1. Introduction to Semax Manufacturing

Semax (Met-Glu-His-Phe-Pro-Gly-Pro) represents a synthetic heptapeptide analog derived from the adrenocorticotropic hormone (ACTH) fragment requiring precise manufacturing protocols to ensure product consistency, purity, and neurological activity preservation. This acetylated peptide consists of seven amino acids with a molecular weight of 813.9 Da and exhibits neuroprotective and cognitive enhancement properties. Manufacturing facilities must maintain stringent quality control measures throughout synthesis, purification, and final product formulation to meet pharmaceutical-grade specifications for research and therapeutic applications.

The manufacturing process for Semax demands specialized equipment, validated procedures, and comprehensive documentation systems compliant with current Good Manufacturing Practices (cGMP) as outlined by the FDA's guidance on cGMP regulations for peptide therapeutics. Production facilities must implement quality management systems that address all aspects of peptide synthesis, from raw material qualification through final product release testing, with particular attention to maintaining the acetylation modification critical to Semax stability and bioactivity.

This technical profile provides detailed specifications for Semax manufacturing operations, including synthesis parameters, purification methodologies, analytical testing requirements, batch documentation standards, stability protocols, storage specifications, and certificate of analysis criteria. Manufacturing personnel and quality control professionals will find comprehensive guidance on process controls, acceptance criteria, and regulatory compliance requirements specific to this neuroprotective heptapeptide analog.

2. Solid-Phase Peptide Synthesis (SPPS) Process

Semax production utilizes Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis methodology, which provides optimal efficiency and purity profiles for short peptide sequences. The synthesis process initiates with resin selection and proceeds through seven iterative coupling and deprotection cycles to assemble the Met-Glu-His-Phe-Pro-Gly-Pro sequence, followed by N-terminal acetylation to complete the active pharmaceutical ingredient structure.

2.1 Resin Selection and Loading

Manufacturing begins with selection of appropriate solid support resin. For Semax, Rink Amide MBHA resin (100-200 mesh, 0.5-0.8 mmol/g substitution) provides optimal performance for C-terminal amidation required for proper biological activity. Resin loading must be verified through quantitative Fmoc determination using UV spectroscopy at 301 nm following piperidine deprotection, with acceptance criteria of 90-110% of theoretical loading capacity.

Pre-swelling procedures require resin exposure to N,N-dimethylformamide (DMF) for minimum 30 minutes prior to first coupling cycle. Resin quality specifications must include:

• Particle size distribution: 100-200 mesh (74-149 μm)
• Swelling volume: minimum 4 mL/g in DMF
• Moisture content: maximum 5% by Karl Fischer titration
• Heavy metal content: less than 10 ppm total
• Functional group loading: 0.5-0.8 mmol/g verified by elemental analysis

2.2 Coupling Cycle Parameters

Each amino acid coupling cycle follows validated protocols using HBTU/HOBt activation chemistry. The standard coupling procedure employs 3-fold molar excess of Fmoc-protected amino acid relative to resin loading, activated with HBTU (3 equivalents) and HOBt (3 equivalents) in presence of DIEA (6 equivalents). Coupling reactions proceed for 45-60 minutes with mechanical agitation, maintaining temperature at 20-25°C.

Critical process parameters for coupling reactions include:

2.3 Sequence Assembly and Critical Residues

Semax synthesis proceeds from C-terminal to N-terminal according to standard SPPS convention. The coupling sequence follows this order: Pro-Gly-Pro-Phe-His-Glu-Met. Specific considerations for challenging amino acids include:

Proline Residues (positions 5 and 7):

• Extended coupling time: 90-120 minutes due to secondary amine steric hindrance
• Double coupling recommended for positions following proline
• Increased activator concentration (4 equivalents) may improve efficiency
• Temperature elevation to 30°C can enhance coupling kinetics

Histidine Residue (position 3):

• Trityl (Trt) side-chain protection for imidazole nitrogen
• Careful monitoring to prevent Trt migration or incomplete deprotection
• Avoid excessive base exposure to minimize racemization
• Verify coupling completion through Kaiser test before proceeding

Methionine Residue (position 1):

• Susceptibility to oxidation requires careful handling
• Use freshly prepared amino acid solutions
• Minimize oxygen exposure during synthesis
• Consider addition of antioxidants to coupling solutions

2.4 Deprotection and Monitoring

Fmoc deprotection employs 20% piperidine in DMF (v/v) for two sequential treatments: initial 5-minute treatment followed by 15-minute treatment. Deprotection completion must be verified through UV monitoring of dibenzofulvene-piperidine adduct formation at 301 nm, with baseline return confirming complete Fmoc removal prior to subsequent coupling.

Quality control during synthesis requires Kaiser (ninhydrin) testing after each coupling cycle to verify completion. Negative Kaiser test (no blue coloration) confirms successful coupling with free amine consumption. Given the presence of two proline residues, chloranil test (Resin Test) provides an alternative for detecting secondary amines, appearing yellow-green when free secondary amine is present.

2.5 N-Terminal Acetylation

Following assembly of the complete heptapeptide sequence, N-terminal acetylation represents a critical quality attribute for Semax biological activity and stability. The acetylation modification improves enzymatic stability by protecting against aminopeptidase degradation. Acetylation procedure specifications include:

• Reagent: Acetic anhydride in DMF (1:1:8 Ac2O:DIEA:DMF)
• Reaction time: 30 minutes at room temperature
• Repeat treatment: Second acetylation to ensure completeness
• Verification: Kaiser test must remain negative (no free amine)
• Mass spectrometry confirmation: +42 Da mass increase for acetyl group

Acetylation efficiency must exceed 98% as determined by analytical HPLC comparison of acetylated and non-acetylated peptide retention times. This critical quality attribute directly impacts pharmacokinetic properties and requires rigorous process validation according to ICH Q7 guidelines for API manufacturing.

3. Cleavage and Crude Peptide Recovery

Following completion of solid-phase assembly and N-terminal acetylation, the peptide undergoes cleavage from the resin support with simultaneous removal of side-chain protecting groups. This critical operation requires precise control of reagent composition, reaction conditions, and work-up procedures to maximize yield while minimizing oxidation and other degradation pathways, particularly critical for the methionine-containing Semax sequence.

3.1 Cleavage Cocktail Preparation

Semax cleavage employs Reagent K formulation with enhanced antioxidant protection: trifluoroacetic acid (TFA) 82.5%, phenol 5%, water 5%, thioanisole 5%, and 1,2-ethanedithiol (EDT) 2.5% by volume. This scavenger cocktail provides comprehensive protection against cationic species generated during deprotection, with particular importance for protecting the N-terminal methionine residue from oxidation during the acidolysis process.

Cleavage cocktail specifications require:

• TFA purity: minimum 99.5%, peptide synthesis grade
• Phenol: freshly redistilled or analytical grade with BHT stabilizer
• Water: Type I ultrapure, 18.2 MΩ·cm resistivity
• Thioanisole: anhydrous, 99% minimum purity
• EDT: 98% minimum purity, used within 6 months of opening
• Volumetric verification: ±2% of target volumes

3.2 Cleavage Process Parameters

The cleavage reaction proceeds at ambient temperature (20-25°C) for 2-3 hours with periodic mixing. Given Semax's short sequence and limited side-chain protection requirements (primarily Glu t-butyl ester and His trityl), shorter cleavage times compared to longer peptides are acceptable. Reaction scale typically employs 10-15 mL cleavage cocktail per gram of resin.

Process controls during cleavage include:

3.3 Precipitation and Washing

Following resin filtration, crude peptide precipitation occurs through addition of the TFA solution to cold diethyl ether (10-fold volume excess) maintained at -20 to 0°C. The precipitate forms immediately and requires collection through centrifugation at 3000-5000 × g for 10 minutes at 4°C. Multiple ether wash cycles (minimum four for Semax to ensure complete scavenger removal) remove protecting group residues and TFA.

For Semax, particular attention to washing is critical due to methionine oxidation susceptibility. Each wash supernatant should be visually inspected for complete scavenger removal (thioanisole appears as oily yellow residue). Incomplete scavenger removal may lead to oxidation during subsequent processing or storage.

Crude peptide drying proceeds under vacuum at room temperature with nitrogen purge, or through lyophilization from dilute acetic acid solution (0.1%). The resulting crude powder typically exhibits 40-70% purity by analytical HPLC, with the target peptide as the major component. Crude yield calculations based on resin loading typically achieve 50-80% for well-optimized Semax synthesis protocols, reflecting the favorable kinetics of short peptide assembly.

3.4 Crude Peptide Characterization

Prior to purification, crude peptide undergoes preliminary analytical characterization to guide purification strategy:

• Analytical HPLC: Purity assessment and retention time determination
• Mass spectrometry: Molecular weight confirmation (expected: 813.9 Da)
• UV spectroscopy: Concentration estimation using phenylalanine absorption
• Visual inspection: Appearance should be off-white to tan powder
• Oxidation assessment: Specific HPLC monitoring for Met-sulfoxide formation

Crude peptide storage prior to purification requires protection from moisture, light, and oxidation. Storage at -20°C in sealed containers with desiccant and under nitrogen or argon atmosphere maintains stability for up to 6 months, though immediate processing to purification minimizes potential methionine oxidation pathways.

4. High-Performance Liquid Chromatography Purification

Semax purification employs preparative reversed-phase HPLC to achieve pharmaceutical-grade purity specifications of ≥98%. The purification process must remove truncated sequences, deletion peptides, incompletely acetylated species, oxidation products (particularly methionine sulfoxide), and residual synthesis reagents while maintaining product integrity and maximizing recovery yield.

4.1 Column Selection and Preparation

Preparative purification utilizes C18 reversed-phase columns with specifications optimized for small peptide separation:

• Column dimensions: 250 × 30 mm (small scale) or 250 × 50 mm (production scale)
• Particle size: 10 μm for preparative applications
• Pore size: 100-120 Å suitable for 813.9 Da molecular weight
• Carbon load: 15-18% for optimal hydrophobic interaction
• Endcapping: complete for reduced silanol interactions
• pH stability: 2.0-7.5 operational range

Column conditioning requires equilibration with mobile phase for minimum 5 column volumes prior to sample injection. Column performance qualification must demonstrate theoretical plate count exceeding 8000 plates/meter for short peptides and peak asymmetry factor between 0.9-1.5 for standard heptapeptide markers.

4.2 Mobile Phase Systems

The purification employs binary gradient elution with acidified aqueous and organic phases. Standard mobile phase composition consists of:

Mobile Phase A (Aqueous):

• Water: HPLC-grade, filtered through 0.22 μm membrane
• TFA: 0.1% v/v (pH approximately 2.0)
• Conductivity: <5 μS/cm prior to TFA addition

Mobile Phase B (Organic):

• Acetonitrile: HPLC-gradient grade, minimum 99.9% purity
• TFA: 0.1% v/v for ion-pairing consistency
• Water content: <0.05% verified by Karl Fischer

Alternative mobile phase systems employing 0.1% formic acid provide milder acidic conditions that may reduce methionine oxidation risk during purification, though TFA generally provides superior peak resolution for acetylated peptides.

4.3 Gradient Optimization and Method Parameters

Purification method development establishes gradient conditions that provide baseline resolution between Semax and closely-related impurities, particularly non-acetylated peptide and methionine sulfoxide variants. A typical optimized gradient profile includes:

Detection occurs at 214 nm and 254 nm wavelengths simultaneously. The 214 nm channel monitors peptide bond absorption for quantitation, while 254 nm provides selectivity for phenylalanine aromatic absorption. Column temperature control at 25 ± 2°C ensures reproducible retention times and separation efficiency. Semax typically elutes at approximately 18-22 minutes under these conditions, with non-acetylated peptide eluting earlier and Met-sulfoxide variants showing altered retention.

4.4 Fraction Collection and Pooling Strategy

Automated fraction collection targets the Semax peak with defined collection windows based on retention time and UV threshold criteria. Collection parameters must balance purity requirements against yield considerations:

• Collection threshold: 10-15% of peak maximum on ascending edge
• Collection termination: 10-15% of peak maximum on descending edge
• Fraction volume: 30-50 mL per fraction tube for analytical verification
• Peak tracking: automated retention time adjustment ±0.3 minutes
• Oxidation monitoring: Specific attention to Met-sulfoxide separation

Individual fractions undergo analytical HPLC testing to determine purity profiles. Fractions meeting the minimum purity specification (typically 98% by area at 214 nm) qualify for pooling. Edge fractions with 95-98% purity may undergo re-purification or polishing chromatography to maximize overall yield while maintaining final product specifications exceeding 98% purity.

4.5 Desalting and Final Processing

Purified fractions contain TFA and acetonitrile that require removal prior to lyophilization. For Semax, selection of desalting method considers methionine oxidation risk:

Option 1: Size-Exclusion Chromatography (Preferred)

• Column: Sephadex G-10, 2.5 × 50 cm minimum dimensions
• Mobile phase: 0.1% acetic acid in water, degassed
• Sample concentration: Concentrate HPLC fractions prior to loading
• Flow rate: 2-3 mL/min, gravity-driven or peristaltic pump
• Detection: UV absorbance at 214 nm for peptide-containing fractions
• Advantages: Gentle conditions, minimal oxidation risk, excellent TFA removal

Option 2: Lyophilization with Buffer Exchange

• Add 0.1M ammonium bicarbonate buffer (pH 8.0) to neutralize TFA
• Concentrate under reduced pressure at ≤30°C
• Repeat dilution and concentration cycles (3 times minimum)
• Adjust final pH to 4.0-5.0 with dilute acetic acid
• Proceed directly to lyophilization

Both desalting approaches provide TFA removal exceeding 99% as verified by ion chromatography. Size-exclusion chromatography represents the preferred method for Semax due to lower oxidation risk compared to concentration/dilution cycles that may expose methionine to oxygen. All desalting operations should occur under nitrogen atmosphere when possible.

4.6 Oxidation Control During Purification

Methionine oxidation represents the primary degradation pathway during Semax purification. Control strategies include:

• Mobile phase degassing: Helium sparging for 15 minutes before use
• Antioxidant addition: 0.01% ascorbic acid in aqueous mobile phase (analytical verification required)
• Minimize sample exposure: Process collected fractions immediately
• Cool collection: Maintain fraction collector at 4°C during operation
• Nitrogen blanketing: Purge collection vessels with nitrogen
• Metal chelation: EDTA addition (0.1 mM) to mobile phases prevents metal-catalyzed oxidation

5. Lyophilization and Final Product Formulation

Lyophilization (freeze-drying) converts purified Semax solution into stable solid form suitable for long-term storage and distribution. The lyophilization cycle must remove water and volatile solvents while maintaining peptide structural integrity, acetylation modification, and methionine residue oxidation state, ensuring consistent reconstitution properties and stability profiles.

5.1 Pre-Lyophilization Formulation

Prior to lyophilization, purified Semax solution undergoes formulation with appropriate excipients to ensure optimal cake structure, stability, and handling characteristics. Standard formulation includes:

• Semax concentration: 2-5 mg/mL in final solution
• Mannitol: 2-3% w/v as bulking agent and cryoprotectant
• Acetic acid: 0.1% to maintain pH 4.5-5.5
• Methionine (free amino acid): Optional, 0.1-0.5% w/v as sacrificial antioxidant
• EDTA: Optional, 0.01% w/v to chelate trace metals
• Fill volume: Calculated to achieve target final peptide content per vial

Addition of free methionine as a sacrificial antioxidant protects peptide methionine residues from oxidation during lyophilization and storage. This approach, commonly employed for methionine-containing therapeutic peptides, preferentially oxidizes free methionine while preserving peptide integrity.

Solution preparation requires sterile-filtered water (0.22 μm filtration) and pharmaceutical-grade excipients. Solution pH verification and adjustment precedes sterile filtration through 0.22 μm PES membranes into pre-sterilized fill vessels. Bioburden control throughout formulation operations maintains counts below 10 CFU/100 mL prior to terminal sterilization or aseptic processing.

5.2 Vial Filling Operations

Aseptic filling procedures in ISO Class 5 (Grade A) environments prevent microbial contamination during vial filling. Process parameters include:

5.3 Lyophilization Cycle Parameters

The lyophilization cycle consists of three phases: freezing, primary drying, and secondary drying. Cycle development uses differential scanning calorimetry (DSC) to determine the glass transition temperature (Tg') and eutectic temperature of the formulation, establishing the maximum product temperature during primary drying. For Semax with mannitol formulations, Tg' typically occurs at -40 to -35°C, while mannitol eutectic temperature is -1.5°C.

Phase 1: Freezing

• Initial shelf temperature: +5°C
• Ramp rate: -1°C per minute to -45°C
• Hold at -45°C for minimum 3 hours
• Annealing step optional: -10°C for 2 hours to improve crystal structure
• Ensures complete ice crystallization before primary drying

Phase 2: Primary Drying

• Chamber pressure: 50-100 mTorr (6.7-13.3 Pa)
• Shelf temperature: -30 to -25°C (10-15°C below Tg')
• Duration: 18-30 hours depending on fill volume
• Endpoint determination: Pirani gauge pressure equals capacitance manometer
• Product temperature monitoring: Thermocouples in representative vials
• Conservative conditions to prevent collapse or melt-back

Phase 3: Secondary Drying

• Shelf temperature ramp: +0.2°C per minute to +25°C
• Chamber pressure: 50-100 mTorr maintained
• Hold at +25°C for 4-6 hours
• Final residual moisture target: <2% by Karl Fischer titration
• Extended secondary drying reduces oxidation risk during storage

5.4 Stoppering and Crimping

Following completion of secondary drying, vials undergo automated stoppering under vacuum or controlled atmosphere. For Semax, nitrogen backfill (750-950 mbar) provides inert atmosphere reducing methionine oxidation during storage. Stopper insertion occurs within the lyophilizer chamber to maintain sterility and prevent moisture re-absorption. Aluminum crimp seals secure stoppers immediately following removal from the lyophilizer.

Critical quality attributes of the lyophilized cake include:

• Appearance: Uniform white to off-white cake without collapse
• Cake integrity: No melt-back, shrinkage, or separation from vial wall
• Reconstitution time: Complete dissolution within 30-60 seconds with gentle swirling
• Clarity after reconstitution: Clear, colorless to slightly yellow solution
• Residual moisture: <2% by Karl Fischer, target <1.5%
• Oxygen content: <3% in headspace for nitrogen-backfilled vials
• pH after reconstitution: 4.5-5.5 in water

Post-lyophilization cake appearance directly correlates with stability performance and requires careful process optimization according to FDA guidance on lyophilization of parenteral products.

6. Quality Control Testing and Release Specifications

Comprehensive analytical testing validates that each Semax batch meets predetermined quality specifications prior to commercial release. The quality control testing program encompasses identity confirmation, purity assessment, potency determination, and safety testing in accordance with ICH guidelines and pharmacopeial requirements, with particular emphasis on acetylation confirmation and methionine oxidation monitoring.

6.1 Identity Testing

Multiple orthogonal methods confirm Semax identity:

Amino Acid Analysis (AAA)

• Method: Acid hydrolysis (6N HCl, 110°C, 24 hours) followed by ion-exchange chromatography
• Acceptance criteria: Each amino acid within ±15% of theoretical molar ratio
• Expected composition: Met, Glu, His, Phe, Pro(2), Gly (1:1:1:1:2:1)
• Limitation: Acetyl group is cleaved during hydrolysis, confirmed by other methods
• Sample size: 50-100 μg peptide per analysis

Mass Spectrometry

• Method: ESI-MS or MALDI-TOF MS
• Acceptance criteria: Observed molecular weight 813.9 ± 0.3 Da
• Acetylation confirmation: Mass difference of 42 Da from non-acetylated peptide (771.9 Da)
• MS/MS sequencing confirms amino acid sequence and acetylation position
• Oxidation monitoring: Met-sulfoxide appears at +16 Da (829.9 Da)

Reversed-Phase HPLC with UV Detection

• Method: Analytical HPLC with gradient elution on C18 column
• Acceptance criteria: Retention time matches reference standard ±2%
• Co-injection with reference standard shows single peak
• UV spectrum (210-300 nm) matches reference standard showing Phe absorption

N-Terminal Acetylation Verification

• Method: Edman degradation or MS/MS sequencing
• Acceptance criteria: No free N-terminal amino group detected
• Acetyl-Met confirmed as N-terminal residue
• Essential for confirming critical quality attribute

6.2 Purity Determination

Multi-dimensional purity assessment employs complementary analytical techniques:

Reversed-Phase HPLC Purity

• Column: C18, 4.6 × 250 mm, 5 μm particle size, 120 Å pore
• Mobile phase: Water/acetonitrile with 0.1% TFA gradient (10-40% B over 30 min)
• Detection: 214 nm (quantitation), 254 nm (aromatic verification)
• Acceptance criteria: ≥98.0% main peak by area at 214 nm
• Individual impurity limit: ≤1.0%
• Total impurities: ≤2.0%
• Specific monitoring for non-acetylated peptide and Met-sulfoxide variants

Critical Impurity Identification

• Non-acetylated Semax: ≤0.5% (elutes earlier than acetylated form)
• Methionine sulfoxide (Met(O)-Semax): ≤0.5% (retention time shift)
• Deletion sequences: ≤1.0% total (various retention times)
• Aggregates: ≤0.5% by SEC-HPLC

Ion-Exchange HPLC (Optional)

• Orthogonal purity assessment based on charge differences
• Column: Weak cation exchange suitable for pH 4-6
• Detects deamidation products if present during storage
• Acceptance criteria: ≥95% main peak, complements RP-HPLC data

6.3 Content and Potency Assays

Peptide content determination employs multiple validated methodologies:

6.4 Physical and Chemical Properties

Additional quality control tests characterize physical and chemical properties:

• Appearance: White to off-white lyophilized powder
• Solubility: Freely soluble in water, pH 4-6 solution
• pH (1 mg/mL solution): 4.5-5.5
• Water content: ≤3.0% by Karl Fischer titration
• Residual solvents: TFA ≤0.5%, acetonitrile ≤410 ppm, DMF ≤880 ppm per ICH Q3C
• Heavy metals: ≤10 ppm by ICP-MS
• Bacterial endotoxins: ≤10 EU/mg by LAL assay (for research applications)
• Sterility: Passes USP <71> sterility test (if labeled as sterile)
• Particulate matter: Meets USP <788> requirements after reconstitution
• Optical rotation: Specific rotation determination (contains D-amino acids in some preparations)

6.5 Stability-Indicating Methods

Analytical methods must demonstrate stability-indicating capability through forced degradation studies. Semax samples undergo stress conditions including:

• Acid hydrolysis: 0.1N HCl, 60°C, 24 hours (monitors peptide bond stability)
• Base hydrolysis: 0.01N NaOH, 40°C, 24 hours (deacetylation monitoring)
• Oxidation: 0.3% hydrogen peroxide, 25°C, 4 hours (methionine oxidation)
• Thermal stress: 50°C, dry heat, 7 days (accelerated degradation)
• Photolytic degradation: ICH Q1B light exposure (photostability assessment)
• Metal exposure: Copper sulfate (10 ppm), 25°C, 24 hours (metal-catalyzed oxidation)

Validated analytical methods demonstrate resolution of degradation products from the main peak, with method specificity confirmed through peak purity assessment using photodiode array detection or mass spectrometry. Primary degradation products include methionine sulfoxide, deacetylated peptide, and peptide bond hydrolysis products. Method validation follows USP <1225> validation of compendial procedures and ICH Q2(R1) guidelines.

7. Batch Documentation and Manufacturing Records

Comprehensive batch documentation provides complete traceability from raw materials through final product release. The documentation system must comply with FDA 21 CFR Part 211 requirements and support regulatory inspections, customer audits, and internal quality reviews specific to Semax heptapeptide production.

7.1 Master Batch Record Structure

The Master Batch Record (MBR) defines all manufacturing operations, in-process controls, and specifications for Semax production. MBR components include:

• Product identification: Semax (Met-Glu-His-Phe-Pro-Gly-Pro-OH, N-acetylated)
• Batch size range: Minimum and maximum synthesis scale
• Complete list of raw materials with specifications and quantities
• Fmoc-protected amino acids: Met, Glu, His, Phe, Pro, Gly specifications
• Resin specifications: Type, loading, particle size, vendor qualification
• Equipment identification and qualification status
• Step-by-step manufacturing instructions with process parameters
• In-process control tests with acceptance criteria
• Acetylation verification requirements
• Sampling procedures and sampling plans
• Yield calculations and acceptance ranges
• Packaging and labeling instructions
• Storage conditions: -20°C, nitrogen atmosphere, light protection

7.2 Batch Production Record Execution

Each manufacturing batch generates a Batch Production Record (BPR) that documents actual execution of MBR procedures. The BPR captures:

Raw Material Documentation

• Identity and lot number of each Fmoc-amino acid
• Resin lot number, loading verification, and acceptance
• Acetic anhydride lot and purity verification
• Coupling reagents (HBTU, HOBt, DIEA) lot numbers
• Actual quantities dispensed with dual verification signatures
• Certificate of analysis review and approval
• Retest or expiration date verification

Process Execution Records

• Synthesis cycle log: Each coupling and deprotection cycle documented
• Kaiser test results for each coupling (negative result required)
• UV monitoring data for each deprotection (baseline return confirmed)
• Acetylation reaction parameters and completion verification
• Cleavage time, temperature, and cocktail composition
• Crude yield calculation and comparison to expected yield
• HPLC purification chromatograms and fraction pool decisions
• Lyophilization cycle data with temperature/pressure traces

7.3 In-Process Control Strategy

Critical process parameters require real-time monitoring and documentation throughout Semax manufacturing:

7.4 Yield Calculations and Material Balance

Yield calculations at each manufacturing stage verify process efficiency:

• Crude yield: (Crude peptide weight / theoretical yield from resin loading) × 100
• Expected crude yield: 50-80% for optimized Semax synthesis
• Purification yield: (Pure peptide weight / crude peptide weight) × 100
• Expected purification yield: 40-60% depending on crude purity
• Overall yield: (Final product weight / theoretical yield) × 100
• Expected overall yield: 20-40% for pharmaceutical-grade Semax
• Acceptance ranges: Typically ±20% of expected historical yields

Material balance accounting reconciles all inputs, outputs, samples, and waste. Short peptides like Semax generally show higher yields than longer sequences due to fewer synthetic steps and reduced cumulative deletion sequence formation.

7.5 Batch Release Criteria

Semax batch release requires satisfactory completion of all specifications:

• All identity tests pass (MS confirms 813.9 Da, amino acid composition correct)
• Purity ≥98.0% by RP-HPLC at 214 nm
• Acetylation complete (no free N-terminal detected)
• Methionine sulfoxide <0.5%
• Water content ≤3.0%
• All residual solvents within ICH Q3C limits
• Bacterial endotoxins ≤10 EU/mg
• Sterility test passes (if required)
• Batch documentation complete and approved
• Stability program initiated (if required)

8. Stability Studies and Shelf-Life Determination

Comprehensive stability programs establish storage conditions, retest dating, and shelf-life specifications for Semax. Stability studies follow ICH Q1A(R2) guidelines for stability testing of new drug substances and provide data supporting commercial storage recommendations, with particular focus on methionine oxidation and acetyl group stability as critical degradation pathways.

8.1 Stability Study Design

Formal stability programs encompass multiple study types:

Long-Term Stability Studies

• Storage condition: -20°C ± 5°C (recommended long-term storage)
• Alternative condition: 2-8°C (refrigerated storage evaluation)
• Duration: Minimum 12 months, extending to 24-36 months
• Testing frequency: 0, 3, 6, 9, 12, 18, 24, 36 months
• Sample configuration: Final product in commercial packaging (nitrogen atmosphere)
• Batch selection: Minimum 3 batches from different manufacturing campaigns

Accelerated Stability Studies

• Storage condition: 25°C ± 2°C / 60% RH ± 5% RH
• Duration: 6 months minimum
• Testing frequency: 0, 1, 3, 6 months
• Purpose: Predict degradation kinetics and shelf-life modeling
• Critical for methionine oxidation rate determination

Stress Testing

• Temperature stress: 40°C, 50°C for solid; various temperatures for solution
• Humidity stress: 75% RH for moisture sensitivity assessment
• Oxidative stress: Oxygen atmosphere exposure at 25°C
• Light exposure: ICH Q1B photostability testing (particularly important for Semax)
• pH stress: pH 3-9 solution stability to establish reconstitution pH range
• Freeze-thaw cycling: -20°C to +25°C, 3 cycles minimum
• Reconstituted solution stability: 2-8°C and 25°C time course studies

8.2 Stability-Indicating Test Methods

Stability samples undergo comprehensive analytical testing using validated stability-indicating methods:

8.3 Degradation Pathway Characterization

Semax exhibits several primary degradation pathways requiring monitoring:

Methionine Oxidation (Primary Pathway)

• Site: N-terminal methionine residue
• Product: Methionine sulfoxide (MW +16 Da, 829.9 Da)
• Mechanism: Free radical or metal-catalyzed oxidation, photo-oxidation
• Temperature dependence: Accelerated at elevated temperatures
• Control strategy: Nitrogen atmosphere, light protection, EDTA addition, free methionine as sacrificial antioxidant, -20°C storage

Deacetylation

• Site: N-terminal acetyl group
• Product: Non-acetylated heptapeptide (MW -42 Da, 771.9 Da)
• Mechanism: Base-catalyzed or neutral hydrolysis
• pH dependence: Accelerated at pH >6
• Control strategy: Formulation pH 4.5-5.5, low temperature storage

Peptide Bond Hydrolysis

• Sites: Peptide bonds, particularly adjacent to proline residues
• Products: Fragmented peptides of various lengths
• Mechanism: Acid or base catalyzed hydrolysis, elevated temperature
• Control strategy: Neutral pH formulation, low temperature storage

Aggregation

• Types: Non-covalent aggregation (minimal for small peptides)
• Factors: Concentration, temperature, agitation, freeze-thaw
• Detection: SEC-HPLC, dynamic light scattering, turbidity
• Control strategy: Bulking agents, single-use vials, proper reconstitution

8.4 Shelf-Life Determination

Statistical analysis of stability data establishes retest dates and expiration dating. The analysis employs linear regression or appropriate non-linear models for each quality attribute. Shelf-life represents the time point where the 95% confidence interval intersects the acceptance criterion.

Typical Semax shelf-life specifications based on degradation kinetics:

• Lyophilized powder at -20°C (nitrogen atmosphere): 24-36 months
• Lyophilized powder at 2-8°C (nitrogen atmosphere): 12-18 months
• Lyophilized powder at 25°C: 3-6 months (limited stability)
• Reconstituted solution at 2-8°C (protected from light): 7-14 days maximum
• Reconstituted solution at 25°C: 24 hours maximum

Methionine oxidation represents the primary shelf-life limiting factor for Semax. Formulations with sacrificial methionine and nitrogen atmosphere packaging demonstrate significantly extended stability compared to air-packaged material.

8.5 Photostability Testing

Given Semax susceptibility to photo-oxidation, comprehensive photostability studies follow ICH Q1B guidelines:

• Light exposure: Option 2 (cool white fluorescent and near UV lamp)
• Overall illumination: 1.2 million lux hours (visible), 200 watt hours/m² (UV)
• Sample presentation: Both exposed and dark control samples
• Packaging configurations: Final product in commercial packaging and unprotected
• Analytical testing: Full quality control battery with emphasis on oxidation products
• Result: Amber glass vials or opaque secondary packaging required

8.6 Reconstituted Solution Stability

Stability of reconstituted Semax solutions provides critical guidance for end-users:

• Reconstitution vehicle: Sterile water for injection, 0.9% saline, or specified buffer
• Storage temperature: 2-8°C in original vial
• Light protection: Aluminum foil wrapping or amber vial
• Stability duration: 7-14 days maximum
• Degradation monitoring: Daily HPLC analysis for first week, then periodic
• Primary degradation: Methionine oxidation increases during liquid storage
• pH effect: Optimal stability at pH 4.5-5.5
• Freezing stability: Not recommended due to potential aggregation

9. Storage, Handling, and Distribution Requirements

Proper storage, handling, and distribution practices maintain Semax quality from manufacturing release through end-user receipt. Temperature control, light protection, oxygen exclusion, and environmental monitoring ensure product stability throughout the distribution chain, with particular attention to preventing methionine oxidation during storage and transport.

9.1 Storage Conditions and Requirements

Semax storage specifications depend on physical form and packaging configuration:

Lyophilized Product Storage (Recommended)

• Temperature: -20°C ± 5°C for long-term storage (optimal)
• Alternative: 2-8°C for intermediate-term storage (up to 12 months)
• Atmosphere: Nitrogen or argon backfill in vials (oxygen <3%)
• Humidity: Ambient (product protected by primary packaging and desiccant)
• Light: Protect from direct light exposure; amber vials or opaque secondary packaging
• Orientation: Upright storage prevents stopper contact with product

Bulk Intermediate Storage

• Purified peptide (pre-lyophilization): -20°C in sealed containers, nitrogen atmosphere, maximum 14 days
• Crude peptide: -20°C in sealed containers with desiccant and nitrogen, maximum 90 days
• Resin-bound peptide: -20°C under nitrogen or argon, maximum 3 days (cleave promptly to minimize degradation)

Reconstituted Solution Storage

• Temperature: 2-8°C immediately after reconstitution
• Stability: Maximum 7-14 days when protected from light
• Container: Original vial with aseptic technique maintained
• Light protection: Aluminum foil wrapping or storage in dark
• Freezing: Not recommended; may cause aggregation and precipitation
• Multiple withdrawals: Minimize oxygen exposure with each access

9.2 Environmental Monitoring Programs

Storage areas require continuous environmental monitoring to verify condition maintenance:

Temperature excursion investigations follow formal protocols with particular emphasis on Semax oxidation risk assessment based on time-temperature exposure modeling.

9.3 Light Protection Requirements

Given Semax photosensitivity, comprehensive light protection strategies are critical:

• Primary packaging: Amber Type I borosilicate glass vials (UV transmission <10% at 290-450 nm)
• Secondary packaging: Opaque cartons or boxes preventing light penetration
• Storage environment: Minimize ambient light exposure, <200 lux recommended
• Handling procedures: Minimize exposure to laboratory lighting during weighing/transfer
• Photostability labeling: "Protect from light" prominent on all labels

9.4 Shipping and Distribution Qualification

Distribution operations maintain cold-chain integrity and light protection through qualified shipping containers and procedures:

Shipping Container Qualification

• Thermal qualification testing: Summer (35°C ambient), winter (-10°C ambient), and spring/fall (20°C ambient) conditions
• Duration testing: Minimum 72 hours (2× expected maximum transit time)
• Temperature mapping: Data loggers at multiple locations throughout container volume
• Acceptance criteria: Maintain -20°C to -10°C throughout qualification duration for frozen shipment
• Alternative: 2-8°C for refrigerated shipment with cold packs
• Light protection: Opaque insulated containers preventing light penetration
• Requalification: Annual or after significant design changes

Shipping Procedure Components

• Product pre-conditioning: Verify -20°C storage temperature before packaging
• Dry ice quantity: Sufficient for transit duration plus 24-hour contingency (typically 5-10 lbs per shipment)
• Temperature monitoring: Data logger included with each shipment or validation batches
• Packaging materials: Insulated containers with validated thermal performance
• Light protection: Amber vials placed in opaque secondary packaging before shipping container
• Package labeling: "Keep frozen at -20°C," "Protect from light," "Do not expose to direct sunlight"
• Transit time limits: Maximum 48-72 hours for domestic shipments

9.5 Handling Procedures and Training

Personnel handling Semax require training on proper procedures to prevent product degradation:

• Minimize light exposure: Work in subdued lighting when possible, especially during weighing
• Minimize temperature exposure: Return to -20°C storage within 5-10 minutes of removal
• Avoid repeated freeze-thaw: Aliquot bulk solutions immediately after reconstitution if multiple uses planned
• Use clean, dry equipment: Contamination and moisture compromise stability
• Wear appropriate PPE: Gloves prevent product contamination and personal exposure
• Gentle mixing only: Avoid vigorous shaking that causes foaming/aggregation
• Aseptic technique: Essential when reconstituting for biological applications
• Oxygen-free handling: Reconstitute with degassed, nitrogen-purged solvents when possible
• Immediate return to storage: After sample removal or reconstitution

9.6 Customer Receiving and Acceptance

Customer receiving procedures verify product integrity upon receipt:

• Immediate inspection: Check package condition and dry ice presence upon delivery
• Temperature verification: Review data logger if included, confirm dry ice remains
• Product inspection: Examine vials for damage, discoloration, or unexpected appearance
• Documentation review: Verify Certificate of Analysis matches product received
• Prompt storage: Transfer to -20°C storage immediately (within 15 minutes maximum)
• Light protection maintenance: Keep vials in secondary packaging until use
• Damage reporting: Contact supplier immediately if temperature excursion, light exposure, or damage noted
• Acceptance testing: Consider performing identity and purity verification for critical applications

End-users should maintain receiving logs documenting condition at delivery and any deviations from expected shipping conditions. Given Semax oxidation sensitivity, any temperature excursions above -10°C during shipping should trigger supplier notification and consideration of analytical verification before use.

10. Certificate of Analysis and Release Documentation

The Certificate of Analysis (CoA) provides comprehensive quality documentation supporting each Semax batch release. This critical document certifies product conformance to specifications and enables customer quality assessment and regulatory compliance verification, with particular emphasis on acetylation confirmation and methionine oxidation status.

10.1 Certificate of Analysis Components

A complete Semax Certificate of Analysis includes the following sections:

Header Information

• Manufacturer name, address, and contact information
• Product name: Semax or N-Acetyl-Met-Glu-His-Phe-Pro-Gly-Pro
• Catalog or product code number
• Batch or lot number with full traceability
• Manufacturing date and expiration/retest date
• CoA issue date and document version number
• Storage conditions: Store at -20°C, protect from light, nitrogen atmosphere
• Handling recommendations: Minimize light exposure during use

Physical and Chemical Characteristics

• Appearance description: White to off-white lyophilized powder
• Molecular formula: C₃₇H₅₁N₉O₁₀S
• Molecular weight: 813.9 Da (theoretical, monoisotopic mass)
• CAS number: 80714-61-0
• Sequence: Ac-Met-Glu-His-Phe-Pro-Gly-Pro-OH (N-terminal acetylation)
• Alternate nomenclature: ACTH(4-10) analog, heptapeptide

10.2 Analytical Test Results Table

Test results appear in tabular format with specification ranges and actual results:

10.3 Chromatogram and Spectral Data

Representative analytical data appendices strengthen CoA documentation:

• HPLC chromatogram: Annotated trace showing Semax main peak with retention time labeled, identified impurities including any Met-sulfoxide or deacetylated peptide peaks
• Mass spectrum: ESI-MS spectrum showing M+H ion at 814.9 m/z and molecular ion confirmation
• Amino acid analysis: Tabular results showing theoretical vs. observed molar ratios for Met:Glu:His:Phe:Pro:Gly
• Overlay with reference standard: HPLC co-injection demonstrating single peak and retention time match

10.4 Storage and Handling Instructions

The CoA specifies proper storage and handling to maintain stated quality:

• Storage temperature: Store at -20°C upon receipt (optimal long-term stability)
• Alternative storage: May store at 2-8°C for up to 12 months
• Protect from light: Keep in original amber vial and secondary packaging until use
• Inert atmosphere: Vials contain nitrogen headspace; minimize air exposure
• Avoid repeated freeze-thaw: Prepare aliquots upon first opening if multiple uses planned
• Minimize light exposure: Handle under subdued lighting conditions when possible
• Reconstitution instructions: Dissolve in sterile water, 0.9% saline, or pH 4.5-5.5 buffer
• Reconstituted stability: Use within 7-14 days when stored at 2-8°C protected from light
• Do not freeze reconstituted solution: May cause aggregation
• Shipping conditions: Product shipped on dry ice with light-protective packaging

10.5 Quality Assurance Certification

The CoA footer includes quality assurance certification:

• Statement of compliance: "This batch has been manufactured and tested in accordance with current Good Manufacturing Practices and meets all established specifications. N-terminal acetylation and methionine integrity have been verified."
• QA approval signature: Authorized quality assurance personnel signature
• Approval date: Date of final batch release
• Regulatory status: "For research use only - not for human or veterinary use" or as applicable
• Technical support contact: Phone and email for technical questions
• Retest date: Date by which material should be retested if used beyond initial period

10.6 Supplementary Documentation

Additional documentation available upon request supports customer quality systems:

• Material Safety Data Sheet (MSDS/SDS): Safety information per GHS requirements
• Stability summary: Summary data supporting storage recommendations and shelf-life
• Photostability data: Light exposure study results justifying light protection requirements
• Oxidation study summary: Methionine oxidation kinetics and control strategies
• Sequence confirmation report: MS/MS fragmentation data confirming complete sequence
• Acetylation verification: Detailed analytical data confirming N-terminal modification
• Residual solvents report: Complete ICH Q3C solvent panel results
• Heavy metals detailed report: Individual element quantitation by ICP-MS
• Manufacturing flow diagram: Simplified SPPS process overview
• Handling best practices guide: Detailed procedures for oxidation minimization

10.7 Special Notations for Semax

Given Semax unique characteristics, CoAs should include specific notations:

• Acetylation status: "N-terminal acetylation confirmed by mass spectrometry (MW 813.9 Da vs. 771.9 Da for non-acetylated peptide)"
• Oxidation state: "Methionine sulfoxide content: [X]% - below specification limit of 0.5%"
• Storage criticality: "CRITICAL: This peptide is highly sensitive to light and oxidation. Strict adherence to storage and handling instructions is essential for maintaining product quality."
• Reconstitution pH: "Recommended reconstitution pH: 4.5-5.5 for optimal stability"
• Packaging atmosphere: "Vial headspace: Nitrogen (oxygen content <3%)"

CoA approval represents the final quality gate before product release, confirming compliance with all applicable regulations including FDA cGMP requirements and customer specifications specific to Semax quality attributes.

Conclusion: Manufacturing Excellence for Semax Production

Semax manufacturing demands rigorous process control, comprehensive analytical testing, and systematic quality assurance throughout the production lifecycle, with particular attention to N-terminal acetylation verification and methionine oxidation prevention. The integration of validated solid-phase synthesis protocols, optimized purification strategies employing gentle oxidation-minimizing conditions, and stability-indicating analytical methods ensures consistent delivery of pharmaceutical-grade heptapeptide meeting or exceeding ≥98% purity specifications.

Manufacturing facilities implementing these technical specifications achieve several critical quality outcomes: batch-to-batch consistency through validated seven-cycle SPPS parameters, complete N-terminal acetylation verified by mass spectrometry and HPLC, methionine sulfoxide impurities maintained below 0.5% through comprehensive oxidation control strategies, extended shelf-life through optimized formulation with nitrogen atmosphere packaging and light protection, and complete traceability supporting regulatory compliance and customer confidence in neuroprotective peptide applications.

Success in Semax production requires cross-functional expertise spanning synthetic peptide chemistry with special attention to acetylation chemistry, analytical method development for oxidation-state monitoring, photostability assessment, and quality systems addressing unique short-peptide manufacturing challenges. Organizations must invest in specialized equipment including automated peptide synthesizers with real-time coupling monitoring, preparative HPLC systems with oxygen-free mobile phase delivery, lyophilizers with nitrogen backfill capability, and comprehensive analytical instrumentation including high-resolution mass spectrometry for acetylation and oxidation verification.

Personnel qualifications represent an equally critical investment. Manufacturing and quality control staff require specialized training in short peptide synthesis optimization, proline coupling challenges, N-terminal modification chemistry, methionine oxidation mechanisms and prevention strategies, photostability principles, and cGMP regulations specific to peptide APIs. Regular competency assessment, continuing education in oxidation control techniques, and participation in peptide manufacturing working groups ensure teams remain current with evolving best practices for oxidation-sensitive peptide handling.

The manufacturing profile presented herein provides the technical foundation for Semax heptapeptide production operations. Facilities should customize these specifications based on their specific equipment capabilities, target purity grades (research vs. pharmaceutical), regulatory requirements, and customer needs while maintaining alignment with fundamental quality principles of acetylation completeness, oxidation prevention, and photostability protection. Regular process review, continuous improvement initiatives focused on oxidation minimization, and proactive adoption of technological advances in peptide manufacturing and packaging will drive ongoing optimization and competitive advantage in this specialized neuroprotective peptide market.

For additional technical resources on peptide manufacturing, consult complementary profiles available through PeptideForge.com covering related topics including ipamorelin synthesis optimization, TB-500 acetylation protocols, sermorelin purification methodologies, oxidation-sensitive peptide handling, and short peptide lyophilization cycle development. Additional resources on modified peptide characterization and stability testing programs for neuropeptides provide complementary technical guidance.

Manufacturing excellence in Semax production ultimately serves the broader objective of providing researchers, neuroscientists, and pharmaceutical developers with reliable, high-quality neuroprotective peptide tools for advancing scientific understanding of cognitive enhancement mechanisms and therapeutic development. The technical rigor detailed throughout this manufacturing profile, particularly regarding acetylation verification and oxidation control, reflects the industry's commitment to quality, stability, and regulatory compliance as foundational principles guiding modern peptide production operations for oxidation-sensitive, bioactive heptapeptides.

References and Regulatory Resources

1. U.S. Food and Drug Administration. (2023). Current Good Manufacturing Practice (CGMP) Regulations for Drugs. 21 CFR Parts 210 and 211. Available at: https://www.fda.gov/drugs/pharmaceutical-quality-resources/current-good-manufacturing-practice-cgmp-regulations
2. International Council for Harmonisation (ICH). (2023). ICH Q7: Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients. Available at: https://www.ich.org/page/quality-guidelines
3. U.S. Food and Drug Administration. (2022). Guidance for Industry: Lyophilization of Parenteral Products. Center for Drug Evaluation and Research. Available at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-lyophilization-parenteral-products
4. United States Pharmacopeia. (2024). USP <1225> Validation of Compendial Procedures. Available at: https://www.usp.org/validation
5. International Council for Harmonisation (ICH). (2023). ICH Q1A(R2): Stability Testing of New Drug Substances and Products. Available at: https://www.ich.org/page/quality-guidelines
6. International Council for Harmonisation (ICH). (2023). ICH Q1B: Photostability Testing of New Drug Substances and Products. Available at: https://www.ich.org/page/quality-guidelines
7. International Council for Harmonisation (ICH). (2023). ICH Q3C(R6): Impurities: Guideline for Residual Solvents. Available at: https://www.ich.org/page/quality-guidelines
8. United States Pharmacopeia. (2024). USP <788> Particulate Matter in Injections and USP <790> Visible Particulates in Injections. Available at: https://www.usp.org
9. U.S. Food and Drug Administration. (2023). Facts about the Current Good Manufacturing Practices (CGMPs). Available at: https://www.fda.gov/drugs/pharmaceutical-quality-resources/facts-about-current-good-manufacturing-practices-cgmps
10. Ashmyansky, I., et al. (2015). "Semax and Pro-Gly-Pro activate the transcription of neurotrophins and their receptor genes after cerebral ischemia." Cerebral Cortex, 25(10), 3638-3644. DOI: 10.1093/cercor/bhu189