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,Uncapped_Percent,Cap0_Percent,Cap1_Percent,Hypercap_Percent,Avg_PolyA_Length,PolyA_Homogeneity_Index,Full_Length_Transcript_Percent,Truncated_Transcript_Percent
MRNA-CAP-2026-001,SARS-CoV-2 Spike mRNA,1.0,2.0,96.0,1.0,120,0.9,85.0,5.0
MRNA-CAP-2026-002,Flu HA mRNA,1.5,2.5,95.0,1.0,110,0.85,82.0,6.0
MRNA-CAP-2026-003,Therapeutic Enzyme mRNA,0.5,1.0,97.5,1.0,130,0.95,90.0,3.0

input.csv fields

Utility description

mRNA Cap & Poly(A) Integrity Checker — mRNA Cap and Poly(A) Integrity Analyzer

mRNA Cap & Poly(A) Integrity Checker — mRNA Cap and Poly(A) Integrity Analyzer

ℹ️ Utility checks critical structural elements of mRNA:
• Cap1 efficiency (≥95%)
• Uncapped RNA level (≤2%)
• Poly(A) tail length (100-150 nt)
• Full-length transcript percentage (≥80%)

⚠️ CRITICAL: Lack of Cap1 drastically reduces protein translation!
Uncapped mRNA activates innate immunity.

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

Input format:
BatchNumber,ProductName,Uncapped_Percent,Cap0_Percent,Cap1_Percent,Hypercap_Percent,Avg_PolyA_Length,PolyA_Homogeneity_Index,Full_Length_Transcript_Percent,Truncated_Transcript_Percent

Example:
MRNA-CAP-2026-001,SARS-CoV-2 Spike mRNA,1.0,2.0,96.0,1.0,120,0.9,85.0,5.0

— WHY IS THIS NEEDED?
mRNA structure control is critical for therapy efficacy (Moderna, BioNTech, CureVac):
• 5' Cap (Cap 1) is required for ribosome recognition and protection from exonucleases
• Poly(A) tail stabilizes mRNA and promotes ribosome recycling
• Uncapped mRNA is perceived by cell as viral threat (RIG-I pathway)
• Truncated transcripts may encode non-functional proteins

⚠️ CRITICAL:
• Cap1 ≥95% — high purity co-transcriptional capping standard
• Uncapped ≤2% — minimization of immune response and degradation
• Poly(A) 100-150 nt — optimal length for stability and translation
• Full-length transcripts ≥80% — guarantee of target protein production
• Poly(A) homogeneity — important for expression reproducibility

Key features:
• Separate accounting of cap types (Cap 0, Cap 1, Uncapped)
• Poly(A) tail length and homogeneity assessment
• Transcript integrity control (Full Length vs Truncated)
• Support for LC-MS and capillary electrophoresis data

Critical parameters:
• Cap1 Efficiency: ≥95%
• Uncapped RNA: ≤2%
• Poly(A) Length: 100–150 nt
• Full-Length Transcript: ≥80%
• Truncated Transcript: ≤10%

💡 Usage tips:
1. Use enzymatic digestion for precise mRNA end analysis
2. Cap 1 is preferred over Cap 0 due to lower immunogenicity
3. Too short Poly(A) (<100 nt) leads to rapid degradation in cytoplasm
4. Compare profiles with reference standard for each synthesis batch
5. For therapeutic mRNA, purity requirements may be even stricter

⚠️ Note: Unlike DNA, mRNA is a single-stranded and unstable molecule. Capping and polyadenylation quality directly determine molecule lifespan in cell and amount of synthesized protein. The utility helps identify enzymatic reaction defects at IVT (in vitro transcription) stage.

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,Uncapped_Percent,Cap0_Percent,Cap1_Percent,Hypercap_Percent,Avg_PolyA_Length,PolyA_Homogeneity_Index,Full_Length_Transcript_Percent,Truncated_Transcript_Percent
MRNA-CAP-2026-001,SARS-CoV-2 Spike mRNA,1.0,2.0,96.0,1.0,120,0.9,85.0,5.0
MRNA-CAP-2026-002,Flu HA mRNA,1.5,2.5,95.0,1.0,110,0.85,82.0,6.0
MRNA-CAP-2026-003,Therapeutic Enzyme mRNA,0.5,1.0,97.5,1.0,130,0.95,90.0,3.0

Output JSON

{
  "Header": {
    "UtilityName": "mRNA_Cap_PolyA_Integrity_Checker",
    "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
        }
      ]
    }
  ]
}