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,Empty_Capsids_Percent,Partial_Capsids_Percent,Full_Capsids_Percent,Total_Particles_per_mL,Genome_Titer_GC_per_mL,Aggregates_Percent,Fragment_DNA_Percent
CGT-AAV-2026-001,AAV9-CNS-Gene,25.0,5.0,70.0,1.0E+13,7.5E+12,2.0,1.0
CGT-AAV-2026-002,AAV2-RPE65,40.0,10.0,50.0,5.0E+12,2.8E+12,3.5,2.0
CGT-AAV-2026-003,AAV8-FactorIX,15.0,3.0,82.0,2.0E+13,1.7E+13,1.0,0.5

input.csv fields

Utility description

AAV Empty/Full Capsid Ratio Checker — AAV Capsid Fill State Analyzer

AAV Empty/Full Capsid Ratio Checker — AAV Capsid Fill State Analyzer

ℹ️ Utility analyzes capsid fill profile (AUC/HPLC/CE):
• Full capsids (≥60%)
• Empty capsids (≤30%)
• Partial capsids (≤10%)
• Aggregates (≤5%)

⚠️ CRITICAL: Empty capsids cause immune response without therapeutic effect!
Full/Empty ratio determines dose efficacy and safety.

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

Input format:
BatchNumber,ProductName,Empty_Capsids_Percent,Partial_Capsids_Percent,Full_Capsids_Percent,Total_Particles_per_mL,Genome_Titer_GC_per_mL,Aggregates_Percent,Fragment_DNA_Percent

Example:
CGT-AAV-2026-001,AAV9-CNS-Gene,25.0,5.0,70.0,1.0E+13,7.5E+12,2.0,1.0

— WHY IS THIS NEEDED?
Capsid ratio control is critical for AAV-based gene therapy (Novartis, BioNTech, Oxford Biomedica):
• Only full capsids contain therapeutic gene and deliver it to cell nucleus
• Empty capsids compete for entry receptors, reducing delivery efficiency of full vector
• Empty capsids are potent immunogens, triggering neutralizing antibody production
• Purification process (chromatography, ultrafiltration) aims to maximize full capsid fraction

⚠️ CRITICAL:
• Full capsids ≥60% — quality standard for clinical batches
• Empty capsids ≤30% — safety limit to reduce immunogenicity
• Partial capsids ≤10% — genome packaging quality control
• Aggregates ≤5% — prevention of embolism and immune reactions
• GC/VP ratio (genome copies/viral particles) must correlate with % Full

Key features:
• Analytical ultracentrifugation (AUC) or capillary electrophoresis (CE) data analysis
• Full/Empty/Partial ratio calculation
• Product purity assessment from aggregates
• Physical titer (particles) and functional titer (genome copies) correlation

Critical parameters:
• Full capsids: ≥60%
• Empty capsids: ≤30%
• Partial capsids: ≤10%
• Aggregates: ≤5%

💡 Usage tips:
1. Use AUC-SV (sedimentation velocity) as gold standard for form separation
2. For routine control, apply HPLC-MALS or CE-SDS
3. High level of empty capsids may indicate plasmid expression issues during production
4. Compare Full/Empty ratio of different batches to monitor process stability
5. Account that different AAV serotypes may have different migration profiles

⚠️ Note: Unlike traditional vaccines where antigen concentration matters, in AAV gene therapy "packaging density" is critical. A product with high total titer but low % Full will be ineffective and dangerous. The utility helps optimize downstream purification process.

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,Empty_Capsids_Percent,Partial_Capsids_Percent,Full_Capsids_Percent,Total_Particles_per_mL,Genome_Titer_GC_per_mL,Aggregates_Percent,Fragment_DNA_Percent
CGT-AAV-2026-001,AAV9-CNS-Gene,25.0,5.0,70.0,1.0E+13,7.5E+12,2.0,1.0
CGT-AAV-2026-002,AAV2-RPE65,40.0,10.0,50.0,5.0E+12,2.8E+12,3.5,2.0
CGT-AAV-2026-003,AAV8-FactorIX,15.0,3.0,82.0,2.0E+13,1.7E+13,1.0,0.5

Output JSON

{
  "Header": {
    "UtilityName": "AAV_Empty_Full_Capsid_Ratio_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
        }
      ]
    }
  ]
}