Microbial fermentation · Process map

Filamentous fungi

This reference process map organizes production and analysis with filamentous fungi (Aspergillus, etc.) in the order fermentation → harvest → purification → characterization. The best method depends on the host, secretion level and protease management, so each step also lists alternative and complementary methods.

19 steps in 6 phases · the reference method for each step, the equipment and makers behind it, and the alternatives used in practice

Editor's note. What really matters in submerged culture of filamentous fungi is management of hyphal morphology rather than the control setpoints themselves, because pellet versus dispersed form changes both DO supply and downstream filterability. Raising agitation to gain dissolved oxygen shears the hyphae and ends up lowering productivity and separability, a classic stumbling block at transfer. The reason the rotary drum vacuum filter with precoat remains in use for primary separation on the shop floor is also a matter of physics: the viscous, compressible mycelial cake cannot be pressed dry by centrifugation. If CFU counting and microscopic morphology checks are dismissed as "old methods," seed variability is noticed only after it has been amplified in the production culture.

This map assumes Secreted protein (industrial enzyme) recovery. Steps marked ◇ change with Organic acid (citric acid) recovery. Even in the same filamentous fungus (Aspergillus, etc.) culture, the downstream splits into two branches depending on whether the target is a "secreted enzyme (high-molecular-weight protein)" or a "low-molecular-weight organic acid such as citric acid (about 192 Da)." For enzymes, UF/DF concentration → ion exchange chromatography → activity standardization and formulation is the reference method. Organic acids cannot be retained by membranes, so the process switches to a precipitation and crystallization system of calcium salt precipitation with lime → release with sulfuric acid → activated carbon decolorization → ion exchange desalting → crystallization, and the specification also changes to FCC/USP acid-base titration (purity 99.5–100.5%).

At a glance

Cell line and seed
1. 01 Production strain identification and cell bank management (MCB/WCB) (cell bank management)
2. 02 Seed culture (seed train) and control of inoculum size
Cell culture and production (upstream)
Harvest and clarification
Purification (downstream)
Formulation and fill-finish
Quality attributes and analytics

Phase 1Cell line and seed

Production strain identification and cell bank management (MCB/WCB) (cell bank management)

Toxin and metabolite production capacity differs from strain to strain, so assuring the identity of the starting strain is the foundation of safety and reproducibility.

Standard method: Master/working cell bank system
Equipment: Sanger sequencing, Ultra low freezer, LN2 storage tank
Makers: Thermo Fisher, Eppendorf, Merck, BD, IDT, Illumina, bioMérieux
Reagents and materials: Filamentous fungi media, Cryopreservation medium

Why it is the standard. The ITS region is established as the universal barcode for fungi (Schoch et al., PNAS 2012, PMID:22454494). The cell bank system follows the thinking of ICH Q5D (derivation and characterization of cell substrates), and assurance of production strain identity is also a premise of GRAS/JECFA review for industrial enzymes.

Check also. The ITS region (the standard barcode sequence for fungi) is strong for genus-level identification, but it cannot discriminate closely related species or reveal even the presence of toxin biosynthesis genes. For applications where safety is at issue, the absence of toxin-producing capacity is confirmed by other methods such as whole-genome sequencing.

About this step and the alternatives

Production with filamentous fungi begins with fixing "which strain to use" down to a single one. This is because, even within the same genus, the ability to produce toxins and metabolites differs greatly from strain to strain.

The fixed inoculum is cryopreserved in two tiers: a master bank (MCB), which serves as the parent, and a working bank (WCB), which is used in daily manufacturing. For each manufacture, material is taken from the WCB.

Why go this far? To assure that the same strain can always be the starting point across generations. If this wavers, every step wavers, so it is laid down first as the foundation.

Even within the same genus, filamentous fungi differ from strain to strain in their ability to produce toxins and metabolites. So the foundation is a system that fixes the starting strain genetically and morphologically and assures that it is the same strain across generations.

Alternatives in practice

Strain-level identification and confirmation of toxin gene clusters by whole-genome sequencing (WGS) · Illumina NextSeq 2000/MiSeq (short reads) + Oxford Nanopore GridION (long reads, genome completion) — High-precision identification that goes as far as discriminating closely related species and the presence of toxin biosynthesis genes (e.g., aflR)
Rapid species identification by MALDI-TOF MS · Bruker MALDI Biotyper sirius + Filamentous Fungi Library (cutoff 1.7)/bioMérieux VITEK MS — Species-level identification in minutes, suited to routine rapid screening (pretreatment is the key for filamentous fungi)
Morphological observation (macroscopic and microscopic) and subculture purity confirmation · Olympus BX53 upright microscope + slide culture, Leica M205 C stereo microscope — Confirms colony characteristics and conidiophore morphology; supports identification and purity assessment

Seed culture (seed train) and control of inoculum size

Variation in seed generation number, morphology and concentration directly governs the macromorphology and productivity of the main culture.

Standard method: Stepwise seed train (flask → seed tank) + viable count (CFU) plate counting and microscopic morphology confirmation
Equipment: Orbital shaking incubator, Benchtop bioreactor, Stainless tank
Makers: Merck, Sartorius, Eppendorf, Logos Biosystems, Beckman Coulter
Reagents and materials: Filamentous fungi media, Surfactants

Why it is the standard. Inoculum size and seed age are key parameters governing the macromorphology (pellet/dispersed mycelia), viscosity and mass transfer of filamentous fungi (Fungal Biol Biotechnol 2021, doi:10.1186/s40694-021-00115-6). CFU plate counting is widely adopted as the standard method for counting viable cells.

About this step and the alternatives

Stored inoculum is not put straight into a large tank. This is the "run-up" step in which cells are expanded stepwise from flask to seed tank to match the amount added to the main culture.

At each stage the number of living cells (viable count, CFU) is counted on plates and their shape is checked under a microscope. Variation in generation number, concentration and vigor is held down so that it is not carried into the production run.

If this is disrupted, the morphology and productivity of the main culture fluctuate. It is the starting point for the uniform start-up in which a fixed amount of cells with uniform vigor is added.

To start the main culture uniformly, a fixed amount of cells with uniform vigor must be added. If seed generation number, morphology or concentration varies, the morphology and productivity of the main culture fluctuate.

Alternatives in practice

Optical counting and automated image analysis of spore concentration · Beckman Coulter Multisizer 4e (Coulter principle)/Logos Biosystems LUNA-FX7 automated cell counter — More reproducible than a hemocytometer and advantageous for standardizing inoculum size
Direct inoculation from frozen spore vials (seed tank omitted) · Prepared in Thermo Fisher Mr. Frosty controlled-rate freezing containers; -80 °C stored vials are put directly into the seed tank — Shortens the seed train and improves lot-to-lot uniformity; omits the flask stage

Phase 2Cell culture and production (upstream)

Sterilization of medium and air and contamination prevention (medium and air sterilization)

In long cultures lasting several days a single contamination is fatal, so reliable sterilization of medium and aeration is the premise of yield.

Standard method: Continuous sterilization (HTST, about 140 °C, 30–120 seconds) + sterilizing filtration of aeration (0.2 µm)
Equipment: Continuous media sterilizer HTST, Vent filter (0.2 µm)
Makers: Sartorius, Pall, Getinge, GEA, SPX FLOW
Reagents and materials: Antifoam defoamer

Why it is the standard. At large scale, continuous HTST sterilization is the standard adopted because it achieves reliable sterilization while limiting thermal damage to nutrient components (ScienceDirect review "Medium Sterilization"). Aeration filtration at 0.2 µm is the industry standard for sterile aeration.

About this step and the alternatives

Filamentous fungal cultures last several days, and once even a single contaminant gets in, the culture is quickly lost. That is why this step reliably sterilizes both medium and air before the production run.

The medium is sterilized by heating for a short time only, by continuous sterilization (HTST, about 140 °C for 30–120 seconds). The air sent in is filtered through a 0.2 µm sterilizing filter to make sterile aeration.

Being thorough here is the premise of yield (the amount finally obtained). A culture whose sterility has been lost cannot be recovered later no matter how hard one tries.

Filamentous fungal cultures last several days, and once contaminants get in, the culture is quickly lost. Reliably sterilizing the medium and aeration before the main culture and assuring sterility is the premise of yield.

Alternatives in practice

Batch sterilization (in-tank 121 °C, specified time) · SIP cycle of the stirred-tank bioreactor (direct steam injection), Getinge steam sterilizer — For small to medium scale and batch media; simple equipment, but medium components degrade easily from the heat load
Filter integrity test (bubble point/diffusive flow) · Sartorius Sartocheck 5 Plus/Pall Palltronic Flowstar V — Automatically judges sterilizing filter integrity before and after use; supports GMP records

Main culture (submerged culture) and control of basic process parameters (main culture)

Temperature, pH, DO and agitation govern the amount of enzyme secreted, and holding setpoints determines lot-to-lot reproducibility and yield.

Standard method: Stirred-tank (STR) fed-batch culture + cascade control of pH, temperature, DO and aeration
Equipment: Microbial fermentation bioreactor, Inline pH and DO sensors and probes, SCADA/MES
Makers: Cytiva, Sartorius, Repligen, Siemens, Emerson
Reagents and materials: Fermentation carbon sources inducers, Filamentous fungi media, Antifoam defoamer, pH adjustment system

Why it is the standard. pH and DO significantly affect the amount of enzyme secreted by filamentous fungi, and a configuration that makes the substrate rate-limiting in fed-batch operation is the industrial standard (Bioresour Technol / Aspergillus nidulans STR paper S0960852420310646). STR fed-batch is the mainstream platform for industrial enzymes.

About this step and the alternatives

The central step in which the inoculum is put into sterilized medium and made to produce the target enzyme or metabolite in a stirred tank. It is the main event where the filamentous fungus "does its work."

The key to how it is run is fed-batch, in which nutrients are not added all at once but topped up little by little along the way. Temperature, pH (degree of acidity/alkalinity), dissolved oxygen and aeration are kept at their setpoints.

These conditions greatly affect the amount produced. Lot-to-lot reproducibility and how much is finally obtained are largely decided here.

Production of enzymes and metabolites varies greatly with conditions such as temperature, pH, dissolved oxygen and agitation. Keeping these at their setpoints determines lot-to-lot reproducibility and yield.

Alternatives in practice

Solid-state fermentation (SSF) · Solid-state fermentation equipment from Bioengineering/rotating drum and tray SSF reactors — Traditional for koji and enzyme production; low water activity suits high enzyme production by filamentous fungi
Rocking (wave) culture · Cytiva WAVE Bioreactor 25/200 (compatible with Sartorius BIOSTAT RM) — Single-use, low shear; for seed expansion and small-volume production
Continuous/perfusion culture · Sartorius BIOSTAT STR+ATF perfusion (Repligen XCell ATF) — Improves productivity by maintaining steady state; for long-term operation and continuous production

Monitoring of cell mass and cell morphology (online cell mass monitoring)

Grasps the viable cell mass and morphology of filamentous fungi, whose clumping makes turbidity unusable, so viscosity and oxygen supply problems are handled ahead of time.

Standard method: Online viable cell mass monitoring by the capacitance (permittivity) method
Equipment: Capacitance biomass probe
Makers: Sartorius, Endress+Hauser, Hamilton, Aber Instruments, Bruker
Reagents and materials: None (in situ electrode, permittivity measurement)

Why it is the standard. Even for filamentous fungi, where light scattering is hard to use, Aber has reported measured data showing that the permittivity method correlates highly with dry cell mass (R²>0.9, >0.98 with the annular type). The advantage is that it reflects only viable cells in situ.

Check also. Filamentous fungi form pellets and clumps, so turbidity cannot measure cell mass accurately, and the permittivity (capacitance) method is the standard. However, it does not distinguish live from dead cells and is affected by morphology, so confirmation by dry cell weight or microscopic observation is needed at key points.

With Organic acid (citric acid) recovery

For enzymes, pH adjustment near neutral plus cationic polymer flocculation removes cells and contaminants while protecting the protein, but for citric acid this is pretreatment for adding lime (slaked lime) directly to precipitate tricalcium citrate tetrahydrate.
Method: Alkalinization for calcium citrate precipitation by adding lime (Ca(OH)₂ milk) (to the clarified liquid after cell removal by filtration)
Reagents: Slaked lime (calcium hydroxide). The cationic flocculating polymer used on the enzyme side is not used
Check also: The direction is the opposite: in enzyme recovery the protein is kept soluble rather than precipitated

About this step and the alternatives

During culture we want to know how much the fungus has grown. But filamentous fungi form clumps and pellets, so the degree of cloudiness (turbidity) cannot measure it accurately.

So the capacitance (permittivity) method is used. It captures an electrical property that only living cells have, and so can track the amount of viable cells online.

Changes in shape (dispersed hyphae, clumps or pellets) are watched at the same time. Knowing this makes it possible to deal early with excessive viscosity or oxygen shortage.

Filamentous fungi form clumps and pellets, so turbidity cannot measure cell mass accurately. If viable cell mass and morphology (dispersed hyphae, clumps, pellets) can be grasped, viscosity and oxygen supply problems can be dealt with early.

Alternatives in practice

Dry cell weight (DCW) + offline automated image-based morphology analysis · Sartorius Cubis II precision balance + Olympus BX53/oCelloScope image analysis — Reference value for absolute amount; effective for monitoring pellet/filament morphology but offline
Estimation of metabolic activity from dissolved oxygen and off-gas (O2/CO2) (OUR/CER) · BlueSens BlueInOne Ferment off-gas analyzer/Hamilton VisiFerm DO Arc — Estimates growth and metabolic activity in real time from the oxygen consumption rate
In situ substrate and product monitoring by near-infrared/Raman spectroscopy · Bruker MATRIX-F NIR/Endress+Hauser (Kaiser) Raman RXN2 — Non-destructive simultaneous measurement of multiple components such as glucose and metabolites, for PAT/feedback control

Phase 3Harvest and clarification

Conditioning of the culture broth (pH adjustment, flocculant addition, inactivation of the production strain) → pretreatment before solid-liquid separation (conditioning)

The viscous broth of filamentous fungi clogs vacuum filtration without flocculation and pH adjustment, and the production strain also remains

Standard method: pH adjustment with acid/alkali + flocculation with inorganic salts and cationic polymers and inactivation of the production strain
Equipment: Stainless tank, Inline pH adjustment, Inline pH and DO sensors and probes
Makers: Merck, Hamilton, Alfa Laval
Reagents and materials: Filter aids and flocculants

Why it is the standard. In industrial enzyme production with filamentous fungi, floc formation by pH adjustment + flocculation before vacuum drum filtration is essential; it is the standard step that secures filtration rate and filter cake release while inactivating the production strain.

Check also. The optimum for flocculant type and dose and for treatment pH and temperature shifts with the host strain and broth properties. The conditions for inactivating the production strain are fixed after actually confirming, in downstream microbial testing, that surviving strain is not detected.

About this step and the alternatives

When culture is finished, the liquid containing the target is separated from the mass of cells. This step is the "preparation" before that.

The broth (culture liquid) of filamentous fungi is thick and sticky, and as it is, the filter cloth clogs quickly in the downstream filtration. So the pH is adjusted, and salts and polymers are added to flocculate the hyphae and make the broth easy to filter.

At the same time, the production strain is inactivated with a biocide or heat. Contaminants and residual live cells are reduced, and a modified strain is not released into the environment. It is the safety valve before the clean separation.

The mycelial broth of filamentous fungi is highly viscous, and unless it is flocculated with inorganic flocculants (calcium chloride/ferric chloride/cationic polymers) and adjusted in pH and temperature, the filter cloth clogs in the downstream rotary drum vacuum filtration and yield falls. At the same time, it is the standard pretreatment that inactivates the production strain by adding a biocide or heating, reducing bioburden and residual viable cells before clarification.

Alternatives in practice

Filter aid precoat by diatomaceous earth (body feed) addition · Imerys Celite (diatomaceous earth)/EP Minerals Celatom dosing system — Improves filterability of high-solids, viscous slurries; used together with drum filter pretreatment
Heat flocculation (cell flocculation and removal of contaminating proteins by heat treatment) · Short-time heat treatment line with plate heat exchanger (Alfa Laval) — Reduces flocculant use and, when the target is heat-stable, also denatures and removes contaminating proteins at the same time

Separation of cells/solids from the culture broth (primary separation) (primary solid-liquid separation and harvest)

The recovery strategy branches by whether the target is a secreted enzyme or intracellular, and removing solids to obtain a clarified liquid is the starting point.

Standard method: Cell removal by rotary drum vacuum filtration (precoat) or centrifugation
Equipment: Rotary drum vacuum filter and decanter centrifuge, Harvest centrifuges
Makers: Sartorius, Pall, Alfa Laval, GEA
Reagents and materials: Filter aids and flocculants

Why it is the standard. For fibrous cell mass such as that of filamentous fungi, rotary drum vacuum filtration with a filter aid is regarded as low in power use and rich in track record (Biology Discussion / downstream review). Widely adopted for primary separation of fermentation broths with many solids.

About this step and the alternatives

The primary separation step in which mycelial lumps and solids are removed together from the prepared broth to obtain a clear liquid to send downstream.

It is done by rotary drum vacuum filtration (suction through a rotating drum) or centrifugation. Both have the same aim: to separate solids and liquid cleanly.

The strategy splits here. Is the target an enzyme dissolved into the liquid, or is it inside the cells or in precipitate? This step of separating solids first is the starting point of downstream purification.

The recovery strategy changes depending on whether the target is an enzyme secreted into the culture broth or is intracellular or precipitated. The starting point is to remove mycelial mass and solids first and obtain a clear liquid that can go on to downstream steps.

Alternatives in practice

Clarification by microfiltration (crossflow membrane) · Pall Microza hollow fiber MF/Sartorius Sartocon Hydrosart 0.2 µm crossflow — No filter aid; high clarity and easy to run continuously (watch cell recovery rate and membrane fouling)
Two-stage separation by decanter + disc centrifuge · Flottweg decanter + GEA Westfalia disc separator — Finish-removes fine solids after coarse separation; effective for high-solids slurries
Cell disruption/acid dissolution and precipitate recovery (when the target is intracellular or in precipitate) · GEA Ariete high-pressure homogenizer (NS3015H, etc.) — When the target accumulates inside cells, disrupt to solubilize and recover

Secondary clarification (removal of fine solids and cell fragments by depth filtration) → polishing clarification of the filtrate (clarification/depth filtration)

Fine hyphal fragments and colloids remain in the filtrate after vacuum filtration and foul the downstream UF membrane and column

Standard method: Two-stage clarifying filtration (coarse → fine) with cellulose-based depth filters
Equipment: Depth filter, Capsule filter
Makers: Merck, Sartorius, Pall, Repligen, Alfa Laval
Reagents and materials: Cellulose + diatomaceous earth/perlite depth filter media: Pall Seitz P-Series (grades such as K250P/K900P)/Merck Millistak+ DE grade media

Why it is the standard. In fermentation broth clarification, depth filtration captures fine solids and colloids in depth and is the industry-standard secondary clarification means that protects UF and chromatography.

With Organic acid (citric acid) recovery

Citric acid is a low-molecular-weight compound of about 192 Da and passes through ultrafiltration membranes (several to several tens of kDa), so UF concentration and DF buffer exchange cannot work in principle. Concentration shifts to evaporation and crystallization.
Method: Vacuum concentration with a multiple-effect evaporator (UF/DF not applicable). Desalting is done with ion exchange resin
Equipment: Vacuum evaporation equipment (multiple-effect evaporator). UF/DF spiral membranes are not used
Check also: UF/DF (ultrafiltration + diafiltration) on the enzyme side presupposes protein retention and cannot be transferred to organic acids

About this step and the alternatives

Large solids can be removed by primary separation, but fine hyphal fragments and colloidal turbidity remain in the liquid. This is the polishing clarification step that removes them as a finish.

The liquid is passed in two stages, from coarse filtration to fine filtration, through cellulose-based depth filters (filters that capture particles through their thickness). The image is of removing ever finer particles progressively.

Why is it needed? If these are left in and sent to the next ultrafiltration, the membrane clogs quickly (fouling). It is a move that protects membrane life and flux.

Fine hyphal fragments and colloidal contaminants remain in the primary filtrate from rotary drum vacuum filtration, and applying it directly to ultrafiltration causes membrane fouling. Secondary clarification with depth filters (cellulose + diatomaceous earth/activated carbon layer) is the standard step inserted between primary separation and concentration to secure UF membrane life and flux.

Alternatives in practice

Removal of fine solids by disc stack centrifugation · Alfa Laval Clara/GEA Westfalia disc separator — No consumables; for large-volume processing; also reduces the load ahead of the depth filter
Microfiltration (0.2–0.45 µm hollow fiber/flat sheet) · Pall Microza hollow fiber MF/Repligen TangenX SIUS flat sheet — Final adjustment of clarity; stabilizes as pretreatment for downstream sterile filtration

Concentration of the target and buffer exchange (UF/DF) (concentration and buffer exchange/UF, DF)

Concentrates the dilute, large-volume clarified liquid with a membrane and removes salts and small molecules, making downstream purification and formulation efficient.

Standard method: Concentration by ultrafiltration (UF) + desalting and buffer exchange by diafiltration (DF)
Equipment: TFF system
Makers: Cytiva, Merck, Sartorius, GEA, Buchi
Reagents and materials: TFF cassette

Why it is the standard. Centrifugation + UF is the standard approach for concentrating microbial enzyme liquids, demonstrated with β-mannanase and others (ResearchGate 318471435). Matching the MWCO to the target's molecular weight achieves concentration and removal of small molecules simultaneously.

Check also. For ultrafiltration of low-molecular-weight targets, a wrong membrane choice (MWCO) lowers yield, so the membrane cutoff and recovery are measured for each product before deciding.

About this step and the alternatives

A clear liquid has been obtained, but the target is dilute and the volume is too large to handle as it is. This step makes it small and concentrated and also adjusts the composition.

Concentration is by ultrafiltration (UF, in which the membrane retains only the target and water is removed). Diafiltration (DF) then washes out salts and low-molecular-weight impurities and replaces the buffer with one that is easy to handle.

Compacting the intermediate liquid here makes the downstream chromatography purification and formulation proceed far more efficiently. It is the bridge to the next step.

The clarified liquid has a dilute target and a large volume, so it cannot be handled as is. Concentrating with a membrane and washing out salts and low-molecular-weight impurities makes later purification and formulation efficient.

Alternatives in practice

Concentration by salting out/organic solvent precipitation · Stirred crystallization/precipitation tank + decanter centrifuge (ammonium sulfate, ethanol/acetone addition) — Allows large-volume concentration without membranes; desalting and solvent recovery steps are needed after recovery
Vacuum concentration (evaporation) · GEA/Buchi vacuum thin-film (falling film) evaporator — High-ratio concentration of heat-tolerant products; low temperature suppresses enzyme inactivation

Phase 4Purification (downstream)

Germ reduction (bioburden reduction filtration) → pretreatment before chromatography load (bioburden reduction filtration)

Lowers viable cells and microbial load of the concentrate before column loading to protect the resin and the process

Standard method: Bioburden reduction filtration with a 0.2 µm sterilizing-grade membrane
Equipment: Sterile filter (0.2 µm), Capsule filter
Makers: Merck, Sartorius, Pall, Merck Millipore
Reagents and materials: PES (polyethersulfone) double-layer membrane cartridge (Sartopore 2)/cellulose acetate double-layer membrane (Sartobran P, high recovery)

Why it is the standard. Bioburden reduction filtration of concentrated enzyme liquid is a standard bioburden control step that lowers microbial load and protects the chromatography resin and the process.

With Organic acid (citric acid) recovery

Enzymes are separated and polished by ion exchange chromatography based on charge differences, but the main axis of capture and purification for citric acid is not chromatography but a precipitation and crystallization system of calcium salt precipitation → acid release → decolorization → crystallization.
Method: Calcium citrate precipitation → release with sulfuric acid (gypsum by-product) → activated carbon decolorization → desalting with strong acid/weak base ion exchange resin → crystallization (corresponds to [11]). Column chromatography purification with charge-based separation is not the main step
Equipment: Precipitation/filtration tank, activated carbon tower, ion exchange resin tower, crystallizer. Ion exchange chromatography columns for proteins are not used
Reagents: Sulfuric acid, activated carbon, ion exchange resin. Salt gradient buffers for protein elution are not needed
Check also: In recent years there are also alternative routes that replace the lime-sulfuric acid method with solvent (reactive) extraction (amine extractants such as trilaurylamine + diluent)

About this step and the alternatives

The concentrated intermediate liquid is full of nutrients and is in a state where bacteria multiply easily. If it is run straight onto chromatography (column purification), it will foul the resin.

So before it goes onto the column, it is passed through a 0.2 µm sterilizing-grade membrane to remove microorganisms and lower the amount of bacteria (bioburden). This is called sterilizing (germ reduction) filtration.

In industrial enzyme purification, inserting this step before the column is standard. It is a precaution that makes the resin last longer and keeps downstream steps clean.

The intermediate liquid concentrated by ultrafiltration is nutrient-rich and microorganisms grow easily in it, and loading it as is onto chromatography causes resin fouling and a rise in bioburden. Bioburden reduction (germ reduction) filtration with a 0.2 µm membrane to lower bioburden before column load is the standard step before entering the purification and polishing steps of industrial enzymes.

Alternatives in practice

Two-stage germ reduction filtration with depth + 0.45/0.2 µm membrane · Pall SUPRAcap depth + Sartopore 2 0.2 µm capsule in series — Extends membrane life and avoids clogging under high bioburden/particle load
Removal of pigments and low-molecular-weight contaminants by combined activated carbon treatment · Cabot Norit activated carbon column/powdered activated carbon addition + depth filtration — Simultaneously reduces pigments, odor and low-molecular-weight impurities; improves enzyme liquid quality

Chromatographic purification and polishing (for high-purity products) (chromatographic purification and polishing step)

For high-value uses such as food and diagnostics, contaminating proteins and aggregates are removed, and the separation mode is chosen to suit the target.

Standard method: Ion exchange
Equipment: Chromatography system, Empty column hardware, Membrane chromatography
Makers: Cytiva, Sartorius
Reagents and materials: AEX resin, CEX resin, HIC resin, Preparative SEC resin

Why it is the standard. Combining IEX, HIC and SEC is a standard configuration in industrial enzyme purification and was also used in a comparative laccase purification study (PMC6751154). SEC is the standard for aggregate removal and polishing.

Check also. Industrial crude enzymes are often stopped here, and chromatographic purification is an option for uses that need high purity, such as food and diagnostics. The separation mode (ion exchange, etc.) is reselected according to the isoelectric point and hydrophobicity of the target protein.

With Organic acid (citric acid) recovery

This precipitation-crystallization step is the core recovery and purification step on the organic acid (citric acid) side and is not performed for secreted enzymes (enzymes skip this step and are purified by chromatography/UF systems).
Check also: This step does not apply to the enzyme recovery route. Citric acid is the representative case, but calcium salt precipitation and reactive extraction are also used for other filamentous fungal/microbial organic acids such as gluconic acid and lactic acid

About this step and the alternatives

The chromatographic purification and polishing step that removes contaminating proteins and aggregates and finishes the target enzyme at high purity.

A separation mode such as ion exchange is chosen to match properties of the target protein such as its charge. The image is of holding back only what is wanted and letting the unneeded pass through, thereby separating them.

However, industrial crude enzymes are often stopped before this stage and not taken this far. This finish works in food, diagnostic and high-value uses where purity is demanded.

Industrial crude enzymes are often stopped here, but for food, diagnostic and high-value uses, contaminating proteins and aggregates must be removed. The separation mode is chosen according to the properties of the target protein.

Alternatives in practice

Affinity chromatography (tag/substrate affinity) · Cytiva Capto resin family (e.g., Ni Sepharose excel for His-tag)/substrate analog immobilized resins — High purity and high selectivity in a single step; effective for specific enzymes and tagged recombinants
Membrane chromatography/mixed mode · Sartorius Sartobind Q/S membrane adsorbers/Cytiva Capto MMC, Capto adhere (mixed mode) — High flow rate and disposable; advantageous for flow-through purification and removal of hard-to-separate impurities
Fractional precipitation · Stirred precipitation tank + centrifugation (stepwise addition of ammonium sulfate/ethanol) — Low cost and large-volume processing; reduces resin load as crude purification or pretreatment

(For metabolites) purification by precipitation, crystallization and decolorization (crystallization and decolorization purification)

Low-molecular-weight metabolites such as citric acid take a route separate from proteins, reaching specification purity through precipitation, decolorization and crystallization.

Standard method: Calcium citrate precipitation with lime → release with sulfuric acid → activated carbon decolorization → crystallization (citric acid as the representative example)
Equipment: Crystallizer, Laboratory centrifuges, Reaction and extraction equipment
Makers: DuPont, GEA, Andritz, Sulzer
Reagents and materials: Activated carbon decolorizing adsorbents, AEX resin

Why it is the standard. The classical recovery method for citric acid is established as lime precipitation → sulfuric acid treatment → decolorization → crystallization (Bioresour Technol 2020 S0960852420317004; J Ind Microbiol Biotechnol 2025 kuaf007). A reference process followed for many industrial organic acids.

Check also. Crystallization by the lime-sulfuric acid method is the classical standard, but in recent years there are also alternative routes replacing it with reactive extraction using amine extractants. The premise is confirming that the precipitant and treatment chemicals do not remain in the product.

With Organic acid (citric acid) recovery

Enzymes are made into liquid formulations by adding stabilizers, standardizing in activity units and bioburden-reducing filtration so as not to inactivate them, but citric acid is a dry solid product specified by crystal purity, and activity standardization and stabilizers are unnecessary.
Method: Citric acid is made into a crystalline product (anhydrous/monohydrate) by crystallization, centrifugation and drying. Stabilizer addition, activity standardization and bioburden-reducing filtration, as for enzymes, are not performed
Reagents: Stabilizers (polyols, salts, preservatives) are unnecessary. The product is crystalline citric acid itself
Check also: The stabilizers, preservatives and activity titer adjustment of liquid enzyme formulations are unnecessary for organic acid crystals

About this step and the alternatives

If the target is a low-molecular-weight metabolite such as citric acid, the story changes. This is a step that raises purity by a route different from that for proteins.

In the representative example of citric acid, lime is first added to precipitate it as a calcium salt, which is then released again with sulfuric acid. Activated carbon then removes the color, and it is taken out as crystals (crystallization).

Separate as a salt, decolorize, crystallize. These three stages bring the product closer to a purity that meets specification.

For low-molecular-weight metabolites such as citric acid, purity is raised by a route separate from protein purification. The product is precipitated as a salt, pigments are removed with activated carbon, and it is taken out as crystals, approaching specification purity.

Alternatives in practice

Direct recovery by ion exchange/electrodialysis · DuPont Amberlite resin columns/PCCell, Eurodia electrodialysis stacks — Recovers organic acids directly without lime or sulfuric acid, reducing waste gypsum (clean process)
Solvent extraction (reactive extraction) · Countercurrent extraction column (Sulzer/Koch-Glitsch)/mixer-settler, tertiary amine extractants — Selective recovery and concentration at the same time; suited to continuous operation

Phase 5Formulation and fill-finish

Liquid formulation (stabilization, standardization) (liquid formulation and filling)

Adds stabilizers to enzymes that readily lose activity and standardizes activity so customers can use the product reproducibly at a consistent titer.

Standard method: Stabilizer addition + activity standardization + bioburden reduction filtration (liquid enzyme formulation)
Equipment: Single-use mixers, Sterile filter (0.2 µm), pH meter
Makers: Merck, Sartorius, Pall, Hamilton, Roquette
Reagents and materials: Excipients and stabilizers

Why it is the standard. Stabilization and titer standardization of liquid enzymes with polyols, salts and preservatives is the standard approach for industrial enzyme preparations. For purity and microbial specifications, the Enzyme Preparations monograph of the Food Chemicals Codex (published by USP; the current edition is the 14th, available online since the 13th edition of 2022) provides the framework. FDA shows how to present data in "Enzyme Preparations: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices" (a recommendation, not a requirement).

About this step and the alternatives

A step that turns the purified enzyme into a liquid product that can be shipped as is. The trouble with enzymes is that they tend to lose activity before they are used.

So stabilizers are added to preserve activity, and the liquid is diluted and standardized to the target activity value. Bioburden reduction filtration is applied last to finish.

The aim is that customers can use the product with the labeled performance in the same way even when the lot changes. Making the shipping titer (strength of effect) uniform is the quality promise.

Enzymes tend to lose activity before use, and the shipping titer must also be made uniform. Adding stabilizers and diluting/standardizing to the target activity value lets customers use the product reproducibly.

Alternatives in practice

Sterilization and final concentration adjustment by crossflow · Sartorius Sartoflow/Merck Pellicon TFF + 0.2 µm sterilizing filter — Integrates concentration, desalting and sterilization; maintains activity without heat
Activity blending with diluents/carriers · Inline metering and mixing skid (mass flow meter + static mixer) — Standardizes to specified activity; supports blended formulations of multiple enzymes

Powderization of the concentrated enzyme liquid by spray drying → drying stage before solid formulation (spray drying and powderization)

The concentrate must be turned into a stable dry powder by spray drying before fluid bed granulation

Standard method: Dry granulation of the concentrate by rotary disc/two-fluid nozzle spray drying
Equipment: Spray dryer
Makers: Roquette, GEA, Martin Christ, SPX FLOW, Glatt
Reagents and materials: Excipients and stabilizers

Why it is the standard. Spray drying is the standard drying means for solidifying industrial enzymes, giving a powder that can be granulated while preserving enzyme activity at an inlet of around 140 °C and outlet of around 55 °C.

About this step and the alternatives

To make a powder or granule product, the liquid must first be dried into powder. Spray drying is the entry to that.

Carriers and stabilizers are added to the UF concentrate, which is sprayed as a mist from a rotary disc or nozzle. The droplets are dried instantly by hot air and formed into a dry powder (core particles).

This powder becomes the starting material for the next fluid bed granulation and coating. It is the indispensable intermediate drying that connects liquid concentration and granulation.

As a premise for solid (granule) formulation, a step that adds carriers and stabilizers to the UF concentrate and spray dries it into a dry powder (core particles) is normally included. Because fluid bed granulation and coating (existing [10]) start from this dry powder, spray drying is an essential intermediate step between liquid concentration and granulation.

Alternatives in practice

Fluid bed spray granulation (drying and granulation integrated) · Glatt GPCG/SPX FLOW Anhydro spray granulator — Gives low-dust granules in a single step; low-temperature drying favors preserving activity
Freeze drying (for heat-labile and high-value enzymes) · GEA/Martin Christ Epsilon industrial freeze dryers — Minimizes heat inactivation; for high-value, small-quantity products (high cost)

Solid formulation (granulation, drying, coating, dust control) (solid formulation and granulation)

Powdered enzymes can be inhalation allergens, so low-dust granulation and coating achieve both safety and storage stability.

Standard method: Low-dust enzyme granulation by fluid bed granulation + coating (spray drying as needed)
Equipment: Fluid bed granulation coating, Spray dryer
Makers: Roquette, GEA, SPX FLOW, Glatt
Reagents and materials: Excipients and stabilizers

Why it is the standard. Fluid bed granulation and coating is an established technology for low-dust enzyme particles, detailed in a family of patents (US6933141 "Enzyme granulate," US6410287 "Enzyme particles"). Spray drying is the representative drying method for turning liquid into particles.

With Organic acid (citric acid) recovery

Citric acid is not an enzyme protein, so a substrate-specific enzyme activity assay (activity units) cannot be defined. Potency is specified by molar purity (content).
Method: Content of 99.5–100.5% is confirmed by acid-base titration with sodium hydroxide standard solution (phenolphthalein indicator, 1N NaOH 1 mL = 64.03 mg citric acid). Conforms to the FCC/USP citric acid monographs
Equipment: Automatic potentiometric titrator. A spectrophotometer for colorimetric enzyme assays is not needed
Reagents: 0.1–1N NaOH standard solution, phenolphthalein TS. Enzyme substrates and chromogenic substrates are not used
Check also: The enzyme side's "defined activity unit (FCC colorimetric/titrimetric enzyme assay)" does not apply to organic acids

About this step and the alternatives

A step that finishes the dry powder into easy-to-handle granules. What matters here is the industry common sense of "not generating dust."

Powdered enzymes can become allergens if inhaled. So fluid bed granulation consolidates the powder into low-dust granules, and a protective coat on the surface suppresses dust generation.

Coating also serves to raise storage stability. Safe, long-lasting and easy to handle. This step decides the final form of the product.

Powdered enzymes can be inhalation allergens, so making low-dust granules is common sense in the industry. A protective coat further raises storage stability and gives a particle that is easy to handle.

Alternatives in practice

Granulation by spray drying alone · GEA PHARMA-SD (PSD series) — Fewer steps, but granule strength and dust reduction are inferior to fluid bed
Extrusion granulation (extrusion/marumerization) · LCI/Caleva extruder + spheronizer (marumerizer) — Dense, spherical granules of uniform diameter; high mechanical strength

Phase 6Quality attributes and analytics

Quantitation and standardization of enzyme activity (potency testing)

An enzyme's value is determined by activity, not mass, and measuring reaction rate with a specified substrate and conditions assures the labeled potency.

Standard method: Substrate-specific enzyme activity assay (FCC-specified colorimetric/titration methods, defined activity units)
Equipment: UV-Vis spectrophotometer, Plate reader
Makers: Agilent, Shimadzu, Molecular Devices, Metrohm, BMG LABTECH
Reagents and materials: Specified substrates: soluble starch (for α-amylase), p-nitrophenyl derivatives (pNP-glucoside, etc., Sigma-Aldrich), casein (for protease, Hammarsten), Megazyme substrate kits (Ceralpha, etc.), standard enzymes, reaction buffers (acetate/phosphate buffer at the specified pH)

Why it is the standard. The Enzyme Preparations monograph of the Food Chemicals Codex gives how activity is measured and how units are defined, and potency labeling of industrial enzymes follows it (the current edition is the 14th). Substrate-specific assays are the core of potency assurance.

Check also. The value of an enzyme is determined by activity, not mass, but activity assays depend on measurement conditions such as substrate, temperature and pH. When comparing labeled potencies, the definition of the specified activity unit and the measurement method must be aligned.

With Organic acid (citric acid) recovery

For low-molecular-weight organic acids, SDS-PAGE and total protein quantitation are out of scope. Purity is confirmed by chemical impurity tests such as oxalate, sulfate, heavy metals, residue on ignition and water.
Method: Oxalate limit test, sulfate/chloride tests, heavy metals (≤0.001%), residue on ignition (≤0.05%), water (Karl Fischer/loss on drying, anhydrous product ≤0.5%). SDS-PAGE/BCA not applicable
Equipment: Karl Fischer moisture meter, muffle furnace for residue on ignition, ICP/colorimetric methods. Electrophoresis equipment is not used
Reagents: Zinc and hydrochloric acid (oxalate test) and others. Coomassie/silver staining reagents and BCA/Bradford reagents are not needed
Check also: Replaced by the impurity tests of the citric acid monographs, whose specifications are harmonized across FCC/USP/EP

About this step and the alternatives

The value of an enzyme product is determined not by weight but by "how well it works." This step measures and standardizes the strength of that effect (potency).

The speed of the reaction is measured with colorimetric or titration methods under a specified substrate and conditions. The point is to use a dedicated assay (measurement method) for each substrate.

Here the product is assured to have the labeled potency. It is the backing that lets customers use it trusting that "this product works this much."

The value of an enzyme product is determined not by mass but by "activity." Reaction rate is measured with a reference substrate and conditions to assure that the product matches its labeled potency.

Alternatives in practice

Continuous photometric kinetic assay · Agilent Cary UV-Vis (kinetics mode)/kinetic measurement on a plate reader — Accurately quantifies specific activity from the initial rate; more reproducible than endpoint methods
Relative potency measurement against an international standard/in-house standard · Plate reader + standard curve (reference standard enzyme) — Standardizes potency between lots as relative values; used for conversion to FCC units

Confirmation of identity, purity and contaminating proteins (protein products) (purity and contaminating protein confirmation)

Visualizes, by molecular weight pattern and purity, the presence of the target enzyme and the low level of contaminating proteins, backing manufacturing consistency.

Standard method: SDS-PAGE (with silver staining as needed) + total protein quantitation (BCA/Bradford)
Equipment: CE-SDS, Plate reader
Makers: Bio-Rad, Agilent, Bruker, Thermo Fisher, Tosoh
Reagents and materials: Precast gels: Bio-Rad Mini-PROTEAN TGX/Thermo Fisher Invitrogen NuPAGE Bis-Tris, staining: Bio-Rad Coomassie R-250/Thermo Fisher SilverQuest silver stain, quantitation: Thermo Fisher Pierce BCA Protein Assay (P/N 23225)/Bio-Rad Bradford, molecular weight markers: Bio-Rad Precision Plus Protein

Why it is the standard. SDS-PAGE (with silver staining if needed) is the standard for evaluating contaminating proteins and purity, alongside CE and RPLC (QC review). BCA/Bradford are the reference methods for total protein quantitation. CE-SDS is an alternative with high quantitative reproducibility.

With Organic acid (citric acid) recovery

For citric acid, the safety specification centers not on multi-analyte mycotoxin analysis but on conformity to food additive monographs built on chemical impurities (oxalic acid, heavy metals, etc.) and microbial specifications.
Method: Specification tests of the FCC/JECFA citric acid monographs (oxalate, heavy metals, residual solvents, etc.). LC-MS/MS multi-analyte mycotoxin analysis is not the main safety test
Check also: Because crystallization, decolorization (activated carbon) and acid treatment remove secondary metabolites, mycotoxin analysis carries less weight than for enzyme preparations (however, safety confirmation of the production strain is common to both)

About this step and the alternatives

The target enzyme is there properly and unwanted proteins are few. For a protein product, this has to be confirmed.

SDS-PAGE (electrophoresis that lines proteins up by size) makes the molecular weight pattern and purity visible, and the BCA or Bradford method counts total protein.

This shows how much contaminating protein there is and whether manufacturing has varied. It is, so to speak, a transcript that shows the product's identity and consistency.

It must be shown that the target enzyme is correctly present and that unwanted proteins are few. Visualizing the molecular weight pattern and purity backs manufacturing consistency and quality.

Alternatives in practice

Purity and molecular weight by CE-SDS (capillary electrophoresis) · Sciex PA 800 Plus/Agilent 5200 Fragment Analyzer — Excellent quantitation and reproducibility and can be automated; purity evaluation more precise than SDS-PAGE
Purity and aggregate evaluation by reversed-phase/SEC-HPLC · Agilent 1290 Infinity II Bio LC + Agilent AdvanceBio SEC/Tosoh TSKgel columns — Separates and quantifies aggregates and fragments; effective for stability testing
Identity confirmation by peptide mapping/mass spectrometry · Thermo Fisher Orbitrap Exploris/Bruker timsTOF + trypsin digestion — Identity confirmation at the primary structure level; the most reliable identification method

Safety testing for mycotoxins and secondary metabolites (mycotoxin safety testing)

In filamentous fungi that can produce toxins depending on strain and environment, confirms at the detection-limit level that no toxins are detected in the final preparation.

Standard method: Mycotoxin multi-analyte analysis by LC-MS/MS
Equipment: LC-MS (liquid chromatography mass spectrometry), Mycotoxin analysis
Makers: Merck, Agilent, Waters, Sigma-Aldrich
Reagents and materials: Mycotoxin standards: Romer Labs/Merck (Sigma-Aldrich) certified standards, immunoaffinity columns: R-Biopharm Rhône EASI-EXTRACT/11+Myco MS-PREP, Vicam AflaTest, extraction solvents (acetonitrile/water/methanol), 13C isotope-labeled internal standards (Romer Labs Biopure)

Why it is the standard. JECFA requires that fungal-derived food enzymes be free of detectable aflatoxin B1, ochratoxin A, sterigmatocystin, T-2 and zearalenone (Toxicol Appl / related review PMID:15041150). LC-MS/MS is the reference for multi-analyte confirmatory analysis.

Check also. Aspergillus and others can produce toxins depending on strain and culture environment, so the absence of toxins is confirmed to the detection-limit level by LC-MS/MS (liquid chromatography mass spectrometry). The toxin items targeted are set according to use and regulation (food, feed).

About this step and the alternatives

Filamentous fungi such as Aspergillus may produce mycotoxins (fungal toxins) depending on strain and environment. That is why we confirm "no toxin remains."

LC-MS/MS (liquid chromatography + mass spectrometry) analyzes multiple toxins together and confirms that they are "not contained" down to levels right at the detection limit.

Because the product is used in food and feed, this cannot be compromised. Showing that no toxin remains is the most important linchpin of safety assurance.

Aspergillus and others can produce toxins depending on strain and environment. Confirming at the detection-limit level that the final preparation contains no toxins is the linchpin of safety assurance for food and feed uses.

Alternatives in practice

Single-analyte screening by ELISA (immunoassay) · R-Biopharm RIDASCREEN/Romer Labs AgraQuant ELISA kits + plate reader — Rapid on-site, high-sample-count screening; confirmation performed by LC-MS/MS
HPLC with fluorescence detection (derivatization) · Agilent 1260 Infinity II HPLC + FLD, post-column derivatization (KOBRA cell/photochemical reactor) — Established for quantitation of a single class such as aflatoxins; for facilities without MS

Confirmation of microbiological quality (viable count, pathogens, absence of production strain) (microbiological quality testing)

Shows the absence of contaminants, pathogens and residual live production strain, securing hygiene safety and prevention of environmental release of modified strains.

Standard method: Total viable count, coliforms/E. coli and Salmonella tests + confirmation of production strain inactivation and non-detection (FCC/pharmacopoeia compliant)
Equipment: CO2 incubator, Real-time PCR systems, Bioburden test
Makers: Merck, Memmert, bioMérieux, Thermo Fisher, Applied Biosystems
Reagents and materials: Selective/differential media: Oxoid/Merck XLD, RVS (for Salmonella), VRBA, VRBG (for coliforms), TBX (E. coli), diluent (buffered peptone water, BPW), PCR detection kits: Thermo Fisher SureTect/bioMérieux GENE-UP (Salmonella, E. coli)

Why it is the standard. Upper limits for viable count, Salmonella and coliforms, and confirmation of the absence of antibiotics and toxins, fall within the scope covered by the Enzyme Preparations monograph of the Food Chemicals Codex and national specifications. Specific numerical values differ by edition and national specification, so confirm the edition that applies. If the production strain is a modified organism, confirmation of inactivation and non-detection is a premise.

Check also. It must be shown not only that contaminants and pathogens are absent but that no living production strain remains (for a modified strain, prevention of environmental release). Inactivation and non-detection of the production strain are most reliably confirmed with a detection system specific to the culture-derived strain.

About this step and the alternatives

Are there contaminants, pathogens or still-living production strain left in the preparation? This is the microbiological quality check that confirms it.

Total viable count, coliforms/E. coli and Salmonella are tested. In addition, it is confirmed against criteria such as FCC that the production strain is properly inactivated and not detected.

There are two aims: hygiene safety, and not releasing modified strains into the environment. It is the final gate before shipment that secures both at once.

It must be shown that no contaminants, pathogens or living production strain remain in the preparation. This concerns both hygiene safety and prevention of environmental release of modified strains.

Alternatives in practice

Specific detection of production strain and pathogens by real-time PCR · Thermo Fisher QuantStudio 5/bioMérieux GENE-UP system — Specifically and rapidly confirms non-detection of the production strain and detects pathogens; complements culture methods
Rapid microbiological methods (automated luminescence, impedance) · bioMérieux TEMPO (MPN)/Neogen Soleris (optical) — Delivers total viable count and coliform results in a short time; for routine quality control

The reference (“gold standard”) method is the one broadly recognized in published practice and guidelines; your process, product and regulator may justify another. Makers and products are examples, not endorsements. This map is translated from the Japanese edition, where it is updated first (Japanese version).