{"id":788,"date":"2025-11-17T11:34:29","date_gmt":"2025-11-17T11:34:29","guid":{"rendered":"https:\/\/chromocan.test\/technology\/n-rich\/"},"modified":"2026-05-11T11:21:52","modified_gmt":"2026-05-11T09:21:52","slug":"n-rich","status":"publish","type":"technology","link":"https:\/\/www.chromacon.com\/en\/technologies\/n-rich\/","title":{"rendered":"Continuous Enrichment (N-Rich\u00ae)"},"content":{"rendered":"
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\n TWIN-COLUMN ENRICHMENT <\/p>\n \n

\n Continuous Enrichment Chromatography with N-Rich\u00ae<\/strong>\n <\/h1>\n \n
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Automated Trace Impurity Isolation.<\/strong> Accumulate what’s barely there \u2014 cyclic enrichment for reference standard preparation, charge variant isolation, and ICH Q3A\/Q6B characterization: 100\u20131,000\u00d7<\/strong> Enrichment factor | 80\u201395%<\/strong> Purity overnight | fully automated via ChromIQ\u00ae N-Rich\u00ae Wizard.<\/p>\n

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Sources: Bigelow et al. <\/em>(BMS), J. Chromatogr. <\/em>A 2021; Weldon & M\u00fcller-Sp\u00e4th, J. Chromatogr. A 2022<\/em><\/strong><\/p>\n <\/div>\n <\/div>\n\n

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\n Request a Webinar<\/span><\/a><\/span> <\/div>\n
\n N-Rich Tutorial Paper<\/span><\/a><\/span> <\/div>\n <\/div>\n <\/div>\n <\/div>\n\n
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\n Request a Webinar<\/span><\/a><\/span> <\/div>\n
\n N-Rich Tutorial Paper<\/span><\/a><\/span> <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n
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\n The Isolation Bottleneck <\/h2>\n \n
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Regulatory agencies require pure, isolated impurity reference standards \u2014 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\u20135% of the feed, co-eluting with a dominant main peak that is 10\u20131000\u00d7 more abundant.<\/p>\n

Conventional batch preparative HPLC has no mechanism to address this ratio. Hundreds of injections, progressive pooling of dilute fractions, weeks of processing time \u2014 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.<\/p>\n <\/div>\n \n <\/div>\n\n

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N-Rich\u00ae Changes What’s Possible<\/h2>\n\n\n
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N-Rich\u00ae does not try to isolate a trace compound in a single pass. Instead, it accumulates it on-column across many cycles \u2014 progressively building concentration in the system until enough pure material is available to collect in a single, fine-fractionated elution.<\/p>\n

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 \u2014 overnight, or over multiple days for very low-abundance targets.<\/p>\n

The results across validated applications are consistent:<\/p>\n\n \n <\/div> \n\n\n

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\n 95% purity <\/h2>\n \n

\n mAb charge variants in 10h vs. 300h by HPLC (BMS) <\/p>\n \n \n <\/div>\n\n<\/div>\n\n <\/div><\/section><\/div>\n\n\n\n

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\n 3 days <\/h2>\n \n

\n vs. 30+ days biosimilar mAb, 9 isoforms
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\n 79\u00d7 <\/h2>\n \n

\n Productivity gain peptide impurity isolation
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\n Solvent reduction vs. analytical HPLC
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Sources: Bigelow et al. (BMS), J. Chromatogr. A 2021<\/a> \u00b7 Weldon & M\u00fcller-Sp\u00e4th, J. Chromatogr. A 2022<\/a><\/em><\/p>\n

N-Rich\u00ae is a fundamentally different tool from MCSGP. MCSGP purifies a main product by removing flanking impurities \u2014 its starting material is already relatively pure. N-Rich\u00ae accumulates toward<\/em> 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\u00ae wizard structure; the problem they solve is different.<\/p>\n\n \n <\/div> <\/div>\n<\/div>\n\n\n

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\n How N-Rich\u00ae Works…. Part 1 <\/h2>\n \n
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N-Rich\u00ae runs as a single uninterrupted procedure through four sequential stages:<\/p>\n

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Stage<\/th>\nWhat happens<\/th>\nDuration<\/th>\n<\/tr>\n<\/thead>\n
Design<\/strong><\/td>\nBatch chromatogram loaded, recycling boundaries set, method generated<\/td>\nOne-time setup<\/td>\n<\/tr>\n
Enrichment<\/strong><\/td>\nn cycles of cyclic accumulation \u2014 target compound builds on-column, main compound removed each cycle<\/td>\nn cycles until sufficient feed is processed<\/td>\n<\/tr>\n
Depletion<\/strong><\/td>\nOne switch without fresh feed \u2014 remaining main compound removed, target concentrated before elution<\/td>\n1 switch<\/td>\n<\/tr>\n
Fractionated Elution<\/strong><\/td>\nShallow gradient across both columns in series \u2014 target eluted and fine-fractionated for collection<\/td>\nSingle elution event<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n <\/div>\n \n <\/div>\n\n
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N-Rich\u00ae Process Principle (.pptx)<\/p>\n <\/div>\n <\/div>\n <\/section>\n\n<\/section>\n\n

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\n How N-Rich\u00ae Works…. Part 2 <\/h2>\n \n
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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:<\/p>\n

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Phase<\/th>\nUpstream column<\/th>\nDownstream column<\/th>\n<\/tr>\n<\/thead>\n
P1 – Non-target elution (Parallel)<\/strong><\/td>\nWeakly adsorbing non-targets eluted to waste<\/td>\nRegenerated and re-equilibrated<\/td>\n<\/tr>\n
P2 – Weak (W) recycling (Interconnected)<\/strong><\/td>\nWeakly adsorbing target compounds transferred with in-line dilution to downstream column<\/td>\nReceives W fraction \u2014 target re-adsorbs<\/td>\n<\/tr>\n
P3 – Main compound removal (Parallel)<\/strong><\/td>\nNon-target (main) compound eluted to waste<\/td>\nLoaded with fresh feed \u2014 more target enters system<\/td>\n<\/tr>\n
P4 – Strong (S) recycling\u00a0 (Interconnected)<\/strong><\/td>\nStrongly adsorbing target compounds transferred with in-line dilution to downstream column<\/td>\nReceives S fraction \u2014 target re-adsorbs<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n <\/div>\n \n <\/div>\n\n
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\n How N-Rich\u00ae Works…. Part 3 <\/h2>\n \n
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At the end of each switch, the two columns swap positions by valve switching and the cycle repeats. The feed volume per cycle<\/strong> is calculated automatically by the N-Rich Wizard from the batch chromatogram.<\/p>\n

The Depletion stage<\/strong> (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.<\/p>\n

The Fractionated Elution<\/strong> uses a shallow gradient run across both columns in series \u2014 at half the normal flow rate with a flatter gradient than the batch method \u2014 to maximize resolution between enriched compounds. This is where the concentrated, pre-enriched target material is collected as fine fractions.<\/p>\n <\/div>\n \n <\/div>\n\n

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Method Design with the N-Rich\u00ae Wizard<\/h2>\n

N-Rich\u00ae methods are designed in the N-Rich\u00ae Wizard<\/strong> in ChromIQ\u00ae 9 \u2014 a tab-based interface that takes a single-column batch chromatogram from the Contichrom\u00ae 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.<\/p>\n <\/div>\n \n

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\n Step 1 \u2014 Load the chromatogram and define recycling boundaries <\/h3>\n
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Load the single-column batch design run from the CUBE into the Batch Chromatogram<\/strong> tab. Drag five boundary markers (t1\u2013t5) 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.<\/p>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n

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\n Step 2 \u2014 Configure washing, regeneration, and Product Removal Control <\/h3>\n
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Set washing and regeneration steps in the Washing & Regeneration<\/strong> tab \u2014 the same protocol as your existing batch process. In the “AutoPeak\u00ae” tab<\/strong>, activate the Product Removal Control<\/strong> if needed. When activated, the start of main-compound removal is UV-triggered rather than time-based \u2014 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.<\/p>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n

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\n Step 3 \u2014 Set up the final fractionation <\/h3>\n
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The Final Fractionation<\/strong> 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 \u2014 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.<\/p>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n

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\n Step 4 \u2014 Set number of cycles and generate <\/h3>\n
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In the Method Settings & Performance<\/strong> tab, set the number of enrichment cycles and review switch and cycle durations. Hit Save<\/strong>: The wizard generates the method files and a procedure file that runs them in the correct sequence. Load the procedure in ChromIQ\u00ae and run. The process runs fully unattended from here.<\/p>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n\n \n

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