iPS cells

This is the starting point of everything. Cells from the blood or skin of a patient or healthy donor are wound back to a stem cell state in which they "can become anything". This is the first move in making iPS cells.

How is it done? The cells receive 4 genes (OCT4, SOX2, KLF4, c-MYC), which switch the young-cell program back on. Sendai virus is used as the carrier.

The crucial point is choosing a carrier that "leaves no damage on the chromosomes". This virus does not integrate into genes and disappears naturally as cells proliferate. The safety of later steps is largely decided by this choice.

The starting step that returns somatic cells from a patient or healthy donor to a pluripotent state. Choosing a method that leaves no damage in the genome greatly affects the safety of later steps. Blood or fibroblasts are reprogrammed by introducing the 4 factors (OCT4, SOX2, KLF4, c-MYC).

Alternatives in practice

• Episomal plasmid method (oriP/EBNA1) · Thermo Fisher Epi5 Episomal iPSC Reprogramming Kit (A15960) + Lonza 4D-Nucleofector (P3 Primary Cell Kit) — No virus used. Requires electroporation, and initial efficiency is somewhat lower than Sendai
• Synthetic modified mRNA method · Stemgent/Reprocell StemRNA-NM Reprogramming Kit (00-0076) — Smallest footprint/integration risk. Daily transfection is required and the workload is large
• Integrating retroviral/lentiviral method · Merck/Sigma STEMCCA Lentivirus Reprogramming Kit (SCR510) family — High efficiency, but genomic integration occurs, so unsuitable for clinical iPSC. Limited to research use

When reprogramming succeeds, many clumps of cells (colonies) appear on the dish. This is the step of picking out only the best group that has remained undifferentiated.

The selected cells are expanded, and keeping them undifferentiated while growing them is the difficult part. Using defined media and scaffolds free of animal-derived components is a prerequisite for clinical use.

The expanded cells are frozen and stored in two tiers: the parent stock (MCB) and the working stock for daily use (WCB). The source of all lots made from now on is here, so this one move lays the foundation for both quality and production volume.

Good-quality clones are selected from the colonies that appear after reprogramming and expanded while remaining undifferentiated. A cell bank that will be the source of all later lots is made and cryopreserved. Using defined media and substrates free of xenogenic components is a prerequisite for clinical use.

Alternatives in practice

• Laminin-521 single-component matrix · BioLamina rhLaminin-521 (CT521, xeno-free) — Single component with high reproducibility, suited to clonal culture. High cost
• Alternative inhibitor / ROCK-free · STEMCELL CloneR2 (100-0691, survival-promoting supplement) — Raises single-cell survival more than Y-27632 alone. Does not depend on antibodies
• Automated culture / closed-system platforms · Cytiva/Sartorius Ambr series, or Lonza Cocoon / Thermo Fisher CTS Rotea — Reduces human-to-human variability in a closed system. Large capital investment

An allogeneic product given to other people is made from 1 lot for many patients. Planar culture with rows of dishes cannot reach the numbers. This is the step that gains volume in one go.

The method is agitated culture in which cells are formed into small clumps (aggregates) and kept floating in the liquid. In a reactor with an ingeniously shaped impeller, cells are expanded uniformly while the shear of the water flow that damages cells is suppressed.

Whether this goes well decides the scale of commercial production. It is the decisive point that bridges a few flasks in the lab to lots for many patients.

Because an allogeneic product makes doses for many patients from 1 lot, mass expansion beyond planar culture is required. Agitated systems that limit shear stress while keeping aggregates uniform are used. This is the key step for commercial production.

Alternatives in practice

• Microcarrier suspension culture · Cytiva Cytodex 1 / Corning Synthemax II microcarriers + stirred tank — Effective for adherence-dependent cells. A carrier separation step is needed at harvest
• Impeller-type stirred-tank bioreactor · Sartorius Ambr 250 High Throughput / Univessel SU, Eppendorf BioBLU 3c — The standard in process development. Shear stress is somewhat higher than in Vertical-Wheel
• Hollow fiber / perfusion bioreactor · Terumo BCT Quantum Flex Cell Expansion System — High-density, closed-system perfusion. Limited by the scale ceiling and suitability for aggregate culture

The "getting ready" before the clumps of cells grown in the reactor are sent on to the next differentiation or freezing. The culture fluid and spent reagents are washed away and the cells are brought to a tight, concentrated state.

A closed-system centrifuge with no contact with outside air is used. In counterflow centrifugation, where opposing liquid flows meet, the cells are recovered gently while the liquid they were in is replaced with the target solution.

It is unglamorous but effective. Because no strong force is needed, living cells are not lost and high recovery and high viability are both achieved. It is the key preparation for handing the cells to the next step in a clean state.

iPSC aggregates expanded in a Vertical-Wheel bioreactor need to have the culture supernatant and residual reagents removed, and the cells concentrated and buffer-exchanged, before proceeding to differentiation induction or freezing. Closed-system counterflow centrifugation is the standard step that handles this with low shear, high recovery and high viability.

Alternatives in practice

• Counterflow centrifugal elutriation cell washing · Beckman Coulter JE-5.0 elutriation rotor (Avanti J-26S XP) — Size fractionation is also possible, but it leans toward an open system and GMP closure takes ingenuity
• Hollow fiber tangential flow filtration (TFF) · Repligen KrosFlo KR2i / Spectrum hollow fiber modules (0.65 µm) — Suited to slow concentration and buffer exchange. Shear and clogging must be managed
• Collection, washing and resuspension by benchtop centrifugation · Thermo Fisher Sorvall X Pro / Eppendorf 5910 Ri — Simple for small scale and development. Open handling makes it unsuitable for large volumes or closed systems

This is where the iPS cells are given a "role". The step directs cells that can become anything toward the target cells, such as retinal pigment epithelium of the eye, cardiomyocytes, immune NK cells or neurons.

The way to do it is to add medium, small molecules and growth factors stepwise according to a fixed recipe. The course of development that occurs in the body is traced on a dish to guide the cells.

The difficulty is finishing the recipe so that it is robust enough to give the same result no matter who runs it, can also be mass-produced, and meets manufacturing standards (GMP). The core of clinical translation is concentrated here.

iPSC are differentiated stepwise into the target cells (retinal pigment epithelium, cardiomyocytes, NK cells, neurons, etc.). Cytokines and small molecules mimic the developmental pathway. For clinical use, a robust, scalable, GMP-compatible defined protocol is essential.

Alternatives in practice

• Differentiation via embryoid body (EB) formation · STEMCELL AggreWell 400 (34415) + AggreWell Medium — Mass-produces size-uniform EBs. Lineage specificity is lower than with directed protocols
• Forced differentiation by gene transfer (transcription factor overexpression) · Doxycycline-inducible piggyBac / lentiviral systems (System Biosciences) — Fast and high purity (e.g., NGN2 neuronal differentiation). Transduction handling and transgene control are required
• 3D organoid / bioreactor differentiation · PBS Biotech Vertical-Wheel / Corning spheroid plates — Gives more physiology-like maturation. Longer protocols and heterogeneity

The target cells that have finished differentiating are still in lumps and clusters. As they are, they cannot go to the next selection. So they are loosened into individual, separate cells.

The way to do it is to gently loosen the adhesion between cells with a recombinant enzyme and pass them through a filter to remove fine clumps and leftover debris. The result is a clear single-cell liquid.

Why is it needed? The next MACS selection requires that the cells be "one by one". The more carefully they are loosened here, the more cleanly the accuracy of selection rises.

Target cells after directed differentiation exist as aggregates and clusters, so to subject them to magnetic-activated cell sorting (MACS) they must be dissociated enzymatically and prepared as a single-cell suspension free of clumps and debris. MACS presupposes single cells.

Alternatives in practice

• Recombinant trypsin-like enzyme + EDTA dissociation · Gibco TrypLE Select (12563011) + Versene/EDTA (15040066) — Animal-component-free. Aggregates that need strong dissociating power are treated multiple times
• Manual trituration + mesh filtration · pluriStrainer 40 µm / Corning cell strainer (352340) — Simple, but operator variability is large and attention is needed to the drop in viability from shear

The differentiated population contains not only the target cells but also extra cells. This is, so to speak, the sorting step that takes out only the wanted cells at high purity.

The marker is a cell surface marker. Beads that stick to a magnet are bound to it, and the cells are separated by attracting them with a magnetic field or letting them pass through (magnetic selection). Collecting what you want and discarding what you do not are equally possible.

What must be removed here is the undifferentiated cells left over after incomplete differentiation. If left, they could become the source of tumors after transplantation. Work that raises purity leads directly to safety.

Only the target cells are taken out of the differentiated population at high purity. Residual undifferentiated cells and by-products pose tumorigenicity and impurity risks, so they are removed. Magnetic/fluorescence separation based on surface markers is central.

Alternatives in practice

• Fluorescence-activated cell sorting (FACS) · BD FACSAria Fusion / Sony MA900 / Miltenyi MACSQuant Tyto (closed-system cartridge type) — High purity with multiple parameters. Low throughput limits clinical large-volume processing. Tyto is a closed system suited to GMP
• Metabolic selection (lactate medium, etc.) · Glucose-depleted, lactate-supplemented medium (Tohyama method for cardiomyocyte purification) — Inexpensive, with no antibodies or beads. Application is limited to specific lineages (cardiomyocytes, etc.)
• Adhesion / gravity separation and filtration · pluriStrainer size-selection strainers / differential adhesion plates — Simple, but low separating power, suited to crude purification

The cells that have finished selection still carry the magnetic beads and labeling reagents that were used, and the leftover debris from the process. They are also in a diluted state. This step brings them into the form of the final product.

The procedure is to wash away the remaining reagents by closed-system counterflow centrifugation and concentrate the cells tightly to the determined density. At the same time, the liquid is replaced with a protective solution that lets the cells withstand freezing.

This is an essential step peculiar to cell therapy. Unlike a protein drug, the product cannot be filter-sterilized afterwards, so the purity and concentration just before administration must be built in here.

Cells after MACS selection contain magnetic beads/labeling reagents and process-derived residues and are in a diluted state, so before final dispensing and freezing they are washed to remove residual reagents, concentrated to the specified cell density and buffer-exchanged into the cryoprotectant solution. This is an essential downstream step specific to cell therapy.

Alternatives in practice

• Automated cell wash and concentration system · Thermo Fisher CTS Rotea Counterflow Centrifugation System (A50757) / Cytiva Sefia — Closed-system, single-use at small to mid scale. Best suited to lower volume ranges than kSep
• Washing and concentration by hollow fiber TFF · Repligen KrosFlo KR2i + hollow fiber (0.65 µm) — Gentle buffer exchange is possible. Shear and recovery must be managed

At last, the finishing of the final product. The completed cells are accurately dispensed into the vials or bags used for administration (fill-finish).

If frozen as they are, ice crystals injure the cells from the inside. So they are frozen together with a DMSO-containing cryoprotectant, with the freezing rate finely controlled in a dedicated freezer.

This is the last gate of manufacturing. Until the moment the product reaches the patient and is administered, the cells are kept asleep with viability and quality preserved. This step seals the value of the product in the frozen state.

The final product is dispensed into vials or bags and frozen while preserving viability. The freezing rate is precisely controlled to limit cell damage from ice crystal formation. It is the final step that maintains quality until administration.

Alternatives in practice

• Low-concentration / DMSO-free cryoprotectant · BioLife Solutions CryoStor CS2 (2% DMSO) / DMSO-free formulations such as HemaCare — Reduces toxicity at administration. Recovery after freezing depends on the formulation and must be verified
• Non-controlled-rate freezing at -80 °C · Thermo Fisher Mr. Frosty / CoolCell (Corning 432138) — Simple and low cost. The cooling rate is not constant, so unsuitable for large volumes or GMP
• Vitrification · High-concentration protectant + direct immersion in liquid nitrogen (Cryotop-type) — Avoids ice crystal formation. Suited to small volumes and special uses; scale-up is difficult

Are the cells that were made really the "correct line" from the registered donor? Mix-ups and contamination with another line are the serious incidents that cell medicine most wants to avoid. This is the check that eliminates them.

The method is DNA fingerprinting. The pattern of short tandem repeats (STR), unique to each individual, is matched to confirm that the starting material and the final product derive from the same person.

It is not flashy, but it is the identity guarantee of the product itself. If this wavers, no other data has any meaning, so it is the test that forms the backbone of the whole quality control.

Assures that the manufactured iPSC or product are the correct line derived from the registered donor. Mix-ups and contamination with other lines lead to serious incidents. The DNA repeat-sequence pattern is matched like a fingerprint.

Alternatives in practice

• STR-NGS (next-generation sequencing-based STR) · Verogen/QIAGEN ForenSeq DNA Signature Prep + Illumina MiSeq FGx — High discriminating power at the sequence level. High cost and analysis burden
• SNP genotyping matching · Thermo Fisher TaqMan SNP Genotyping Assays + QuantStudio — Effective for detecting contamination and mix-ups. Less of an international standard than STR
• HLA typing · One Lambda/Thermo Fisher AllType NGS + Illumina — Gives immune-compatibility information in addition to identity. High cost, excessive for routine identity testing

Is the iPS cell bank staying undifferentiated and keeping its high quality? This step confirms that "pluripotency-likeness" with numbers.

The method is flow cytometry, which lights up cells in multiple colors and measures them one cell at a time. Evaluation is from both sides: surface markers such as SSEA-4 and TRA-1-60, and transcription factors inside the nucleus.

The point is to reduce it to a concrete number, the positive rate. Instead of "looks about right", a pass/fail line can be drawn, so it serves as the basis for deciding whether to pass or stop a lot.

Confirms that the iPSC bank is in an undifferentiated, high-quality pluripotent state. The standard is to evaluate both surface antigens and nuclear transcription factors. The positive rate is quantified and used for lot decisions.

Alternatives in practice

• Immunofluorescence staining (ICC/IF) · Molecular Devices ImageXpress / Thermo Fisher CellInsight CX7 high-content imager — Assesses morphology and localization together. Quantitation and throughput are inferior to FC
• Pluripotency gene expression by qPCR / RT-qPCR · Thermo Fisher QuantStudio 5 + OCT4/NANOG/SOX2 TaqMan Assays — Quantifies at the mRNA level. Does not give cell-level information on protein expression or positive rate
• PluriTest (transcriptome matching) · Illumina BeadArray / RNA-seq + PluriTest analysis — Scores pluripotency comprehensively. Does not give a positive rate for single cells

Positive markers alone might be "pretending to be undifferentiated". This step proves by actual function whether the cells can really become anything.

What is checked is whether the cells can differentiate into all three sources that build the body (endoderm, mesoderm, ectoderm). A dedicated assay measures the expression of related genes and supports pluripotency quantitatively.

Previously a test was used in which cells were transplanted into animals to see whether a teratoma formed. This is the center of the move to replace it with an in vitro quantitative method that is highly reproducible and uses no animals.

Shows not only marker expression but the actual function of differentiating into all three germ layers. This is the essential proof of pluripotency. The trend is to replace the former teratoma test with a quantitative in vitro method.

Alternatives in practice

• Teratoma formation assay (in vivo) · Transplantation into immunodeficient mice (NOD/SCID, etc.) + histopathological evaluation — Conclusive proof of three germ layer differentiation. The animal, time and cost burden is large, and its regulatory role is shrinking
• Embryoid body (EB) differentiation + three germ layer marker staining · STEMCELL AggreWell + ICC (AFP/SMA/β-III tubulin) — Confirms the three germ layers simply in vitro. Quantitation is inferior to the Scorecard
• PluriTest / directed differentiation verification · Illumina RNA-seq + PluriTest — Infers pluripotency and differentiation propensity from the transcriptome. Not direct proof of actual differentiation potential

During reprogramming and long culture, cells can quietly accumulate chromosomal abnormalities and copy number changes. This step is like a health checkup that keeps watch for them.

The method is karyotype analysis: the chromosomes of dividing cells are given a striped pattern (G-banding), lined up, and checked by eye for disorder in number or shape. The whole genome can be surveyed at a glance.

Why is it important? Chromosomal disorder can lead to tumorigenesis or functional abnormalities. Starting from here, higher-sensitivity methods are also combined to nip the seeds of abnormality early.

Chromosomal abnormalities, copy number changes and mutations can accumulate through reprogramming and long-term culture. These can lead to tumorigenesis or functional abnormalities, so they are monitored. Karyotype analysis, which gives a whole-genome overview, is the starting point, combined with high-sensitivity methods.

Alternatives in practice

• SNP array (molecular karyotype) · Thermo Fisher Applied Biosystems CytoScan HD Array / Illumina Infinium Global Screening Array — Detects sub-karyotype-level CNV/LOH at high resolution. Cannot detect balanced translocations
• Digital PCR targeted assay (iCS-digital PSC) · Stem Genomics iCS-digital PSC kit (24 probes, detects high-frequency CNVs such as 20q11.21) + Bio-Rad QX200 / Stilla Naica dPCR — Rapid and low cost, covering high-frequency abnormalities. Cannot detect novel off-target abnormalities
• Whole-genome sequencing (WGS) / NGS · Illumina NovaSeq X / NextSeq 2000 — Covers everything from point mutations to structural abnormalities. Heavy burden in cost, analysis and clinical decision criteria

Are the Sendai virus used for reprogramming and the genes introduced still lingering inside the cells? This check proves that they have "really disappeared".

The method is high-sensitivity PCR (RT-qPCR), which catches even trace amounts if any remain. It confirms the outcome that the vector is diluted and disappears as passaging is repeated.

Why is a negative result required? Lingering vector can lead to unintended modification of genes and safety concerns. This test draws the line that keeps the tools used at the start from being carried over into the product.

Confirms that the Sendai virus and introduced factors used for reprogramming do not remain in the cells. Residual vector is a concern for safety and genome modification. Loss over passaging is supported by high-sensitivity PCR.

Alternatives in practice

• SeV protein detection by immunocytochemistry (ICC) · Anti-SeV antibody (MBL PD029, etc.) + fluorescence microscope / high-content imager — Visualizes residual viral protein. Less sensitive than RT-qPCR
• qPCR detection of episomal/plasmid DNA · QuantStudio + oriP/EBNA1-targeted qPCR — For iPSC derived by the episomal method. Not suitable for the Sendai method
• Digital PCR (high-sensitivity quantification) · Bio-Rad QX200 / QIAGEN QIAcuity One — Absolutely quantifies trace residuals. Cost and throughput limit routine use

Are undifferentiated iPS cells that failed to differentiate mixed into the differentiated product? This is the heaviest safety homework specific to iPS products.

Why is it the most important? Even a handful of undifferentiated cells could grow after transplantation and form a tumor. So ultra-high sensitivity, detection down to "the tiniest amounts", is required.

For this purpose, digital PCR targets the undifferentiated marker (LIN28A), dividing a single drop into many fine partitions and counting them. It brings out even grain-of-sand residual cells with sensitivity higher by orders of magnitude.

If undifferentiated iPSC remain in a differentiated product, they can form tumors after transplantation. This is the most important safety issue specific to iPSC products. Extremely small numbers of undifferentiated cells must be detected with high sensitivity.

Alternatives in practice

• RT-qPCR (LIN28A / TRA-1-60 transcripts) · Thermo Fisher QuantStudio 5 + LIN28A TaqMan Assay — Simple and easy to introduce. Its detection limit (0.001% class) is higher than that of ddPCR, so sensitivity is lower
• Culture supernatant miRNA measurement (miR-302b, etc.) · QuantStudio + TaqMan miRNA Assay (miR-302b/367) — Non-destructive and usable for in-process monitoring. Establishing standardization and sensitivity is an issue
• Flow cytometry (TRA-1-60 / SSEA-5-positive cells) · BD FACSCanto II + anti-TRA-1-60 / anti-SSEA-5 — Detects at the cell level. Inferior to ddPCR in detecting rare residuals (below 0.01%)
• In vivo tumorigenicity test (transplantation into immunodeficient animals) · NOG/NSG mouse transplantation + pathological evaluation — Biologically conclusive. Time and cost are large, making it impractical for routine QC

The cells themselves are the product, so they cannot be sterilized afterward by filtration or heat. Any contamination is therefore carried directly into the patient's body.

So three things are confirmed as essential items for shipment: whether living bacteria are absent (sterility), whether the hard-to-see parasite mycoplasma is absent, and whether fever-causing substances are absent. These are the three pillars.

Another constraint is the short shelf life. Results cannot be waited for over many days. Therefore, use of rapid methods that give an answer in a short time, together with conventional methods, is emphasized.

The final product cannot be terminally filtered or heat-sterilized and has a short shelf life, so contamination reaches the patient directly. The 3 items of sterility, mycoplasma and pyrogens are essential release tests. Use of rapid methods alongside is emphasized.

Alternatives in practice

• Rapid mycoplasma qPCR (track record of approval in CAR-T, etc.) · Roche MycoTOOL Real-Time PCR (06495605001) / Thermo Fisher MycoSEQ (4460623) — Result within 5 hours. Accepted by pharmacopoeias as an alternative to culture methods
• Recombinant Factor C (rFC) endotoxin method · Lonza PyroGene rFC Assay (50-658U) / bioMérieux ENDONEXT EndoZyme II — Independent of horseshoe crabs and sustainable. Equivalence verification is needed when switching from LAL
• Rapid microbial detection (ATP bioluminescence / respiration detection) · bioMérieux BACT/ALERT series / Rapid Micro Biosystems Growth Direct — Shortens detection time. Additional verification of species identification and pharmacopoeial compliance is required

Endotoxin, derived from the shell of Gram-negative bacteria, causes fever when it enters the body. For cell products given by injection, it is a hazard that cannot be overlooked.

So every lot is confirmed to fall below the specification (for example, lower than the equivalent of 5.0 units per 1 mL). It is a contamination check on whether anything has entered from the formulation or process.

The measurement uses a reagent derived from horseshoe crab blood (LAL) or its recombinant version. The amount of endotoxin is read out numerically from the progress of turbidity or color development.

Endotoxin derived from Gram-negative bacteria is pyrogenic, and for iPSC-derived cell products given by injection, every lot is confirmed to be at or below the specification (e.g., equivalent to <5.0 EU/mL). An essential safety test for controlling contamination from formulation and process.

Alternatives in practice

• Recombinant horseshoe crab Factor C (rFC) method · Lonza PyroGene Recombinant Factor C Assay (50-658U) / bioMérieux ENDONEXT EndoZyme II GO — No animal-derived material, so supply is stable. A fluorescence plate reader is required
• Gel-clot method (LAL gel clot) · Charles River/Associates of Cape Cod Pyrotell gel-clot LAL — No instrument needed and simple for limit tests. Inferior in quantitation and automation

Mycoplasma is a troublesome microorganism that sneaks into cell culture. It is very small and slips through ordinary sterility tests. That is why a dedicated exclusion test is needed.

For iPS-derived cell products, confirming that it is "not there" is mandatory in every lot. Contamination can unknowingly throw off the properties of the cells and can reach the patient.

To meet the short product life, real-time PCR (NAT), which amplifies nucleic acid to find it, is widely used instead of the classical culture method that has to be waited for. The pharmacopoeias also accept it as an alternative method.

Mycoplasma is not detected by ordinary sterility tests, and exclusion in every lot is mandatory for iPSC-derived cell products. NAT (real-time PCR) methods are widely used to cope with the short product life.

Alternatives in practice

• Culture method (agar plates / liquid medium) · Pharmacopoeial mycoplasma medium (PPLO agar/broth) + indicator cells — The pharmacopoeial reference method, but it takes 28 days and misses non-culturable strains
• Indicator cell / DNA staining (Hoechst) method · Vero/3T3 indicator cells + Hoechst 33258 staining, fluorescence microscopy — Complements the culture method. Judgment is subjective, with issues in sensitivity and objectivity

How purely does the final product contain the intended target cells? This is the central step of the release decision, confirming the substance of the product itself.

The method confirms purity and identity by flow cytometry using a marker that only that cell lineage has. The cell number and viability that form the basis of the dose are measured at the same time.

A specification such as "purity of 90–95% or more" is decided in advance for each product, and pass/fail is judged by whether it is met. It is the gate that guarantees the contents of the single dose that reaches the patient.

Confirms with lineage-specific markers how pure the final product is in the target cells. At the same time, the cell number and viability that form the basis of the dose are measured. A specification is set for each product (e.g., purity >90–95%).

Alternatives in practice

• Automated counting by acridine orange / PI method · Logos Biosystems LUNA-FX7 / Nexcelom Cellaca — Live/dead judged by dual fluorescence with AO/PI. Marker identification is not possible, so another method is needed for purity evaluation
• Immunofluorescence / high-content imaging · Molecular Devices ImageXpress Confocal / Thermo Fisher CellInsight CX7 — Assesses morphology and localization simultaneously. Inferior to FC in throughput and quantitation
• Functional potency assay · Cardiomyocytes: Axion Maestro MEA / NK: Promega ADCC/cytotoxicity assay — Measures function directly, not purity. Complementary as a potency test for lot release