days biosimilar mAb, 9 isoforms

79×

Productivity gain peptide impurity isolation

69×

Solvent reduction vs. analytical HPLC

Sources: Bigelow et al. (BMS), J. Chromatogr. A 2021 · Weldon & Müller-Späth, J. Chromatogr. A 2022

N-Rich® is a fundamentally different tool from MCSGP. MCSGP purifies a main product by removing flanking impurities — its starting material is already relatively pure. N-Rich® accumulates toward a compound that starts at trace abundance and may never be resolvable in a single-column run. Both use the same twin-column platform and similar ChromIQ® wizard structure; the problem they solve is different.

How N-Rich® Works…. Part 1

N-Rich® runs as a single uninterrupted procedure through four sequential stages:

Stage	What happens	Duration
Design	Batch chromatogram loaded, recycling boundaries set, method generated	One-time setup
Enrichment	n cycles of cyclic accumulation — target compound builds on-column, main compound removed each cycle	n cycles until sufficient feed is processed
Depletion	One switch without fresh feed — remaining main compound removed, target concentrated before elution	1 switch
Fractionated Elution	Shallow gradient across both columns in series — target eluted and fine-fractionated for collection	Single elution event

N-Rich® Process Principle (.pptx)

How N-Rich® Works…. Part 2

Within each enrichment cycle, two switches alternate. Each switch consists of four phases running on the upstream (eluting) and downstream (regenerating/loading) columns in parallel:

Phase	Upstream column	Downstream column
P1 – Non-target elution (Parallel)	Weakly adsorbing non-targets eluted to waste	Regenerated and re-equilibrated
P2 – Weak (W) recycling (Interconnected)	Weakly adsorbing target compounds transferred with in-line dilution to downstream column	Receives W fraction — target re-adsorbs
P3 – Main compound removal (Parallel)	Non-target (main) compound eluted to waste	Loaded with fresh feed — more target enters system
P4 – Strong (S) recycling (Interconnected)	Strongly adsorbing target compounds transferred with in-line dilution to downstream column	Receives S fraction — target re-adsorbs

How N-Rich® Works…. Part 3

At the end of each switch, the two columns swap positions by valve switching and the cycle repeats. The feed volume per cycle is calculated automatically by the N-Rich Wizard from the batch chromatogram.

The Depletion stage (one switch, no fresh feed) transfers any remaining enriched material from the upstream to the downstream column before elution, minimizing non-target compound in the final fractions and improving final purity.

The Fractionated Elution uses a shallow gradient run across both columns in series — at half the normal flow rate with a flatter gradient than the batch method — to maximize resolution between enriched compounds. This is where the concentrated, pre-enriched target material is collected as fine fractions.

Method Design with the N-Rich® Wizard

N-Rich® methods are designed in the N-Rich® Wizard in ChromIQ® 9 — a tab-based interface that takes a single-column batch chromatogram from the Contichrom® CUBE and guides you through to a complete, ready-to-run method. As with MCSGP, the batch chromatogram is the essential input from the CUBE.

Step 1 — Load the chromatogram and define recycling boundaries

Load the single-column batch design run from the CUBE into the Batch Chromatogram tab. Drag five boundary markers (t1–t5) to define the five zones: load/wash region, weak recycling zone (W), main compound zone (P), strong recycling zone (S), and strip region. These boundaries define exactly which parts of the chromatogram are recycled to enrich the target compounds and which are sent to waste in each cycle.

Step 2 — Configure washing, regeneration, and Product Removal Control

Set washing and regeneration steps in the Washing & Regeneration tab — the same protocol as your existing batch process. In the “AutoPeak®” tab, activate the Product Removal Control if needed. When activated, the start of main-compound removal is UV-triggered rather than time-based — Front Control (absolute mAU threshold) if the removal boundary falls on the ascending peak slope, Tail Control (% of peak maximum) if on the descending slope. The stop of removal remains time-based.

Step 3 — Set up the final fractionation

The Final Fractionation tab defines the shallow gradient elution and fraction collection for the Fractionated Elution stage. The “Automatic Computation of Elution Conditions” toggle (ON by default) calculates elution parameters automatically from the batch chromatogram — half the normal flow rate, shallower gradient, doubled gradient length across both columns in series. Adjust fraction size and start/stop times. The preview shows the expected final elution profile on two columns in series.

Step 4 — Set number of cycles and generate

In the Method Settings & Performance tab, set the number of enrichment cycles and review switch and cycle durations. Hit Save: The wizard generates the method files and a procedure file that runs them in the correct sequence. Load the procedure in ChromIQ® and run. The process runs fully unattended from here.

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AutoPeak® — Product Removal Control

N-Rich’s AutoPeak® implementation is distinct from MCSGP’s. In MCSGP, AutoPeak® controls up to four fraction collection boundaries around a main product peak. In N-Rich®, AutoPeak® controls a single transition: when to start removing the main compound during the enrichment phase.

During each switch, the main (non-target) compound must be actively diverted to waste at the right moment to prevent it from accumulating. With AutoPeak® off, this diversion happens at fixed times t3 and t4 derived from the batch chromatogram. Fixed timing is appropriate during early method development with stable, well-characterised conditions.

With AutoPeak® on, the start of the removal is UV-triggered. If the removal boundary (t3) falls on the ascending slope of the main compound peak, removal is triggered by an absolute mAU threshold (Front Control). If t3 falls on the descending slope, removal is triggered by a relative threshold as a percentage of the peak maximum (Tail Control). In both cases, removal stops after a fixed time window (t4 − t3) — the stop is time-based, not UV-triggered.

A Threshold Detection Window acts as a safety margin: if the UV trigger is not reached within this extended window, the process freezes before the next phase rather than proceeding blindly — ensuring accumulated material is never compromised by a missed trigger during unattended multi-day runs.

Verifying Results: The Evaluation Center

Every N-Rich® run is captured in the Evaluation Center — ChromIQ®’s integrated data analysis module.

Cycle Overlay — Superimpose all enrichment cycles in one view. The target compound UV signal should grow progressively taller across cycles while the main compound signal diminishes — visual confirmation that accumulation is proceeding as designed
Auto Integration — Quantitatively tracks the enriched fraction areas across every cycle, confirming the rate of accumulation and documenting when the enrichment plateau has been reached
Superimpose — Overlay the batch design chromatogram against the final Fractionated Elution output — the side-by-side comparison shows directly how much the target compound has been enriched relative to the main compound
PDF and Excel export — Per-cycle chromatograms, Auto Integration results, and fractionation tables compiled into a structured report

Platform Advantages at a Glance

Advantage	What it means in practice
100–1,000× enrichment factor	Trace compounds at 0.1–5% of feed concentrated to high purity in a single automated run
Up to 80× faster than analytical HPLC	Hours or days instead of weeks — overnight operation for many applications
3 days vs. 30+ days	Biosimilar mAb isoform isolation: complete in 3 days, 50 analytical samples vs. >600
95% purity — 30× faster than HPLC	mAb charge variants: 95% purity in 10 hours vs. 300 hours by analytical HPLC
Up to 69× solvent reduction	Same enrichment result with dramatically lower mobile phase consumption and waste
Same resin and buffers	No change to stationary phase or mobile phase conditions — your existing batch conditions are the starting point
Fully unattended	Procedure runs overnight or over multiple days without operator supervision — AutoPeak® maintains consistent depletion
Scale-up path	Methods developed on the Contichrom® CUBE transfer to the Contichrom® PILOT 300X and TWIN HPLC for higher throughput

N-Rich®Applications

N-Rich® is applicable wherever a target compound exists at low abundance relative to a dominant co-eluting species and cannot be isolated to useful purity or quantity by conventional batch methods.

Molecule class	Typical target	Use case
Monoclonal antibodies	Acidic and basic charge variants (typically 5–25% of feed)	Reference standards for forced degradation studies, biosimilar comparability, potency assay development
Biosimilar mAbs	Multiple isoforms from 1.5% to 56% of feed	Complete isoform panel in 3 days — all main isoforms of a biosimilar mAb isolated simultaneously
Antibody-drug conjugates (ADCs)	DAR 1 – 7 species from DAR distribution	Reference material for individual DAR-species PK/PD and toxicology characterization
Peptides	Deletion sequences, oxidized variants, closely eluting process impurities	ICH qualification standards — >50× productivity increase and >50× solvent reduction vs. analytical HPLC
Oligonucleotides	n−1 shortmers, n+1 longmers, backbone variants from dsRNA therapeutics	15× purity increase, 20× mass enrichment
Recombinant proteins	PEGylation isoforms, co-eluting process variants	Reference standard preparation at higher purity and concentration than batch methods allow
Nutraceuticals / natural products	Low-abundance bioactive compounds in complex matrices	Selective enrichment from natural extracts without requiring affinity steps

N-Rich Applications Page

Recommended Systems for Continuous Enrichment (N-Rich®)

The Contichrom® platform offers both development and scale-up systems for N-Rich®.

Systems Overview

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 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.

Systems Overview

Isolate the Untouchable. Overnight.

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How is N-Rich® different from MCSGP — aren't they both twin-column processes?

They use the same hardware and the same wizard structure, but they solve opposite problems. MCSGP purifies a main product by internally recycling the impure fractions that flank it — the starting material is typically 50–90% pure and the goal is to push yield and purity simultaneously. N-Rich® accumulates toward a target that starts at 0.1–5% of the feed — the starting material is the impurity itself, and the goal is to enrich it to the point where it can be isolated pure. MCSGP is a manufacturing polishing step; N-Rich® is an analytical and development tool for generating reference standards and characterizing trace species.

Does N-Rich® require a batch chromatogram from the Contichrom® CUBE?

Yes. The N-Rich® Wizard in ChromIQ® loads the batch design run directly from the ChromIQ® measurement database. Chromatograms from other systems use different file formats and cannot be imported. The batch design run on the CUBE is typically a short single-column experiment using your existing method conditions — it is the starting point for N-Rich® development, not a separate preparatory step.

How many enrichment cycles are typically needed?

The number of cycles depends on the starting abundance of the target, the degree of enrichment required, and the resolution between the target and the main compound. The number of required cycles can be calculated based on the content of the target compound in the starting material, known from offline analysis, and the requested target amount. For many applications — mAb charge variants at 5–20% abundance, for example — 10–25 cycles are sufficient for an overnight run. For very low-abundance targets (0.1–1%), more cycles are needed and multi-day runs are typical.

What is the AutoPeak® "Product Removal Control" and when should I activate it?

Product Removal Control is N-Rich’s AutoPeak implementation — it controls when the main (non-target) compound is diverted to waste during each enrichment cycle. When OFF, diversion happens at fixed times derived from the batch chromatogram — appropriate for early development runs with stable conditions. When ON, the start of main-compound removal is UV-triggered (Front Control for an ascending-slope boundary, Tail Control for a descending-slope boundary), making the process robust to retention time shifts across many cycles. For extended, unattended enrichment runs — overnight or multi-day — Product Removal Control ON is recommended to maintain consistent depletion performance as conditions drift.

What is the scale-up path from CUBE development?

N-Rich® runs on the Contichrom® CUBE in its Combined configuration (CUBE + CUBE+). For applications requiring higher flow rates or HPLC pressure (peptides, oligonucleotides, small molecules), methods developed on the CUBE transfer to the Contichrom® PILOT 300X (up to 300 mL/min, GMP-ready) and the Contichrom® TWIN HPLC. Column dimensions change; the ChromIQ® method logic, zone boundary positions, and AutoPeak® trigger values transfer directly.