Severinghaus vs Optical CO2 Sensors: Which to Use in Bioreactors
Severinghaus electrodes remain the installed-base default; optical CO2 sensors are the safer pick for new builds. The Mettler InPro 5000i Severinghaus probe owns the cGMP installed base and the wider 0–1000 mbar range microbial fermentation needs. Solid-state mid-IR optical (Hamilton CO2NTROL) eliminates electrolyte refills and membrane changes for stainless steel mAb fed-batch, and HPTS-dye optical (PreSens) is the only realistic option in single-use bags. The DO version of this comparison goes the same way — see Optical vs Polarographic DO.
Key differences at a glance
- Severinghaus electrode: potentiometric — CO2 diffuses through a membrane into a bicarbonate electrolyte; an internal pH electrode reads the resulting pH shift. Wide range (0–1000 mbar), needs electrolyte refills, ~£3,000–£5,000 per channel.
- Optical CO2 sensor: two flavors — mid-IR absorption (solid-state, no consumables, Hamilton CO2NTROL) or HPTS-dye fluorescence in a bicarbonate buffer (PreSens spots and probes). Maintenance-free or single-use ready, ~£4,000–£7,500 per channel.
- Best for stainless-steel mAb fed-batch: Severinghaus (installed base) or solid-state mid-IR (lifetime maintenance).
- Best for single-use bags (Sartorius / Thermo / Cytiva): optical fluorescence — gamma-sterilised spots or flow-through cells.
- Best for high-pCO2 microbial fermentation (>200 mbar): Severinghaus — wider range and proven lifetime at high CO2 partial pressures.
Side-by-side comparison
| Factor | Severinghaus electrode | Optical CO2 sensor |
|---|---|---|
| Measurement principle | Potentiometric — pH shift in bicarbonate electrolyte | Mid-IR absorption (solid-state) or fluorescence quenching of HPTS dye |
| Internal consumables | Bicarbonate electrolyte + gas-permeable membrane | None (solid-state) or factory-sealed dye |
| Maintenance interval | Electrolyte every 3–6 months, membrane every 6–12 months | Maintenance-free for probe lifetime |
| Typical accuracy | ±10% (10–900 mbar) | ±0.5–1.2 mmHg at physiological pCO2 (HPTS) |
| Working range | 0–1000 mbar (0–760 mmHg) | 10–250 hPa (HPTS) / 5–1000 mbar (mid-IR) |
| Response time (t90) | 60–120 s at 25°C | 60–180 s at 25°C |
| Sterilisation | Autoclave + SIP up to 130°C | SIP to 140°C (mid-IR) / gamma (single-use spots) |
| Single-use bag compatibility | Not available | SP-CD1 spots + FTC-SU-CD1 flow cells |
| Calibration drift between cycles | Moderate — electrolyte dilution and membrane fouling | Low — solid-state has no internal phase to drift |
| Typical capital cost (per channel) | £3,000–£5,000 | £4,000–£7,500 |
Values reflect typical published specifications. Vendor datasheets take precedence for specific instrument specs.
How Severinghaus CO2 electrodes work
The Severinghaus electrode was introduced in 1957 by Dr. John W. Severinghaus and has been the industry-standard dissolved CO2 sensor in bioprocess for more than five decades. The basic principle is indirect: CO2 cannot be measured potentiometrically on its own, but its hydration product carbonic acid can be measured through pH. A thin film of bicarbonate electrolyte is held behind a gas-permeable membrane (typically silicone-reinforced PTFE). CO2 from the culture diffuses through the membrane into the electrolyte film, where it equilibrates with HCO3- ions and shifts the buffer's pH according to the carbonic-acid equilibrium. An internal pH glass electrode reads that pH shift, and the sensor's electronics convert it to a partial-pressure CO2 reading using a logarithmic relationship.
The dominant commercial Severinghaus probe in bioprocess is the Mettler Toledo InPro 5000i, which is integrated with Mettler's Intelligent Sensor Management (ISM) digital platform for predictive diagnostics. The InPro 5000i covers a 0–1000 mbar pCO2 range, has a t90 response time under 120 seconds at 25°C, and is autoclavable and SIP-compatible up to 130°C. The membrane body is field-replaceable on the existing probe shaft, which is the design feature that has kept it competitive against newer optical alternatives — operators can refurbish a probe in place rather than send it out for service.
When Severinghaus wins
Severinghaus dominates three scenarios. First, existing cGMP installed base — a typical commercial mammalian facility built between 2005 and 2020 has Severinghaus probes plumbed into every bioreactor port, validated against the InPro 5000i datasheet. Switching all of them to optical would trigger an IQ/OQ/PQ revalidation campaign that costs more than 5–10 years of electrolyte refills. Second, microbial fermentation at high pCO2 (above 200 mbar) — the InPro 5000i's 0–1000 mbar range covers high-density E. coli and yeast fed-batch where dCO2 routinely climbs above 300 mbar; many fluorescence optical sensors saturate or lose accuracy above 200 mbar. Third, academic and pilot-scale teaching — the lower capital cost per channel and the ubiquity of Mettler's training material make Severinghaus the default in research-grade glass bioreactors.
How optical CO2 sensors work
"Optical CO2 sensor" is a category, not a single technology. Two physical principles share the label, and they have very different operating envelopes.
Mid-IR absorption (solid-state). CO2 has a strong absorption band near 4.26 µm. A small mid-IR LED inside the probe emits at this wavelength, the light passes through a polymer-protected sample volume in contact with the culture, and a photodiode measures the transmitted intensity. The Beer-Lambert relationship between transmittance and CO2 partial pressure gives the reading. This is the principle Hamilton CO2NTROL uses. Because there is no electrolyte and no consumed reagent, the probe is solid-state and maintenance-free — Hamilton specifies it as having no consumables for the rated lifetime. The CO2NTROL covers 5–1000 mbar, mounts in standard PG13.5 bioreactor ports, and is SIP- and CIP-compatible up to ~140°C.
Fluorescence quenching of an HPTS dye in a bicarbonate buffer. This is the original optical Severinghaus principle. A pH-sensitive fluorescent dye (typically 1-hydroxypyrene-3,6,8-trisulfonate, HPTS) is dissolved in a thin bicarbonate buffer film behind a CO2-permeable membrane, with an LED exciting the dye and a photodiode measuring its fluorescence. CO2 from the culture diffuses through the membrane, equilibrates with the buffer, shifts its pH, and changes the dye's fluorescence. PreSens uses Dual Lifetime Referencing (DLR) — a second inert reference dye in the same matrix — to internally correct for intensity drift and ageing of the LED. PreSens offers HPTS-based optical CO2 in three formats: insertable benchtop probes (DP-CD1), gamma-sterilised SP-CD1 sensor spots for single-use bags, and FTC-SU-CD1 single-use flow-through cells for perfusion bypass loops.
When optical wins
Mid-IR optical wins on lifetime maintenance for new stainless-steel cGMP builds — no electrolyte to refill, no membrane to replace, no consumables to qualify, no operator hours per probe per year. Hamilton estimates a roughly 3-year crossover where the avoided maintenance overtakes the higher capital cost. HPTS-dye optical wins decisively on single-use bag compatibility — the PreSens spot is glued to the inside of the bag wall by the bag vendor, gamma-sterilised together with the bag, and read non-invasively through a transparent window. A traditional Severinghaus form factor with a refillable electrolyte chamber simply cannot integrate with a sealed bag. Optical is also preferred in perfusion and small-scale parallel bioreactors (pCO2 mini FTC-CD1, Sartorius ambr) where the small-volume probe footprint matters.
Pros and cons
Severinghaus electrode
Advantages
- Industry standard since 1957 — deep validation heritage
- Wide working range (0–1000 mbar) covers microbial high-pCO2
- Lower capital cost per channel than mid-IR optical
- Field-replaceable membrane body (InPro 5000i) — refurbish in place
- Mature ISM digital diagnostics in Mettler ecosystem
Disadvantages
- Bicarbonate electrolyte requires periodic refilling
- Membrane fouling and pinhole leaks are routine failure modes
- Calibration drift between membrane changes
- No single-use bag form factor exists — incompatible with SUBs
- 10–20 operator-hours per probe per year for membrane/electrolyte service
Optical CO2 sensor
Advantages
- No electrolyte, no membrane changes — maintenance-free (mid-IR)
- Single-use bag-compatible (HPTS spots glued in by bag vendor)
- Higher precision in physiological pCO2 range (40–80 mmHg)
- Lower long-term calibration drift (solid-state, no electrolyte dilution)
- Pre-calibrated from the factory — minimal pre-batch work
Disadvantages
- 1.3–1.5× higher capital cost per channel (mid-IR)
- Smaller installed base — younger validation track record
- HPTS dye saturates above ~250 hPa — limited for microbial high-pCO2
- Single-use spots are consumables tied to the bag (per-batch cost)
- Retrofit into validated process triggers IQ/OQ/PQ revalidation
Which should you choose?
The CO2 sensor decision is dominated by vessel type and process modality, with capital budget as a secondary consideration.
New stainless-steel cGMP facility
Reusable bioreactors at 200–10,000 L for commercial mAb or recombinant protein. Solid-state mid-IR (Hamilton CO2NTROL) eliminates electrolyte refills across 100+ batches per year and integrates with the Hamilton Arc digital ecosystem alongside VisiFerm DO and EasyFerm pH.
Choose Optical (mid-IR)Single-use bag bioreactor
Sartorius Biostat STR, Thermo HyPerforma, Cytiva Xcellerex, or Pall Allegro at 50–2,000 L. PreSens SP-CD1 spots are glued inside the bag at manufacture and gamma-sterilised together with the bag. Severinghaus electrodes have no equivalent form factor.
Choose Optical (HPTS)High-density microbial fermentation
High-OUR E. coli or Pichia with dCO2 climbing above 200–300 mbar during induction. Severinghaus (InPro 5000i) covers the 0–1000 mbar range cleanly; HPTS-dye optical can saturate at the upper end.
Choose SeveringhausValidated legacy cGMP process
Existing commercial process running InPro 5000i for the last 10+ years. Switching probe technology triggers IQ/OQ/PQ revalidation, SOP updates, and cleaning validation refresh — usually more expensive than continuing with electrolyte refills.
Keep SeveringhausReal-world use cases
Four representative deployments and why each team converged on their choice.
Mid-IR optical (Hamilton CO2NTROL)
New build with full Hamilton Arc ecosystem (VisiFerm DO, EasyFerm pH, CO2NTROL). One-time pre-run air-zero calibration, RS485 to DeltaV, no consumable PO line for the lifetime of the probe. Maintenance overhead matched to DO and pH probes — single ISM-style diagnostic dashboard.
Severinghaus (Mettler InPro 5000i)
dCO2 climbs to 350–500 mbar during oxygen-limited induction with continuous glucose feed. The InPro 5000i's 0–1000 mbar range covers the regime cleanly. Membrane refurbished every 6 months, electrolyte refilled at each campaign turnaround.
Optical spot (PreSens SP-CD1, bag-integrated)
Spot pre-glued inside the Sartorius Biostat STR 200 bag, gamma-sterilised together with the bag, ready at installation. Read by an external pCO2 mini optical reader through the transparent window. No Severinghaus form factor was ever in consideration.
Optical flow cell (PreSens FTC-SU-CD1)
Single-use beta-irradiated flow-through cell in a perfusion bypass with C-Flex tubing. Reads pCO2 in the recirculation stream without breaching the main vessel. Perfusion dCO2 routinely accumulates above 100 mmHg, and the FTC's 8–180 mmHg range covers it.
Not sure which CO2 sensor fits your scale and modality?
Answer a few quick questions about scale, vessel, parameters, and budget, and get ranked sensor recommendations covering CO2, DO, pH, biomass, and PAT analytics.
Open the Sensor Selection ToolCost and lifecycle considerations
Severinghaus wins on probe-price alone, but consumables (electrolyte cartridges, replacement membrane bodies, calibration gases) plus operator time for membrane swaps converge the 3-year TCO with mid-IR optical. The decisive factor is operator hours — each membrane change on an InPro 5000i takes 2–4 hours including re-calibration. At 100 batches/year with a change every 4–6 months, that is 4–12 operator-hours per probe per year that solid-state optical eliminates entirely. For single-use bag deployments the comparison is moot — the per-batch consumable cost of an SP-CD1 spot is bundled into the bag price, not a separate line item.
A single 2,000 L mammalian bioreactor deploying solid-state mid-IR CO2 (Hamilton CO2NTROL) costs approximately £4,500–£7,500 for the probe with integrated transmitter, with effectively zero recurring consumable cost over the rated lifetime. The same vessel with a Severinghaus InPro 5000i costs £3,000–£5,000 up front but adds £400–£900/year in consumables (electrolyte cartridges, membrane bodies, calibration gas refills) plus 4–12 hours/year of operator time for replacements.
For single-use deployments the calculation is different. The PreSens SP-CD1 spot itself is inexpensive (~£15–£40 per spot), but it ships pre-integrated in the bag at a per-batch cost rolled into the bag price. The pCO2 mini reader unit (£3,000–£5,000) is reused across batches. Over a 100-batch single-use campaign, the all-in single-use CO2 monitoring cost is comparable to a 2-channel reusable Severinghaus deployment, but with the upside that there is no probe to clean, recalibrate, or revalidate between batches.
| Cost component | Severinghaus (InPro 5000i) | Optical mid-IR (CO2NTROL) |
|---|---|---|
| Probe + transmitter (per channel) | £3,000–£5,000 | £4,500–£7,500 |
| Consumables / year (electrolyte, membrane, cal gas) | £400–£900 | ~£0–£100 (cal gas only) |
| Operator labour / year (maintenance) | 4–12 hours | <1 hour |
| 3-year TCO (per channel, 100 batches/yr) | £4,500–£7,500 | £4,800–£7,800 |
Vendor landscape
The dissolved CO2 sensor market is more concentrated than the DO market — three vendors cover essentially the entire bioprocess installed base.
Severinghaus electrode vendors
- Mettler Toledo InPro 5000i: the dominant Severinghaus probe in cGMP bioprocess. ISM digital ecosystem, field-replaceable membrane body, 0–1000 mbar range, autoclave + SIP to 130°C. The reference design that essentially every other Severinghaus probe in bioprocess is benchmarked against.
- Broadley-James CO2 Probe: niche Severinghaus competitor in research-grade glass bioreactors and pilot vessels; often bundled with Broadley-James pH and DO in academic labs.
- Sentinel Process Systems: distributor of Severinghaus and adjacent CO2 sensing solutions for biopharma and food/beverage; good US service network.
Optical CO2 sensor vendors
- Hamilton CO2NTROL: the only solid-state mid-IR DCO2 probe in commercial bioprocess at the time of writing. RS485 transmitter, EHEDG-approved hygienic design, 5–1000 mbar range, SIP/CIP to ~140°C, no electrolyte. Targets new stainless-steel cGMP builds where the Hamilton Arc ecosystem (VisiFerm DO, EasyFerm pH) is already deployed.
- PreSens optical CO2: the dominant HPTS-dye fluorescence CO2 vendor. Three product families — DP-CD1 insertable benchtop probes, SP-CD1 sensor spots for single-use bags, and FTC-SU-CD1 single-use flow-through cells for perfusion bypass. Covers 1–25% CO2 (8–190 mmHg) with a low-range 0–1% option for trace measurement.
- Scientific Bioprocessing: non-invasive optical CO2 for shake flasks and small-scale parallel bioreactors; competes with PreSens at the screening end of the market.
- Pyroscience: OEM optical CO2 fibre-optic sensors for research and biomedical adjacencies; aggressive pricing relative to PreSens for instrument-builders.
Frequently asked questions
What is the difference between a Severinghaus and an optical CO2 sensor?
Is the Mettler InPro 5000i a Severinghaus sensor?
Why do optical CO2 sensors work better for single-use bioreactors?
How accurate are optical CO2 sensors compared to Severinghaus electrodes?
What is the response time of CO2 sensors in bioreactors?
Can optical CO2 sensors be sterilized in place (SIP)?
Do CO2 sensors need calibration?
Which CO2 sensor is best for CHO mAb fed-batch?
Resources and references
- Xing et al. 2017 — A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors (Biotechnology and Bioengineering 114:1184–1194) — peer-reviewed mass-transfer model validated against 5,000 L and 25,000 L CHO bioreactors. The reference for understanding why dCO2 accumulates with scale.
- Darja et al. 2016 — Responses of CHO cell lines to increased pCO2 (Journal of Biotechnology 219:98–109) — proteomics-level evidence that elevated pCO2 alters CHO metabolism and recombinant protein quality. Why a CO2 sensor reading is a critical process parameter, not a nice-to-have.
- Chaudhry 2025 — Lessons in Bioreactor Scale-Up Part 6: Dissolved CO2 (BioProcess International) — industry overview of dCO2 accumulation patterns, sensor data interpretation, and CO2 stripping strategies in commercial-scale CHO.
- Busse et al. 2017 — Sensors for disposable bioreactors (Engineering in Life Sciences) — peer-reviewed review covering CO2, DO, and pH sensor technology for single-use systems, including the integration challenges that drive optical adoption.