Chemically synthesized APIs

Think of it as inspecting the ingredients in cooking. Before charging to the reactor, this step checks at the door that the raw material or reagent is really the target compound and that its purity is within specification.

The procedure is simple. Infrared spectroscopy (FT-IR) identifies the molecular skeleton like a fingerprint, and high-performance liquid chromatography (HPLC) measures purity. Together, the two pin down both identity and quality.

The outcome of a synthesis is determined by the purity of the starting materials. If poor-quality material gets in at the first step, impurities are carried through to the later steps. The key is therefore to stop quality problems at the entrance.

Synthesis is determined by the purity of the starting materials. If poor-quality material enters the first step, impurities are carried through to the later steps. So before charging to the reaction, confirm 'is this really the target compound?' and 'is the purity within specification?'

Alternatives in practice

• Rapid identification by near-infrared/Raman spectroscopy · Bruker MPA II (NIR)/Thermo Scientific TruScan RM (handheld Raman, raw material identification through the container) — Effective for nondestructive identification through the container at receipt. Discriminating variants requires building a library first.
• Gas chromatography (volatile raw materials and solvents) · Agilent 8890 GC (FID)/Shimadzu Nexis GC-2030 — For confirming the purity/impurities of volatile liquid raw materials and solvents.
• Confirmation of physical constants such as melting point and optical rotation · Mettler Toledo MP90 melting point apparatus/Anton Paar MCP 5100 polarimeter — A simple supporting identification. Low molecular specificity, so unsuitable as a primary method.

A mid-process check to judge when to 'stop' the reaction. Like tasting a simmering dish, you check partway how far the running reaction has progressed.

The method is to draw a small amount of liquid from the reaction vessel and follow the conversion and the appearance of impurities by high-performance liquid chromatography (HPLC). This is called in-process control.

Misjudging the stop point is costly. Too early and unreacted starting material remains; too late and over-reaction increases by-products. Determine the optimal endpoint before moving on to the next operation.

If you do not check how far the reaction has progressed along the way, you will misjudge when to stop. Stop too early and unreacted starting material remains; too late and over-reaction increases by-products. Track the conversion in real time or by sampling, and move on at the optimal endpoint.

Alternatives in practice

• In situ ATR-FTIR (real-time reaction tracking) · Mettler Toledo ReactIR 702L (DiComp/SiComp probes) — Monitors reaction start and endpoint in real time without sampling. Better suited to understanding behavior than to quantitation.
• TLC (simple reaction tracking) · Merck TLC silica gel 60 F254 plates + UV cabinet (CAMAG UV Cabinet 4) — Quick at the bench. Low quantitation, so it complements the primary method.
• In-line/benchtop NMR, NIR · Bruker Fourier 80 (benchtop NMR)/Bruker Matrix-F (process NIR) — Depends on the solvent and reaction system, but allows nondestructive continuous monitoring.

A checkpoint: each time a reaction in a multistep synthesis finishes, the key intermediate or registered starting material (RSM/KSM) is taken out of the liquid, its purity is confirmed, and only then is it passed on.

It is isolated as a solid by crystallization and filtration, dried, and then judged for purity, content and impurities against the specification. If it does not pass, it does not go on to the next reaction.

Why check at every stage? Because the more impurities are removed at the intermediate stage, the more stable the quality of the final API. What is built up along the way determines the final quality.

In the standard multistep synthesis of a small-molecule API, it is usual to isolate and dry the key intermediates and registered starting materials (RSM/KSM) after the reaction ends, and to judge purity and impurity profile against the specification before moving to the next reaction. Removing impurities at the intermediate stage directly affects the quality of the final API.

Alternatives in practice

• Telescoping (carrying the intermediate into the next reaction without isolation) · Mettler Toledo OptiMax 1001 (with ReactIR for in-line concentration monitoring) — Shortens the process and improves yield. The risk is that the quality of each intermediate cannot be controlled directly.
• Intermediate purification by column chromatography · Biotage Selekt (Sfär Silica cartridges)/Teledyne ISCO CombiFlash NextGen — Effective for hard-to-separate intermediates. At large scale, cost and solvent load are high.

The liquid after the reaction is a 'hodgepodge' in which salts, catalyst, unreacted material and solvent are mixed in with the target compound. This is the primary purification that first washes out the bulk of them.

The liquid is washed after being separated into a layer compatible with water (aqueous layer) and a layer compatible with oil (organic layer). Water-soluble impurities and inorganic salts are moved to the aqueous side and discarded, and the target stays in the organic layer.

The more is removed coarsely here, the cleaner the later crystallization proceeds. It is, so to speak, the prep work that makes the finishing crystallization easier.

In addition to the target compound, the reaction liquid contains salts, catalyst, unreacted material and solvent. Separating it into aqueous and organic layers and washing first removes water-soluble impurities and inorganic salts to a large extent. The more is removed coarsely here, the easier the later crystallization becomes.

Alternatives in practice

• Activated carbon treatment + filtration (decolorization and removal of trace impurities) · Cabot NORIT powdered activated carbon + Pall SUPRApak depth filter/Sartorius Sartoclear depth filter — Effective for pigments and polar impurities. Watch for API loss and the handling of carbon.
• Quench + filtration (removal of solid by-products) · Agitated Nutsche Filter (Rosenmund RVNF/De Dietrich Guedu) — A simple way to remove salts and inorganic solids.
• Continuous countercurrent extraction (continuous liquid-liquid extraction) · Sulzer centrifugal extractor/Koch-Glitsch countercurrent extraction column — Suited to large-volume, continuous production. Capital investment and emulsion management are the challenges.

A 'polishing' step done just before crystallization. Activated carbon is added to the crude product solution and stirred, so that colored substances, residual metals and trace impurities are soaked up onto the carbon surface.

After adsorption, clarifying (polish) filtration filters off carbon grains and insoluble foreign matter. This gives a clear, clean solution, ready for crystallization.

Activated carbon treatment before crystallization is the norm in API purification. If color and trace impurities are removed first, the target crystal form can be precipitated cleanly in the crystallization that follows.

In API purification, it is standard to treat with activated carbon immediately before crystallization to adsorb and remove colored substances, residual metals and trace impurities. The clarifying (polish) filtration that follows removes carbon particles and insoluble foreign matter, and the target crystal form is precipitated from a clean solution.

Alternatives in practice

• Removal of residual catalyst (Pd, etc.) with metal scavenger resins · Johnson Matthey QuadraPure TU/Smopex fibers, SiliCycle SiliaMetS Thiol/Thiourea — Selective removal of residual Pd/metals. Decolorization is limited, so choose between it and carbon treatment as appropriate.
• In-line passage through cartridge-type activated carbon · 3M Zeta Plus activated carbon cartridges (CUNO)/Pall Seitz AKS carbon modules — Decolorization in a closed system, avoiding the addition of powdered carbon. Capacity is smaller than with depth-type carbon.

The peak of the synthesis, handling purification and solid-form building at the same time. The solution is cooled or an antisolvent is added so that only the target precipitates as crystals.

With conditions set properly, impurities stay in the mother liquor (the remaining liquid) and only the target comes out as crystals. Seed crystals are added, and precipitation is controlled while being monitored by process analytical technology (PAT).

In addition, which crystal polymorph or salt form is produced is also decided here. It is the most important stage, which also determines the solid form and thereby affects ease of dissolution, stability and ease of manufacture.

Crystallization is the most important step, handling purification and solid form determination at the same time. With conditions set properly, impurities stay in the mother liquor and only the target comes out as crystals. Moreover, which crystal polymorph or salt is produced determines solubility, stability and ease of manufacture.

Alternatives in practice

• Simple cooling recrystallization · Mettler Toledo EasyMax 102 (programmed cooling) — Simple, but control of polymorph and particle size is limited.
• Salt and cocrystal screening · Technobis Crystal16/Crystalline (multi-reactor crystallization screener) — Comprehensive exploration of solubility and polymorphs. Small scale.
• Continuous crystallization (MSMPR/continuous flow) · Alconbury Weston/Cambridge Reactor Design continuous crystallizers, monitored with Mettler Toledo PAT — Uniform particle size and higher productivity. Startup and control are complex.

The crystals produced by crystallization are still in a 'wet' state, containing mother liquor and solvent. They are separated from the liquid and the remaining solvent is driven off to finish a dry API.

After the liquid is removed by vacuum filtration or centrifugal filtration, vacuum drying removes moisture and solvent to within specification. Temperature and time are carefully set in a conical dryer or by tray drying.

Judgment is key here. Inadequate drying puts residual solvent out of specification, and overdoing it can cause the crystal polymorph to transition or decompose.

Crystals obtained by crystallization contain mother liquor and solvent. Filtration removes the liquid, and drying drives off the remaining solvent. Inadequate drying leaves residual solvent and moisture out of specification, and excessive drying can cause polymorph transition or decomposition, so temperature and time are carefully set.

Alternatives in practice

• Fluid bed drying · GEA/Glatt fluid bed dryers (GPCG series) — Fast and uniform drying. Watch for fine-powder scattering and static charge.
• Freeze drying (heat-sensitive/amorphous APIs) · SP VirTis/Millrock freeze dryers (GMP-compliant) — Effective for heat-labile products. High cost and long time.
• Rotary vacuum drying (double cone) · GEA/Italvacuum double-cone vacuum dryers — Gentle mixing-drying. More product-discharge steps than with ANFD.

A step that finely crushes the particles of dry API to the target size. In a jet mill, the API rides an air stream and the particles collide at high speed to form a fine powder.

Particle size is directly tied to how the drug works. For poorly soluble drugs, making the particles smaller increases surface area and makes the drug dissolve more readily (= be absorbed more readily).

But too small is also a problem. Particles pick up static charge and flowability drops, so bringing them to just the right size distribution is where on-site skill shows.

The particle size of an API is directly tied to how readily it dissolves (that is, how it works) and to how easily it can be handled in the formulation. For poorly soluble drugs, the particles are made smaller to increase surface area. On the other hand, particles that are too small cause static electricity and flowability problems, so they are brought to the target size distribution.

Alternatives in practice

• Pin mill/hammer mill (coarse to medium milling) · Hosokawa Alpine UPZ Fine Impact Mill/Quadro Fitzmill (hammer mill) — Down to tens of µm. Insufficient for sub-10 µm micronization.
• Wet bead mill (nanonization) · NETZSCH LabStar/Bühler wet bead mill — Effective for preparing nanosuspensions. A drying step and solvent management are added.
• Particle design by spheronization/crystallization · Controlled crystallization with Mettler Toledo OptiMax (spherical agglomeration crystallization) — No milling needed. The process development burden is heavy.

A finishing step that combines the multiple small portions (sublots) or charges after micronization into one and mixes them uniformly.

They are mixed in a bin (container) blender or tumble blender and homogenized into a single final batch in which content, particle size and impurities are the same wherever you sample.

GMP (ICH Q7, etc.) requires uniformity of the whole batch. Mix and even it out, then proceed to release specification testing. This holds down variability within the lot.

GMP (21 CFR 211 and ICH Q7) requires uniformity of the whole API batch, and it is standard for multiple micronized and dried sublots to be mixed and homogenized by final blending before being submitted to release specification testing. This assures content uniformity and limits within-lot variability.

Alternatives in practice

• Blending after cone mill deagglomeration and sizing · Quadro Comil U20/U30 (conical mill)/Frewitt sizing mill — Breaks up agglomerates and evens out particle size. Improves blend homogeneity.
• Tumble blending (double cone/V-type) · GEA/Servolift double-cone blender, Patterson-Kelley V-Blender — Gentle mixing. Take care with powders prone to segregation.

An identification test that finally proves 'whether what was made is really the target molecule.' Chromatographic retention time alone cannot guarantee structure.

Nuclear magnetic resonance (NMR) reads the skeleton and the arrangement of functional groups, and mass spectrometry corroborates the molecular weight. Layering several spectroscopic methods makes the structure certain.

A step that is the linchpin of consistent 'identity confirmation' from the starting materials to the final API. If this wavers, the foundation for discussing purity collapses.

Finally proves that 'what was made is really the target molecule.' HPLC retention time alone cannot guarantee structure, so the skeleton, functional groups and molecular weight are corroborated by several spectroscopic methods. It is the linchpin of identification from the starting materials through to the final API.

Alternatives in practice

• FT-IR (functional group identification, comparison with reference standard) · Thermo Scientific Nicolet iS20/Bruker ALPHA II — Effective for confirming functional groups and comparing against a reference. Cannot determine the full structure.
• UV-Vis spectroscopy · Agilent Cary 60/Thermo Evolution UV-Vis — A supplementary check for chromophores.
• Elemental analysis (CHN) · Elementar vario MICRO cube/Thermo Flash 2000 CHNS — Supports confirmation of the empirical formula. Cannot determine the structure itself.

The main event of quality testing, measuring the core of API purity. It looks at the 'content (assay)' responsible for efficacy and the related substances that get mixed in, together.

Content is quantified by reverse-phase HPLC/UHPLC, and related substances from synthesis and degradation are separated and tracked one by one. This reveals which impurities are present and how much.

Stability studies also follow how degradation products increase, so a 'stability-indicating method' that can properly separate degradation products is indispensable. The story of purity is told here.

How pure the API is, and which impurities are present and how much, is the core of quality. The content responsible for efficacy is measured, and related substances derived from synthesis and degradation are followed one by one. Stability studies also look at how degradation products increase, so a method that can separate the degradation products is essential.

Alternatives in practice

• LC-MS/MS (assignment and quantitation of trace impurities) · Sciex Triple Quad 7500/Waters Xevo TQ-XS — Effective for impurities with weak UV absorption and for structural assignment.
• Ion chromatography · Thermo Scientific Dionex Integrion/Metrohm 940 Professional IC — For quantifying counterions and inorganic ions.
• CAD/ELSD detection · Thermo Scientific Vanquish Charged Aerosol Detector (CAD)/Agilent 1290 Infinity II ELSD — General-purpose quantitation of compounds without UV absorption.

A special control step that watches for impurities that are carcinogenic even in trace amounts at concentrations orders of magnitude lower. It deals with a range that ordinary purity testing cannot reach.

Alkylating agents and nitrosamines are the leading examples. They are quantified at the parts-per-billion (ppb) level using methods with extremely low detection limits, such as LC-MS/MS.

Since nitrosamine contamination was discovered in the 2018 sartan drug product (antihypertensive) problem, regulation has tightened all at once. It is a test at the front line of safety.

Impurities that carry a carcinogenic risk even at very low levels need to be controlled at ppb levels, orders of magnitude lower than in ordinary purity testing. Alkylating agents and nitrosamines are representative, and regulation has tightened since the 2018 sartan drug product problem. They are reliably controlled with methods that have extremely low detection limits.

Alternatives in practice

• HS-GC-MS (volatile nitrosamines) · Agilent 8890 GC/7000E MS+7697A headspace — Effective for volatile low-molecular-weight nitrosamines.
• GC-ECD (volatile alkyl halides, etc.) · Agilent 8890 GC+ECD detector — High-sensitivity detection of halogenated compounds. Limited versatility.
• HRMS screening/process purge assessment · Thermo Orbitrap Exploris/Waters Xevo G3 QTof (HRMS) — Supports the search for unknown nitroso compounds and measured purge factors.

A safety test that confirms no trace of the solvents used in synthesis and crystallization remains in the API. It also serves as a final check that drying was sufficient.

Quantified by headspace gas chromatography (HS-GC), which warms the sample and captures the vaporized solvent. It efficiently picks up readily volatile solvents.

Toxicity differs by solvent type, so stricter limits are set for the more toxic ones. Pass/fail is judged by comparing the residual amount with the limit.

Solvents used in synthesis and crystallization remain in the API in trace amounts. Toxicity differs by type, so the more toxic the solvent, the stricter the limit set. It also serves as a final check that drying was sufficient, making it a test directly tied to safety.

Alternatives in practice

• HS-GC-MS (when identification is needed) · Agilent 8890 GC/5977B MSD+7697A headspace — Effective for identifying peaks of unknown solvents. Quantitative reproducibility is somewhat inferior to FID.
• Direct injection GC · Agilent 8890 GC-FID (direct liquid injection) — For high-boiling, hard-to-volatilize solvents. Watch for matrix contamination.
• Karl Fischer (water measured separately) · Metrohm 851 Titrando/Mettler Toledo C20S — Residual water cannot be measured by GC, so a separate method is needed.

A step that measures the 'leftovers' of metal catalysts used in synthesis. If palladium, nickel and the like remain in the API even in trace amounts, toxicity becomes a concern.

After pretreatment in which the sample is dissolved in acid, elements are quantified by inductively coupled plasma mass spectrometry (ICP-MS). Even trace metals are picked up accurately.

Permitted amounts are set for each route of administration. The elements specified by regulation are reliably measured, providing final support that catalyst removal was sufficient.

If metal catalysts used in synthesis, such as palladium and nickel, remain in the API even in trace amounts, toxicity becomes a concern. Permitted amounts are set for each route of administration, so the elements specified by regulation are reliably measured. It is also the final check that catalyst removal was sufficient.

Alternatives in practice

• ICP-OES (for high-concentration elements) · Agilent 5800/5900 ICP-OES/Thermo iCAP PRO — Simple and robust for high-concentration elements. Trace-level (ppb) sensitivity is inferior to ICP-MS.
• AAS (atomic absorption) · Agilent 240/280 AA/PerkinElmer PinAAcle 900 — For single-element targets. Simultaneous multi-element measurement is not possible.
• Colorimetric methods/simple kits (limit tests) · Merck heavy metal limit test kits/Agilent Cary 60 UV-Vis — Simple judgment in the manner of a limit test. Low quantitative accuracy and selectivity.

A test to confirm that the molecule that is the mirror image (enantiomer), with left and right reversed as if reflected in a mirror, has not been mixed in on the unwanted side. It is essential for optically active APIs.

Enantiomers, related like left and right hands, can differ in efficacy and safety. The two are separated by HPLC with a chiral stationary phase and quantified as an impurity.

Did asymmetric synthesis or chiral resolution work as intended? A stereochemical quality check that assures the result in the form of enantiomeric excess (ee).

In an optically active API, if the enantiomer (a molecule related like left and right hands) is mixed in, efficacy and safety change. The unwanted enantiomer is separated and quantified as one impurity. This test assures that asymmetric synthesis or chiral resolution worked as intended.

Alternatives in practice

• Chiral SFC (supercritical fluid chromatography) · Waters ACQUITY UPC2/Agilent 1260 Infinity II SFC+Daicel CHIRALPAK columns — Fast and low in solvent, and also advantageous for preparative work. Requires equipment investment.
• Optical rotation measurement · Anton Paar MCP 5100/JASCO P-2000 polarimeters — Simple, but unsuited to high-precision quantitation of enantiomeric excess.
• Chiral GC · Agilent 8890 GC + cyclodextrin-based chiral column (Agilent CP-Chirasil-Dex CB) — Effective for volatile chiral compounds. Limited range of application.

Even for the same molecule, a different arrangement within the crystal (polymorph) makes it behave like a different substance. Ease of dissolution, stability and absorption change.

Powder X-ray diffraction (XRPD) reads the arrangement of the crystal like a fingerprint and confirms that the target polymorph is being made consistently.

Transition to an unwanted polymorph or to the amorphous form leads directly to problems with efficacy and stability. That is why the identity of the crystal is watched lot by lot.

Even for the same molecule, a different arrangement of the crystal (polymorph) changes solubility, stability and absorption. Whether the target polymorph is being produced consistently is confirmed like a fingerprint. Transition to an unwanted polymorph or to the amorphous form leads directly to problems with efficacy and stability, so it is checked lot by lot.

Alternatives in practice

• Solid-state NMR (ss-NMR) · Bruker AVANCE NEO 400 WB+CP-MAS probe — Strong at discriminating local structure of polymorphs and amorphous forms. Instrumentation and analysis are advanced.
• Raman/FT-IR spectroscopy · Thermo DXR3 Raman/Bruker ALPHA II FT-IR — Effective for polymorph screening and mapping. Quantitative polymorph ratios require calibration.
• Polarized light microscopy (PLM) · Olympus BX53P polarizing microscope/Leica DM2700 P — Confirmation of crystallinity and crystal habit. Supports polymorph identification.

A thermal analysis step that reads the 'behavior' of the API when heated. From the melting temperature, transition temperature and weight changes, it probes polymorph stability and whether the material is a hydrate or solvate.

Differential scanning calorimetry (DSC) follows the heat flow in and out, thermogravimetry (TGA) follows the loss of weight, and Karl Fischer titration measures water at the same time.

Water is controlled separately. Because it affects stability and content calculation, it is separated from thermal behavior and measured accurately.

The temperatures of melting and transitions on heating, and changes in weight, tell whether a polymorph is stable and whether it is a hydrate or solvate. Water content is also measured accurately. Water affects stability and content calculation, so it is controlled separately.

Alternatives in practice

• Dynamic vapor sorption (DVS) · Surface Measurement Systems DVS Intrinsic/TA Instruments Q5000 SA — Effective for understanding hygroscopicity and hydrate formation behavior.
• Hot-stage microscopy (thermal microscopy) · Linkam THMS600 hot stage + Olympus polarizing microscope/Mettler Toledo FP90 — Melting and transitions are confirmed visually. Quantitative heat measurement depends on DSC.
• Loss on drying (LOD) · Mettler Toledo HC103 halogen moisture analyzer/Sartorius MA37 infrared moisture analyzer — Simple measurement of volatiles. Cannot distinguish water from solvent.

A step that measures the water contained in the API itself, as an absolute amount. The water content required in the release specification is established here.

Trace water is directly quantified by coulometric Karl Fischer titration. If needed, an oven that heats the sample to drive out the water is used with it, picking up even tightly held water.

Used to judge hydrate stoichiometry, hygroscopicity and compliance with the loss-on-drying specification. Separately from thermal analysis (DSC/TGA), it is a test that treats water as the main subject.

Water (water content) is a mandatory item in API release specifications, and trace water is quantified directly by the Karl Fischer method. Separately from the thermal behavior seen by DSC/TGA, it is used to judge hydrate stoichiometry, hygroscopicity and compliance with the loss-on-drying specification.

Alternatives in practice

• Volumetric Karl Fischer method (for items with high water content) · Metrohm 901 Titrando/Mettler Toledo V20S+Hydranal Composite 5 — Suitable for items with water of 1% or more. Higher upper limit than the coulometric method.
• Loss on drying (LOD) · Mettler Toledo HC103 halogen moisture analyzer/vacuum dryer + electronic balance — Simple, but includes volatile solvents and is not water-specific.

A physical property test that measures variation in particle size. Particle size affects dissolution, flowability and the uniformity of the final drug product, so it is an important quality attribute related to formulability.

The particle size distribution is measured by laser diffraction, and Dv10, Dv50 and Dv90 are read from the cumulative distribution from the small side. It confirms whether micronization gave the target distribution.

Especially for poorly soluble drugs, particle size is directly tied to variation in effect. That is why it is controlled as a critical quality attribute, down to the shape of the distribution.

Variation in particle size affects dissolution, flowability and the uniformity of the final drug product. Measure whether micronization gave the target distribution and, if needed, look at surface area as well. For poorly soluble drugs, particle size is directly tied to variation in effect, so it is controlled as a critical quality attribute.

Alternatives in practice

• BET specific surface area measurement · Micromeritics TriStar II Plus/3Flex (N2 adsorption) — An indicator of fine powder and adsorption surface area. Does not give the particle size distribution itself.
• Dynamic image analysis · Malvern Morphologi 4/Sympatec QICPIC — Particle size analysis accompanied by shape information. Watch sample amount and statistics.
• Sieving/microscopy · Retsch AS 200 sieve shaker/Olympus optical microscope + image analysis — A simple method for coarse particles. Low resolution in the fine-powder range.

An API is a chemical, but this step confirms there is no microbial contamination before it goes on to formulation. The required tests are chosen according to the route of administration.

The microbial limits test measures the viable count (total aerobic microbial count, total yeast and mold count) to determine how many microbes are present. This is the basic check.

When the API becomes a raw material for a sterile drug product such as an injectable, endotoxin, a pyrogen, also becomes a concern. It is confirmed by BET (LAL method), putting a double safety net in place.

An API is a chemical, but before it goes on to formulation, it is checked for microbial contamination. Especially when it becomes a raw material for a sterile drug product such as an injectable, not only the microbial count but also pyrogens (endotoxin) are a concern. The required tests are chosen according to the route of administration of the product.

Alternatives in practice

• Recombinant factor C (rFC) method · Lonza PyroGene rFC Assay/Charles River Endosafe Trillium rCR cartridges — Independent of horseshoe crabs and sustainable. Requires validation against each pharmacopoeia and method.
• Rapid microbiological methods (RMM) · bioMérieux BacT/ALERT/Rapid Micro Biosystems Growth Direct — Speeds up results. An equivalence study is required at introduction.
• Sterility test (for sterile APIs) · Sartorius Sterisart/Merck Steritest Symbio (membrane sterility test) — For sterile APIs. Not applicable to limit testing of non-sterile products.