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,MainIsoform_Percent,AcidicVariants_Percent,BasicVariants_Percent,Deamidation_Asn_Percent,Deamidation_Gln_Percent,IsoAsp_Formation_Percent,pI_Shift,CterminalAmidation_Percent
PEP-ISO-2026-001,Semaglutide API,97.5,1.5,1.0,0.3,0.1,0.2,0.05,99.0
PEP-ISO-2026-002,Liraglutide Bulk,96.0,2.5,1.5,0.5,0.2,0.3,0.08,98.5
PEP-ISO-2026-003,Tirzepatide API,98.0,1.0,0.5,0.1,0.05,0.1,0.02,99.5

input.csv fields

Utility description

Peptide Isoform & Deamidation Monitor — Peptide Isoform and Deamidation Monitor

Peptide Isoform & Deamidation Monitor — Peptide Isoform and Deamidation Monitor

ℹ️ Utility analyzes charge variants and specific degradation:
• Main isoform (≥95.0%)
• Acidic/Basic variants (charge heterogeneity)
• Deamidation (Asn/Gln)
• Iso-aspartate formation
• C-terminal amidation (critical for GLP-1)

⚠️ CRITICAL: Deamidation changes charge and may reduce activity!
Iso-aspartate disrupts peptide backbone.

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

Input format:
BatchNumber,ProductName,MainIsoform_Percent,AcidicVariants_Percent,BasicVariants_Percent,Deamidation_Asn_Percent,Deamidation_Gln_Percent,IsoAsp_Formation_Percent,pI_Shift,CterminalAmidation_Percent

Example:
PEP-ISO-2026-001,Semaglutide API,97.5,1.5,1.0,0.3,0.1,0.2,0.05,99.0

— WHY IS THIS NEEDED?
Isoform control is critical for peptide drugs (Novo Nordisk, Eli Lilly, Gan & Lee):
• Peptides are sensitive to pH and temperature changes, leading to deamidation
• Deamidation of Asn and Gln converts them to Asp and Glu, changing molecule charge
• Iso-aspartate (iso-Asp) formation is an irreversible backbone modification
• For GLP-1 analogs, complete C-terminal amidation is critical for enzyme protection
• Charge heterogeneity affects pharmacokinetics and receptor binding

⚠️ CRITICAL:
• Main isoform ≥95.0% — ensures dominance of active form
• Acidic variants ≤3.0% — indicator of deamidation and oxidation
• Deamidation ≤1.0% — strict stability limit
• Iso-aspartate ≤0.5% — irreversible degradation control
• C-terminal amidation ≥98.0% — critical for in vivo half-life

Key features:
• Separation of acidic and basic charge variants (cIEF/CE-MS)
• Specific quantitative analysis of deamidation
• C-terminal modification quality control
• Isoelectric point (pI) shift assessment

Critical parameters:
• Main isoform: ≥95.0%
• Acidic variants: ≤3.0%
• Basic variants: ≤2.0%
• Total deamidation: ≤1.0%
• Iso-aspartate: ≤0.5%
• C-terminal amidation: ≥98.0%

💡 Usage tips:
1. Use capillary isoelectric focusing (cIEF) for high resolution of charge variants
2. Deamidation accelerates at high pH — control buffer pH
3. Iso-aspartate is difficult to separate from aspartate chromatographically, use MS or specific enzymes
4. For GLP-1 analogs, check amidation degree for each batch
5. Compare isoform profiles after stress tests (heat, light)

⚠️ Note: Unlike standard impurities, isoforms have the same molecular weight (or very close) but different charge or structure. Their control requires special methods (CE, cIEF), not just standard RP-HPLC.

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,MainIsoform_Percent,AcidicVariants_Percent,BasicVariants_Percent,Deamidation_Asn_Percent,Deamidation_Gln_Percent,IsoAsp_Formation_Percent,pI_Shift,CterminalAmidation_Percent
PEP-ISO-2026-001,Semaglutide API,97.5,1.5,1.0,0.3,0.1,0.2,0.05,99.0
PEP-ISO-2026-002,Liraglutide Bulk,96.0,2.5,1.5,0.5,0.2,0.3,0.08,98.5
PEP-ISO-2026-003,Tirzepatide API,98.0,1.0,0.5,0.1,0.05,0.1,0.02,99.5

Output JSON

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