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,Monomer_Percent,HMW_Aggregates_Percent,LMW_Fragments_Percent,Mw_Monomer_kDa,Rg_Monomer_nm,Methionine_Oxidation_Percent,Turbidity_NTU
EYE-AGG-2026-001,Aflibercept Intravitreal,99.1,0.2,0.7,97.0,3.5,1.5,0.8
EYE-AGG-2026-002,Ranibizumab Solution,99.5,0.1,0.4,48.0,2.1,0.8,0.5
EYE-AGG-2026-003,Faricimab IVT,98.8,0.4,0.8,150.0,5.2,2.0,1.2

input.csv fields

Utility description

Anti-VEGF Aggregation SEC-MALS Checker — Anti-VEGF Aggregation Control

Anti-VEGF Aggregation SEC-MALS Checker — Anti-VEGF Aggregation Control

ℹ️ Utility analyzes SEC-MALS data for ophthalmic biologics:
• Monomer purity (≥98.5%)
• HMW Aggregates (≤0.5%)
• LMW Fragments (≤1.0%)
• Methionine oxidation (≤5.0%)
• Turbidity and molecular weight

⚠️ CRITICAL: Aggregates in the eye cause inflammation (uveitis)!
Met oxidation reduces VEGF binding.

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

Input format:
BatchNumber,ProductName,Monomer_Percent,HMW_Aggregates_Percent,LMW_Fragments_Percent,Mw_Monomer_kDa,Rg_Monomer_nm,Methionine_Oxidation_Percent,Turbidity_NTU

Example:
EYE-AGG-2026-001,Aflibercept Intravitreal,99.1,0.2,0.7,97.0,3.5,1.5,0.8

— WHY IS THIS NEEDED?
Aggregation control is critical for intravitreal drugs (Roche, Novartis, Kanghong):
• Eye is an immune-privileged organ, but aggregates can trigger inflammation (uveitis, vasculitis)
• Anti-VEGF drugs are often stored at high concentrations, increasing aggregation risk
• Methionine oxidation in CDR regions critically reduces affinity to VEGF-A
• SEC-MALS allows precise determination of aggregate mass and size without calibration

⚠️ CRITICAL:
• Monomer ≥98.5% — guarantee of high specific activity
• HMW Aggregates ≤0.5% — strict safety limit for the eye
• LMW Fragments ≤1.0% — proteolytic degradation control
• Met Oxidation ≤5.0% — preservation of therapeutic efficacy
• Turbidity ≤2.0 NTU — no precipitation

Key features:
• Combined SEC (separation) and MALS (mass detection) analysis
• Specific methionine oxidation control (CQA for VEGF-traps)
• Intraocular inflammation risk assessment
• Support for various formats (Fab, Fc-fusion, IgG)

Critical parameters:
• Monomer: ≥98.5%
• HMW Aggregates: ≤0.5%
• LMW Fragments: ≤1.0%
• Met Oxidation: ≤5.0%
• Turbidity: ≤2.0 NTU

💡 Usage tips:
1. Use small pore volume columns for better fragment separation
2. MALS detector is critical for distinguishing dimers from larger aggregates
3. Control oxidation upon light and peroxide exposure
4. For high-concentration forms (e.g., 100 mg/mL), check viscosity
5. Compare profiles before and after stress tests (agitation, heat)

⚠️ Note: Unlike systemic biologics, ophthalmic forms have zero tolerance for visible particles and very low tolerance for sub-visible aggregates due to risk of irreversible vision loss. The utility helps ensure the highest level of purity.

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,Monomer_Percent,HMW_Aggregates_Percent,LMW_Fragments_Percent,Mw_Monomer_kDa,Rg_Monomer_nm,Methionine_Oxidation_Percent,Turbidity_NTU
EYE-AGG-2026-001,Aflibercept Intravitreal,99.1,0.2,0.7,97.0,3.5,1.5,0.8
EYE-AGG-2026-002,Ranibizumab Solution,99.5,0.1,0.4,48.0,2.1,0.8,0.5
EYE-AGG-2026-003,Faricimab IVT,98.8,0.4,0.8,150.0,5.2,2.0,1.2

Output JSON

{
  "Header": {
    "UtilityName": "AntiVEGF_Aggregation_SecMals_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
        }
      ]
    }
  ]
}