Purpose

Define user requirements for a standalone FUZKK utility that accepts laboratory CSV data, evaluates the records using limits embedded in code, and produces LabWare-compatible JSON.

Scope

The utility is intended for preliminary QC/QA review, integration testing, LIMS/LabWare flow and evidence-trail preparation. Final release decisions remain under the laboratory's validated procedure and responsible personnel.

Users

QC analyst, QA reviewer, CSV/validation engineer, LIMS/LabWare integration engineer, responsible laboratory specialist.

User requirements

1. The utility shall run without arguments and print its self-description, a built-in input.csv example from GetDemoData(), and demo evaluation for the embedded records.
2. The utility shall run with two arguments: input.csv output.json.
3. The utility shall not read input.csv and shall not write output.json when started without arguments.
4. CSV numeric values shall be parsed using CultureInfo.InvariantCulture.
5. Output shall be generated as LabWare-compatible JSON with Header, Samples, Results, Status, StatusCode, ErrorMessage, Description and DescriptionEN.
6. For PASS records, ErrorMessage shall be an empty string.
7. Embedded limits shall follow this priority: Ph. Eur. → British Pharmacopoeia / UK implementation → EAEU / regional requirements → EMA/ICH/EU guidance → USP fallback.
8. If an exact monograph is not known, strict standard API limits are used where applicable: assay 98–102%, total impurities ≤1.0%, individual impurity ≤0.5%.
9. For biologics and mAb-like products, aggregation, sterility and endotoxin checks shall be included where relevant to the utility purpose.
10. If a parameter may arrive in different units, the unit shall be represented as a separate input field or explicitly reflected in the input.csv field name.

Input CSV

BatchNumber,ProductName,EC50_ng_ml,Emax_Relative_Percent,HillSlope,R_Squared,RelativePotency_Percent,Z_Factor,CV_Percent,Background_Signal,StandardEC50_ng_ml
MRNA-POT-2026-001,SARS-CoV-2 Spike mRNA-LNP,12.5,98.0,1.2,0.992,105.0,0.78,8.5,50.0,13.0
MRNA-POT-2026-002,Flu HA mRNA-LNP,18.0,92.0,1.1,0.985,95.0,0.72,12.0,45.0,17.5
MRNA-POT-2026-003,Therapeutic Enzyme mRNA-LNP,8.0,105.0,1.3,0.995,110.0,0.82,6.5,30.0,8.5

input.csv fields

Utility description

mRNA/LNP Potency Bioassay Reviewer — mRNA/LNP Biological Potency Reviewer

mRNA/LNP Potency Bioassay Reviewer — mRNA/LNP Biological Potency Reviewer

ℹ️ Utility analyzes cell-based functional activity assay data:
• Relative potency (70-130%)
• EC50 (half-maximal expression concentration)
• Emax (maximum protein level)
• Curve fit quality (R²)
• Z-Factor and CV (bioassay quality)

⚠️ CRITICAL: Physicochemical parameters do not guarantee translation!
Only functional test confirms mRNA ability to produce protein.

Usage:
mRNALNPPotencyBioassayReviewer.exe → demo mode (console output)
mRNALNPPotencyBioassayReviewer.exe input.csv output.json → evaluate your data

Input format:
BatchNumber,ProductName,EC50_ng_ml,Emax_Relative_Percent,HillSlope,R_Squared,RelativePotency_Percent,Z_Factor,CV_Percent,Background_Signal,StandardEC50_ng_ml

Example:
MRNA-POT-2026-001,SARS-CoV-2 Spike mRNA-LNP,12.5,98.0,1.2,0.992,105.0,0.78,8.5,50.0,13.0

— WHY IS THIS NEEDED?
Bio-potency control is critical for mRNA drugs (Moderna, BioNTech, CureVac):
• mRNA must be not only chemically pure but also functionally active
• Cell-based assay mimics entire pathway: LNP endocytosis → mRNA cytoplasmic release → protein translation
• Defects in capping, poly(A) tail, or mRNA integrity manifest specifically here
• This is the only test confirming therapeutic efficacy of the product

⚠️ CRITICAL:
• Relative potency 70-130% — acceptable range for bioassays (wider than chemistry)
• EC50 must correlate with standard (±40%)
• Emax ≥80% — confirmation of full translational capacity
• R² ≥0.95 — reliability of dose-response mathematical model
• Z-Factor ≥0.5 — high signal-to-noise separation quality
• CV ≤20% — acceptable variability for cell-based tests

Key features:
• Functional activity assessment (protein translation)
• Bioassay quality control (Z-factor, CV, R²)
• Comparison with reference standard in each run
• Support for various assay types (ELISA, luminescence, flow cytometry)

Critical parameters:
• Relative potency: 70–130%
• EC50: Within 40% of standard
• Emax: ≥80%
• R²: ≥0.95
• Z-Factor: ≥0.5
• CV: ≤20%

💡 Usage tips:
1. Always include reference standard on each plate for normalization
2. Use 4-parameter logistic model for curve fitting
3. High background may indicate transfection or detection issues
4. Compare EC50 of new batches with historical data to detect degradation trends
5. If low potency observed, check mRNA integrity and encapsulation efficiency

⚠️ Note: mRNA bioassays have high intrinsic variability due to use of living cells. The utility accounts for this by setting wider limits (70-130%, CV≤20%) compared to physicochemical tests. However, this test is decisive for batch release.

Traceability and limitations

• The URS is used as the source document for functional specification, CSV review and later validation work.
• This document does not replace an approved pharmacopoeial monograph, validated analytical method or internal product specification.
• For product-specific limits, the approved customer specification takes priority.

The functional specification describes the behaviour of the standalone FUZKK console utility, input-data format, evaluation algorithm and output JSON structure.

Functional flow

1. Main() checks the number of arguments.
2. If no arguments are provided: PrintHello() prints the description and built-in input.csv example, then RunDemoEvaluation() executes Evaluate() over GetDemoData() and prints demo JSON.
3. If two arguments are provided: RunWithFiles(input.csv, output.json) reads CSV, evaluates each record and writes LabWare-compatible JSON.
4. LoadData() uses CultureInfo.InvariantCulture and shall not be called in no-arguments mode.
5. Evaluate() returns a named tuple with BatchNumber, ProductName, Parameters, CriticalFailCount, WarningCount, Recommendation and RecommendationEN.
6. GetIssues() builds messages for ErrorMessage in WARNING/FAIL cases.

Evaluation rules

• PASS: CriticalFailCount = 0 and WarningCount = 0.
• WARNING: CriticalFailCount = 0 and WarningCount > 0.
• FAIL: CriticalFailCount > 0.
• ERROR: exception during reading or processing.
• ErrorMessage remains empty for PASS.
• Limits are embedded in Program.cs; no external limit configuration is required.

Input and fields

BatchNumber,ProductName,EC50_ng_ml,Emax_Relative_Percent,HillSlope,R_Squared,RelativePotency_Percent,Z_Factor,CV_Percent,Background_Signal,StandardEC50_ng_ml
MRNA-POT-2026-001,SARS-CoV-2 Spike mRNA-LNP,12.5,98.0,1.2,0.992,105.0,0.78,8.5,50.0,13.0
MRNA-POT-2026-002,Flu HA mRNA-LNP,18.0,92.0,1.1,0.985,95.0,0.72,12.0,45.0,17.5
MRNA-POT-2026-003,Therapeutic Enzyme mRNA-LNP,8.0,105.0,1.3,0.995,110.0,0.82,6.5,30.0,8.5

Output JSON

{
  "Header": {
    "UtilityName": "mRNA_LNP_Potency_Bioassay_Reviewer",
    "Version": "1.0.0",
    "Timestamp": "UTC",
    "InstrumentID": "FUZKK-QC-WORKSTATION",
    "OperatorID": "Admin"
  },
  "Samples": [
    {
      "SampleID": "from BatchNumber",
      "BatchNumber": "from CSV",
      "ProductName": "from CSV",
      "TestName": "utility-specific test",
      "AnalysisCode": "utility-specific code",
      "Status": "PASS | WARNING | FAIL | ERROR",
      "StatusCode": "1 | 2 | 0 | -1",
      "ErrorMessage": "",
      "Description": "Russian recommendation",
      "DescriptionEN": "English recommendation",
      "Results": [
        {
          "ParameterName": "parameter",
          "ResultValue": 0.0,
          "UnitOfMeasure": "unit",
          "SpecificationLimit": "limit",
          "IsWithinSpec": true
        }
      ]
    }
  ]
}