Article Category: Peptide Purification

@article{eisenhuth2025process,
  title   = {Process Characterization and Performance Qualification of MCSGP},
  author  = {Eisenhuth, Ralf and M{\"u}ller-Sp{\"a}th, Thomas},
  journal = {Processes},
  volume  = {13},
  number  = {12},
  pages   = {3950},
  year    = {2025},
  doi     = {10.3390/pr13123950}
}

MCSGP purifies continuously, recycling impure side fractions between two columns so that high yield and high purity are reached together. It does that by running to predefined recycling and collection windows, cycle after cycle, for hours or days — without the mid-run adjustment to pooling boundaries that an operator can make in batch chromatography. The chromatographic separation therefore has a greater influence on final product quality, and correspondingly more weight falls on the control strategy around it.

That makes validation a more demanding exercise. How do you show, in a regulatory filing, that a process running to fixed windows for days will deliver the right material every cycle?

The study is a collaboration between Bachem AG and YMC ChromaCon, with Bivalirudin as the test system. It sits alongside an earlier characterization and validation study for CaptureSMB, the simpler twin-column capture process, and extends that thinking to gradient purification.

1. Why This Study Matters: A Gap Between Practice and Paperwork

MCSGP is in operation at several companies and research institutes, purifying synthetic peptides and oligonucleotides at production scale. Its appeal is well documented: internal recycling of impure side fractions lets it reach high yield and high purity at the same time, with a high degree of automation, fewer in-process controls and a lower process mass intensity than batch chromatography.

Regulatory validation is a separate matter from process performance, and it has its own structure. The FDA Process Validation guideline sets out three elements:

• Process Design Builds scientific understanding of the process. Process Characterization is the central piece: identifying which parameters could affect critical quality attributes, testing the high-ranked ones experimentally, and defining the ranges within which quality is met.
• Process Performance Qualification Confirms that the equipment and process perform as intended and can manufacture reproducibly at commercial scale. At least three consecutive representative batches are run under normal operating conditions, in the actual facility, with trained operators and qualified equipment.
• Continued Process Verification Ongoing monitoring once routine manufacturing is under way. This study covers the first two elements; the third is outside its scope.

None of that framework is specific to continuous chromatography, and the paper’s finding is that it largely does not need to be. What was missing was a worked example showing how it applies to a process whose defining feature — recycling material between two columns — has no counterpart in batch operation.

Where MCSGP differs, and where it does not

The paper separates the purification into two parts: the chromatographic part, which is identical for batch and MCSGP, and the product collection and recycling part, which is specific to MCSGP. The list of process parameters is therefore largely the same as for a batch process, with a few additions covering the recycling and collection step.

The practical difference is how many elutions it takes to generate meaningful data. For a batch purification, one elution per tested parameter is considered sufficient. An MCSGP experiment needs at least three cycles with two elutions each — enough to confirm that the process has reached cyclic steady state, that impurities are not accumulating through the weak or strong recycling streams, and that changes in column load caused by internal recycling do not affect the outcome.

2. The Study at a Glance

Bivalirudin was used as the test system: a 20-amino-acid direct thrombin inhibitor with a molecular weight of 2,180 g/mol, produced by solid-phase peptide synthesis and supplied as crude material of 81% purity. The target for the product pool was more than 99.0% purity, with the added constraint that the impurity profile had to be comparable to the legacy batch process.

What was run, and on what

3. How Far the Parameters Were Pushed

Most of the 34 identified parameters were never tested experimentally, and the reasoning behind that is worth following. Large-scale MCSGP equipment holds most parameters within narrow limits by construction. A Contichrom TWIN system has four three-head pumps — two for gradient generation, one for feeding, one for in-line dilution, cleaning-in-place and equilibration — and each is controlled by a dedicated flow meter with a calibration tolerance of ±2%.

Whether that ±2% matters depends entirely on what the pump is doing. For equilibration, in-line dilution, cleaning-in-place and chase washes, a 2% variation is negligible. For column load it is not: a 2% change alters the peak shape, which can shift what ends up inside the recycling zones. So the same tolerance makes one parameter well-controlled and another potentially critical, and only the second needs an experiment.

That assessment left four parameters to test. Each was then varied well beyond the range it covers in production.

4. What Process Characterization Found

Every set point produced product above the 99.0% purity specification. Across all the characterization runs the average pool purity was 99.30%, and every individual value fell inside a band of ±3 standard deviations, that is 99.30% ± 0.32%. Since ±3σ captures roughly 99.7% of expected values for a normal distribution, the effect of the parameter variations was statistically indistinguishable from ordinary process noise. On purity alone, all four parameters would have been classified as non-critical, and the proven acceptable range would have been the full characterized range.

Each eluate pool was also examined for individual impurities. Those profiles were consistent across every run but one: at −25% displacer slope, a single impurity rose from below the limit of quantitation to 0.13%. Small in absolute terms, but the specification required the impurity profile to be comparable to the legacy process, and it no longer was.

On that basis gradient slope was designated a critical process parameter, and the variance permitted during routine manufacturing was reduced through organizational measures. Column load, elution flow rate and detection wavelength were designated non-critical. The gross-to-gross yields were also evaluated; excluding the shallow-slope run, they fell between 60% and 65%.

Why one factor at a time, rather than a designed experiment

The choice of OFAT over DoE is argued explicitly, and the reasoning is specific to peptides. Related substances in a peptide tend to behave as regular samples, with similar retention behavior under gradient conditions, so the primary observable effect of parameter variation is the width of the overall peak rather than a change in resolution. Purity changes in MCSGP then tend to come from misalignment between the peak and the fixed recycling and collection zones.

That creates a practical hazard for a designed experiment. Combinations that cumulatively broaden or narrow the peak — a lower column load with a steeper slope and a wavelength shift that lowers the signal, for instance — can push the peak far enough out of alignment with fixed zones to fail the run, forcing repeated iterations of the DoE setup. OFAT does not reveal interactions between parameters, which is a real limitation, but it allows much wider ranges to be tested. For molecules with stringent purity specifications the paper recommends including the combinations that produce the narrowest and broadest peaks as fixed experiments, and considering smaller ranges.

5. Supervising a Process That Runs for Days

An MCSGP process producing eluate continuously for several days presents a sampling problem that batch chromatography does not. Collecting and analyzing every main cut separately is not cost-effective at production scale, but sampling too little leaves the process unsupervised. The answer here is a two-phase strategy.

• Start-up: intensive monitoring Every main cut is analyzed by an in-process control method until three criteria for cyclic steady state are met — comparable impurity profiles for the main cuts, good overlay of the UV traces on each column, and comparable main cut volume on a column basis.
• Steady state: strategic sampling Once those criteria are satisfied, HPLC sampling drops to two samples per day. Supervision then rests on three elements together: the strategic samples, the online UV trace overlay, and the recorded main cut volume per column.

The approach is aligned with the risk management expectations for product availability set out in ICH Q9 (R1).

AutoPeak as Process Analytical Technology

Underpinning the strategy is AutoPeak, which monitors UV at the column outlets and starts and stops the recycling and collection phases from absolute and relative UV triggers rather than from the clock. The main cut borders are set as UV thresholds entered into the control. Because the cut points track the peak rather than elapsed time, retention time shifts that accumulate over a long run do not move the pool composition.

The qualification runs illustrate this. Over PPQ batch 1, a slight peak shift appeared from cycle to cycle; when the traces were realigned to the AutoPeak trigger point marking the start of product collection, they overlaid exactly. On that basis the paper positions AutoPeak as Process Analytical Technology in the sense of the FDA PAT guidance — a framing supported by the FDA Process Validation and PAT guidance documents and by ICH Q8, Q11 and Q13, which explicitly support real-time monitoring as part of a control strategy.

6. The PPQ Campaign: Full Results

A pre-PPQ GMP run came first: eleven elutions, 1.5 kg of crude purified to 0.8 kg of intermediate at 99.6% purity, with a PMI of 1,402 kg/kg for the purification alone. The qualification campaign then ran four multiday MCSGP runs, the last batch split into two parts, to cover a range of batch sizes.

All four produced consistent elution profiles, main cut sizes and product purities. Comparing the third elution — the first at cyclic steady state — with elution 106 of the longest run showed the impurity profile holding across the full duration, and purity for every full elution stayed within the ±3σ band anticipated from characterization.

• Crude in 54.5 kg at 81% purity
• MCSGP 397 elutions over 235 hours, producing 1,576 L of main cut
• SPE 42 elutions over 65 hours on the same system and columns, alternately loading and eluting with a sharp gradient, producing 631 L of main cut
• API out 33.6 kg at 99.2% purity — a gross-to-gross yield of 62%, or 76% effective yield taking product and crude purity into account
• Total 300 hours of chromatography operating time

Against the legacy process

Swipe the table sideways to see the legacy and MCSGP columns.

Source: Eisenhuth and Müller-Späth, Processes 2025, 13, 3950 (CC BY). The legacy figure is the existing Bivalirudin batch process, which includes extensive side-cut re-chromatography.

Experimental conditions

7. What This Means for Peptide Manufacturing

On the evidence here, the regulatory framework does not need reinventing for continuous chromatography. The parameter list is largely the batch list, the risk assessment is the standard one, and the qualification requirement is the standard three-batch expectation. What changes is the number of elutions needed to generate data, and the shift from correcting a process to supervising one.

That is a useful result for anyone weighing continuous purification against the validation work it would imply. Of 34 identified parameters, four warranted experiments and one turned out to be critical — and the paper notes that those same four would also be investigated during characterization of the corresponding batch process. The characterization burden is comparable; the difference is material consumption, since cyclic steady state has to be confirmed from successive identical elution profiles. In this study that extra experimental time was minimized by daisy-chaining the required MCSGP runs and executing them automatically.

The operational advantages appear in the campaign figures rather than in the validation argument. Running uninterrupted over several days, with the control strategy supported by online measurement, produced a substantial reduction in analytical sampling and process mass intensity alongside a higher gross yield. The authors also note that data integrity and electronic batch record handling requirements are identical for batch and MCSGP.

Another crucial topic for commercial manufacturing about “green solvents” for peptide purification is covered in another research highlights article, and the platform view — where MCSGP fits in a peptide workflow — is set out under peptide purification with MCSGP. AutoPeak itself is described in more detail under dynamic process control.

8. Key Terms in This Study (Glossary)

9. Frequently Asked Questions

@article{prestia2026countercurrent,
  title   = {Counter-Current Chromatography Enables Use of Green Solvents
             for Productive Peptide Purification Processes},
  author  = {Prestia, Rosella and Hauri, Damian and Sponchioni, Mattia
             and Vogg, Sebastian and M{\"u}ller-Sp{\"a}th, Thomas},
  journal = {Separations},
  volume  = {13},
  number  = {7},
  pages   = {195},
  year    = {2026},
  doi     = {10.3390/separations13070195}
}

Preparative reversed-phase liquid chromatography is one of the most resource-intensive stages of synthetic peptide manufacturing, and production processes commonly rely on acetonitrile (ACN) with trifluoroacetic acid (TFA). That eluent system is chosen mainly for its high chromatographic resolution — and it is exactly the combination that environmental and safety pressure is now working to remove.

Greener alternatives are well characterized. Ethanol is low-toxicity and bio-derived; dimethyl carbonate (DMC) is an emerging bio-based substitute for acetonitrile; acetic acid is a typical candidate for a green counterion in place of TFA. What has blocked their adoption is not chemistry but process economics: in conventional batch chromatography, they lead to decreased resolution and product loss.

This highlight summarizes the study, its data, and its limitations, and links to the full open-access article.

1. Why This Study Matters: The Solvent Problem in Peptide Purification

Peptide therapeutics are in a demand surge. More than 80 peptide drugs have received regulatory approval since 2023, with over 200 candidates in clinical development. GLP-1 receptor agonists have become the dominant class in the pharmaceutical market, and the global anti-obesity segment is projected to exceed $100 billion by 2030. That demand has exposed manufacturing bottlenecks in synthesis, purification, and fill-finish.

Purification carries a disproportionate share of the burden. Solid-phase peptide synthesis already runs at a process mass intensity of roughly 3,000–15,000 kg of material per kg of active ingredient for peptides in the 1,000–5,000 Da range. Downstream chromatography then adds several hundred to several thousand liters of eluent per kilogram of purified peptide, of which 10–40% is organic modifier.

Three problems with the standard eluent system

• TFA is a PFAS Trifluoroacetic acid belongs to the per- and polyfluoroalkyl substances and is highly persistent in the environment — it is a “forever chemical” with potential toxicity risks. Regulatory and environmental pressure on PFAS in pharmaceutical manufacturing is increasing.
• Acetonitrile is flammable and toxic Replacing the organic modifier is therefore one half of the problem, alongside replacing the acid. Ethanol and dimethyl carbonate are the substitutes tested here.
• The dilution prevents recycling Because the organic modifier is so heavily diluted, the eluent cannot practically be recycled. The paper links that volume directly to waste generation, environmental impact, and limits on process throughput.

The obstacle to switching has been consistent and well documented: replacing ACN/TFA with green alternatives reduces chromatographic resolution, and reduced resolution in a batch process means lower yield and lower productivity. Until now, no industrially scalable process had combined high purity, high yield, and a low environmental footprint.

2. The Study at a Glance

Researchers at YMC ChromaCon and Politecnico di Milano purified two therapeutic peptides using eluent systems containing no acetonitrile and no TFA, first as conventional batch processes and then as continuous multi-column counter-current solvent gradient purification (MCSGP) processes. Method development held the column, the 10 CV gradient volume and the 10 g/L_resin loading fixed, so that the batch comparisons isolate the eluent system; the MCSGP processes were then built from those batch methods.

The two model peptides

Both molecules are conventionally purified with ACN and TFA, and both were run against an optimized ACN/TFA batch process as the reference. Together they bracket the range of therapeutic peptides — a heavily engineered modern construct and a long-established sequence — while sharing the same downstream challenges.

3. Where the Yield Goes

The clearest way to understand the result is to look at how the product pool is defined in each of the three processes. The schematic below redraws the Tirzepatide elution profiles reported in the study; the shaded area is the material actually collected.

4. The Green Solvent Penalty, Quantified

Both peptides were first optimized as conventional batch processes using a common gradient-narrowing procedure, so the green and conventional batches are directly comparable. The results reproduce, with numbers, the trade-off that has kept green eluents out of production.

Tirzepatide with ethanol/acetic acid

Under ACN/TFA, the highest purity appeared along the tail of the main peak and a wide collection window satisfied the 89.0% specification, recovering 81.9% at 5.3 g/L_resin/h. Under ethanol/acetic acid the peak took on a double-peak shape, with high purity only at the front and progressive co-elution with closely related impurities after it. The usable window narrowed sharply and yield fell to 43.2%, with productivity down to 2.9 g/L_resin/h.

Tetracosactide with dimethyl carbonate/acetic acid

The ACN/TFA batch produced a sharp product peak with fractions above 95.0% purity and a maximum in-specification yield of 82.8%. The DMC/AcOH batch produced a very narrow collection window followed by a long tail that was high in purity but low in product concentration. The pool consisted of two fractions, giving 92.3% pool purity at 35.3% yield — a 2.3-fold reduction against the conventional batch — and productivity of 1.6 g/L_resin/h.

The penalties compound rather than trade off. Lower recovery — and, for Tetracosactide, a longer process time from the added counterion exchange — reduced productivity by a factor of two and three respectively, and solvent consumption moved the wrong way at the same time — roughly doubling for Tirzepatide and rising more than three-fold for Tetracosactide. Reducing the loading density would recover some yield, but at the cost of still lower productivity and still more solvent. In batch operation there is no setting that delivers all three.

5. How MCSGP Closes the Gap

MCSGP — multi-column counter-current solvent gradient purification — is a continuous, twin-column form of gradient chromatography. It does not improve the resolution of the separation. It changes what happens to the fractions that resolution fails to resolve.

The operating principle

• Two columns, one repeating switch While one column is eluting, the other is being loaded. The roles reverse at each switch, so neither column sits idle and the process runs continuously.
• The overlap regions are recycled, not discarded The elution is separated into weakly adsorbing impurities, the product fraction, and strongly adsorbing species. The two overlap regions between them — product co-eluting with weak impurities, and product co-eluting with strong impurities — are returned to the loading column instead of being discarded.
• Inline dilution keeps recycled material on the column For Tetracosactide, a 4.5-fold inline dilution was used to ensure the recycled material re-adsorbed completely on the downstream column. For Tirzepatide, a 4-fold dilution served a second purpose: preventing precipitation or gelation during the collection and recycling phases.
• The process converges to a cyclic steady state Recycled material accumulates until the internal concentration profile stabilizes. Here that took three cycles; from cycle four onward the chromatograms overlaid almost exactly, and cycles 4 and 5 were used for the performance calculations.

Holding the cut points: UV-based dynamic control

The switch points were set from the live UV signal rather than from elapsed time, using AutoPeak process control. For the Tirzepatide process, a first threshold at 15 mAU opened weak-side recycling, a second at 200 mAU started product collection, and a fall to 75% of the peak maximum — plus a 20-second delay — began strong-side recycling. Because the cut points track the actual peak, the process maintained reproducible cyclic steady-state operation despite variations in the elution profiles.

Applied to the green eluent systems, that mechanism recovered what the batch processes had been discarding. The ethanol/acetic acid MCSGP process reached 88.1% yield at the 89.0% purity specification and 6.3 g/L_resin/h — higher yield and higher productivity than the conventional ACN/TFA batch, using 3.4 L/g of solvent instead of 4.3 L/g. The DMC/AcOH MCSGP process for Tetracosactide, which used weak-side recycling only, reached 93.8% yield at 94.0% purity, exceeding the conventional batch on both.

6. Full Results: Process Comparison

The table below reproduces the study’s process comparison. For the batch processes, the pool with the maximum achievable yield within specification is reported; for MCSGP, the values are averages of steady-state cycles 4 and 5.

Swipe the table sideways to see purity, yield, productivity and solvent use.

Source: Table 3, Prestia et al., Separations 2026, 13, 195 (CC BY). Green values improve on the conventional ACN/TFA batch benchmark; red values fall short of it.

Experimental conditions

7. What This Means for Peptide Manufacturing

Sustainability and process performance have generally been treated as a trade-off in peptide downstream processing. The practical contribution of this study is to separate them into two independent levers — the eluent chemistry and the operating mode — and to show that the second can pay for the first.

For Tirzepatide, the green continuous process was simply the better process on every measured axis: higher yield, higher productivity, and lower solvent consumption than the conventional batch, while eliminating both TFA and acetonitrile. For that molecule at least, removing a PFAS from the purification step did not have to be traded against process performance. Because eluent consumption is directly associated with the overall waste of the step, and the dilution of the organic solvent prevents recycling, lower consumption means less waste.

The authors also argue that the scalability and operational robustness of MCSGP with AutoPeak support implementation at manufacturing scale, as previously demonstrated for complex biopharmaceutical products such as oligonucleotides, and that the technique has been characterized for process qualification. In that context they identify solvent viscosity and the associated pressure drop as the main additional consideration when using green eluents.

For the wider picture — where MCSGP sits in a peptide purification workflow and what it has delivered at commercial scale — see peptide purification with MCSGP. The regulatory side of the same question is taken up by another study in this series, which works through Process Characterization and Process Performance Qualification on a commercial peptide.

8. Key Terms in This Study (Glossary)

9. Frequently Asked Questions