The Four CHO Growth Phases
The CHO cell growth curve is usually drawn as the classic three-phase sigmoid — exponential, stationary, death — but a more accurate description has four phases. Pan et al. (2017), working with industrial fed-batch data, showed that between the exponential (number-increase, NI) and stationary phases, CHO cells enter a distinct size-increase (SI) phase. During SI, viable cell density (VCD) plateaus but volume and dry weight per cell climb roughly three-fold. Most of the monoclonal antibody titer in a typical 14-day fed-batch is produced during this SI phase, not the NI phase.
This article walks through all four CHO growth phases with the biology that drives them, the numbers you should expect, and the decision each phase demands from the bioprocess engineer.
Growth Curve Fitter
Paste VCD time-series data, auto-fit exponential or logistic models, and extract μmax, lag time, and doubling time for your CHO clone.
Number-Increase (NI) Phase — Exponential Growth
The CHO number-increase phase is the classical exponential growth phase. VCD rises from seeding density (0.3–0.6 × 106 cells/mL) to 15–25 × 106 cells/mL over 5–7 days in a standard fed-batch. Cell volume stays constant around 2,000–2,500 fL per cell for CHO-K1 suspension cultures. The specific growth rate μ is close to μmax — typically 0.029–0.039 h−1 (td 18–24 h) for CHO-K1 and CHO-DG44, a little faster (~0.04–0.05 h−1) for engineered high-productivity lines.
What's happening under the surface in NI:
- Glycolysis-dominant metabolism. Glucose uptake is fast; lactate is produced aerobically (the Warburg-like effect). Lactate rises from ~0 to 2–4 g/L.
- High mAb secretion machinery demand. ER and Golgi capacity is being built in parallel with biomass.
- qP is modest. Specific productivity per cell is 15–30 pg/cell/day for standard lines — later phases are more productive.
- IVCD accumulates linearly because VCD is still rising; daily IVCD increment grows each day.
NI phase — at a glance
: Day 0–5 · : 0.5 → 15–25 × 106/mL · : 0.029–0.039 h−1 · : ~2,000–2,500 fL
Bioprocess decision: start fed-batch feeding by day 3–4; monitor lactate accumulation; do not harvest here.
Size-Increase (SI) Phase — Where Titer Accrues
The size-increase (SI) phase is the hidden middle phase of the CHO growth curve. Pan et al. (2017) showed that between the end of exponential growth and the onset of stationary, CHO cells keep growing — not in number but in size. Mean cell volume rises approximately linearly with time, reaching ~6,000–8,000 fL per cell by day 8–10, roughly three-fold the NI baseline. Dry weight per cell follows the same pattern.
Metabolically, the SI phase is a shift from glycolysis to TCA-dominant flux. Coulet et al. (2022) profiled this transition in detail: specific glucose uptake drops, lactate stops accumulating and starts being consumed (the CHO lactate shift), TCA-cycle intermediates rise, and mitochondrial oxygen consumption peaks. Specific productivity qP often increases 1.5–2× relative to NI, meaning each larger cell is making more mAb per unit time than it did when it was dividing.
For a fed-batch engineer, the SI phase is where most of the final titer is actually produced. Peak VCD on day 7 and peak titer on day 12 are connected by the SI phase in between. Harvesting too early (end of NI) gives up the SI contribution; harvesting too late (deep stationary or death) costs quality.
SI phase — at a glance
: Day 5–9 · : plateau 20–25 × 106/mL · : 2,500 → 6,000–8,000 fL · : rising 1.5–2×
Bioprocess decision: this is the productive window. Maintain feed, watch lactate (shift is a positive signal), begin daily IVCD tracking for harvest prediction.
Harvest Window Predictor
Integrate VCD, viability, glucose, and lactate trajectories to find the optimal CHO fed-batch harvest day — including lactate-shift detection.
Stationary Phase — Productivity Without Growth
The CHO stationary phase begins when cell volume stops increasing and IVCD accumulation slows markedly. VCD is typically holding in the low-20s (× 106 cells/mL) in a standard fed-batch, viability is still >90 %, glucose remains supplemented via feed, and lactate is being consumed or is at a steady low level. This phase typically spans day 9–11 before viability begins to drop.
Productivity during stationary can remain high. Donaldson et al. (2021) reviewed strategies for decoupling growth and protein production in CHO cells — temperature downshift to 31–33 °C, mild hyperosmolality, and growth-arrest engineering all extend stationary productivity. Many commercial mAb processes deliberately bank titer in this phase by holding cells arrested in G1.
Stationary phase — at a glance
: Day 9–11 · : plateau · : 90–95 % · : elevated but declining
Bioprocess decision: if viability holds and IVCD is still accruing, keep running. If viability begins dropping, prepare to harvest within 24–48 h.
Death Phase — Apoptosis and Beyond
The CHO death phase begins when the balance between survival and death tips permanently. Viability falls below ~90 %, lactate may re-accumulate as stressed cells revert to glycolysis, and host cell protein, DNA, and proteases rise in the supernatant. In standard fed-batch this is usually day 11–14.
Historically, the dominant framework for CHO cell death has been apoptosis — the canonical caspase-mediated, annexin-V-positive pathway. Much of cell-line engineering in the 2000s–2010s focused on anti-apoptotic strategies (Bcl-2, Bcl-xL overexpression; caspase-3/7 knockdown) to extend stationary phase.
But Mentlak et al. (2024) showed that industrial CHO fed-batch death is often not apoptosis. Dissecting the cell-death pathways in production-scale runs, they found that the viability decline in these cultures is dominated by parthanatos and ferroptosis — oxidative, caspase-independent death modes that standard annexin V / caspase-3 assays miss entirely. This is a significant finding for anyone using flow-cytometry viability panels to decide harvest: apparently healthy cells may be committed to non-apoptotic death that shows up hours or days later as bulk viability loss.
Death phase — at a glance
: Day 11–14+ · : < 90 %, falling · : rising · : may re-accumulate
Bioprocess decision: harvest. Lactate re-accumulation, viability < 80 %, and daily IVCD increment < 7 % of peak are all triggers.
IVCD, qP and Titer Prediction
Integral viable cell density (IVCD) is the single most useful summary number for CHO fed-batch. Defined as the time-integral of VCD:
IVCD(t) = ∫0t VCD(τ) dτ
In units of 106 cell-days/mL (or 109 cell-hours/L), IVCD tracks the cumulative "cell-time" available for production. Because specific productivity qP (pg/cell/day) is roughly constant over the run in many CHO processes, final titer is approximated by:
Titer ≈ qP × IVCD
Worked example — projecting CHO titer from IVCD
A 14-day CHO fed-batch shows daily VCD of: 0.5, 1.1, 2.5, 5.8, 10.2, 15.1, 19.0, 21.0, 22.0, 22.3, 21.8, 19.5, 15.2, 10.1, 6.2 (all in 106/mL). Your cell line has a historical qP of 20 pg/cell/day. What final titer do you expect?
Step 1. Compute IVCD by trapezoidal integration of VCD over time (daily steps Δt = 1 day):
IVCD = 0.5×(0.5+1.1) + 0.5×(1.1+2.5) + ... + 0.5×(10.1+6.2)
IVCD ≈ 189 × 106 cell-days/mL
Step 2. Compute expected titer:
Titer = 20 pg/cell/day × 189 × 106 cell-days/mL
= 3,780 pg/mL × 106 = 3.78 × 109 pg/mL
= 3.78 g/L
This is a reasonable titer for a modern CHO mAb fed-batch. The Harvest Window Predictor computes IVCD automatically and flags the day beyond which further culture adds little to IVCD.
| Phase | Days | VCD (106/mL) | Cell volume (fL) | qP (pg/cell/day) | Key signal |
|---|---|---|---|---|---|
| NI (exponential) | 0–5 | 0.5 → 20 | ~2,000–2,500 | 15–30 | Lactate rising |
| SI (size-increase) | 5–9 | 20–25 plateau | 2,500 → 6,000+ | 25–50 | Lactate shift |
| Stationary | 9–11 | Plateau | Plateau | Falling, still productive | Viability still high |
| Death | 11–14+ | Falling | Falling | → 0 | Viability drop, HCP spike |
Bioprocess Decisions by CHO Growth Phase
Pulling it together, here is the practical decision framework most CHO fed-batch teams use:
- NI phase (Day 0–5): start bolus or continuous feed by day 3–4. Track glucose (keep 2–6 g/L), monitor acidification. Don't harvest.
- SI phase (Day 5–9): confirm lactate shift has occurred (metabolism turning from glycolysis to TCA-dominant); this is the productive window. Temperature downshift to 31–33 °C, if used in your process, is typically applied at the NI→SI transition.
- Stationary (Day 9–11): monitor daily IVCD increment. When ΔIVCD < 5–7 % of peak daily IVCD, further culture time is rarely worth the quality risk.
- Death (Day 11–14+): harvest. Triggers are viability < 75–80 %, lactate re-accumulation above 3–4 g/L after shift, or ≥ 3 consecutive days of post-peak VCD decline.
For the full monitoring toolkit that tells you which phase you're in, see cell growth monitoring in suspension culture. For day-by-day CHO process troubleshooting, see the CHO troubleshooting guide.
Frequently Asked Questions
What are the growth phases of CHO cells?
CHO cells in fed-batch culture pass through four phases: the number-increase (NI) or exponential phase where VCD doubles every 18–24 h; the size-increase (SI) phase where VCD plateaus but cell volume rises ~3×; the stationary phase where productivity continues but growth stops; and the death phase where viability drops and proteolysis accelerates.
What is the doubling time of CHO cells?
CHO cell doubling time is typically 18–24 h in suspension culture with chemically defined media (μ ≈ 0.029–0.039 h−1). Optimised CHO-K1 or CHO-DG44 sub-lines in fed-batch can reach 16 h doubling. This is 20–40× slower than E. coli and shapes every process decision.
What is the lactate shift in CHO fed-batch?
The lactate shift is the metabolic switch where CHO cells stop producing lactate (glycolysis-dominant) and start consuming it (TCA-dominant). It usually coincides with the NI→SI transition and precedes peak titer by 1–3 days. Re-accumulation of lactate after the shift is an early stress signal.
What is IVCD and why does it matter?
IVCD (integral viable cell density) is the time-integral of VCD, in 106 cell-days/mL. Because qP is roughly constant over the run, final titer ≈ qP × IVCD. IVCD predicts titer better than peak VCD alone because it captures how long cells were productive.
When do CHO cells enter the size-increase phase?
Around day 5–7 in a typical 14-day fed-batch, when the NI phase ends. Pan et al. (2017) characterised this phase quantitatively: volume and dry weight per cell rise ~3× linearly with time, lactate consumption begins, and TCA flux increases while glycolytic flux drops.
References
- Pan X, Dalm C, Wijffels RH, Martens DE. Metabolic characterization of a CHO cell size increase phase in fed-batch cultures. Applied Microbiology and Biotechnology (2017). DOI: 10.1007/s00253-017-8531-y.
- Coulet M, Kepp O, Kroemer G, Basmaciogullari S. Metabolic Profiling of CHO Cells during the Production of Biotherapeutics. Cells (2022) 11(12):1929. DOI: 10.3390/cells11121929.
- Donaldson JS, Dale MP, Rosser SJ. Decoupling Growth and Protein Production in CHO Cells: A Targeted Approach. Frontiers in Bioengineering and Biotechnology (2021) 9:658325. DOI: 10.3389/fbioe.2021.658325.
- Mentlak DA, Raven J, Moses T, Pybus LP, Dickman MJ, Smales CM. Dissecting cell death pathways in fed-batch bioreactors. Biotechnology Journal (2024). DOI: 10.1002/biot.202300257.
- Metze S, Ruhl S, Greller G, Grimm C, Scholz J. Monitoring online biomass with a capacitance sensor during scale-up of industrially relevant CHO cell culture fed-batch processes in single-use bioreactors. Bioprocess and Biosystems Engineering (2020). DOI: 10.1007/s00449-019-02216-4.