TB-500 Manufacturing Profile: Comprehensive Process Documentation and Quality Standards
TB-500, a synthetic peptide fragment corresponding to the active region of thymosin beta-4, represents a critical therapeutic peptide requiring stringent manufacturing controls and quality assurance protocols. This manufacturing profile provides detailed technical specifications for commercial-scale production, encompassing synthesis methodology, purification strategies, analytical testing, batch specifications, stability parameters, storage requirements, and certificate of analysis documentation. Manufacturing facilities producing TB-500 must implement robust quality management systems aligned with current Good Manufacturing Practices (cGMP) to ensure consistent product quality, batch-to-batch reproducibility, and regulatory compliance.
1. Product Identification and Molecular Characteristics
TB-500 is a synthetic peptide fragment derived from thymosin beta-4, specifically representing amino acids 17-23 (LKKTETQ) within the actin-binding domain of the full TB4 sequence. The commercial product typically features N-terminal acetylation (Ac-LKKTETQ) to enhance metabolic stability and biological activity. Understanding the precise molecular structure and physicochemical properties of TB-500 is essential for establishing appropriate manufacturing controls and analytical specifications.
| Parameter | Specification |
|---|---|
| Chemical Name | N-Acetyl-L-leucyl-L-lysyl-L-lysyl-L-threonyl-L-glutamyl-L-threonyl-L-glutamine |
| Sequence | Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln |
| Molecular Formula | C41H76N12O14 |
| Molecular Weight | 963.13 g/mol (calculated as acetate salt) |
| CAS Number | 77591-33-4 (Thymosin Beta-4 Fragment) |
| Fragment Origin | Thymosin Beta-4 (Amino Acids 17-23) |
| Appearance | White to off-white lyophilized powder |
| Solubility | Soluble in water, PBS, and acidic aqueous solutions |
The molecular structure of TB-500 contains multiple functional groups that influence its chemical reactivity, stability profile, and manufacturing requirements. The presence of two lysine residues provides positive charge at physiological pH, while the glutamic acid and glutamine residues contribute to the peptide's polar character. The N-terminal acetylation blocks the free amino group, reducing susceptibility to aminopeptidase degradation and improving shelf-life stability. Manufacturing processes must preserve the integrity of these structural features throughout synthesis, purification, and formulation operations.
2. Solid-Phase Peptide Synthesis (SPPS) Methodology
TB-500 is manufactured using solid-phase peptide synthesis (SPPS), a Nobel Prize-winning technology that enables efficient, scalable production of peptides with precise sequence control. The Fmoc (9-fluorenylmethoxycarbonyl) protection strategy represents the industry standard for commercial peptide manufacturing due to its compatibility with automated synthesis platforms, mild deprotection conditions, and favorable economics at multi-kilogram scale[1].
The SPPS process for TB-500 involves stepwise assembly of the seven-amino-acid sequence on a solid support resin, beginning with attachment of the C-terminal glutamine residue and proceeding through sequential coupling cycles to build the peptide chain from C-terminus to N-terminus. Each synthesis cycle comprises four fundamental operations: Fmoc deprotection of the N-terminal protecting group, activation of the incoming amino acid, coupling of the activated amino acid to the growing peptide chain, and washing to remove excess reagents and byproducts[2].
| Synthesis Step | Reagents/Conditions | Duration | Quality Control |
|---|---|---|---|
| Resin Loading | Fmoc-Gln(Trt)-Wang resin, 0.5-0.8 mmol/g substitution | Pre-loaded resin | Resin substitution verification |
| Fmoc Deprotection | 20-25% piperidine in DMF | 5-10 minutes | UV monitoring at 301 nm (Fmoc-fulvene) |
| Amino Acid Activation | HBTU/HOBt or HATU/HOAt with DIEA | 1-2 minutes | Pre-activation time monitoring |
| Coupling Reaction | 3-5 equivalents Fmoc-amino acid, DMF solvent | 15-60 minutes | Kaiser test or chloranil test for completion |
| Washing | DMF (3x), DCM (3x), or equivalent solvent system | 2-5 minutes per wash | Visual inspection and filtration |
| Capping (if required) | Acetic anhydride/DIEA/DMF (5:6:89) | 5 minutes | Acetylation completion test |
| N-Terminal Acetylation | Acetic anhydride/DIEA following final Fmoc removal | 15-30 minutes | Mass spectrometry confirmation |
| Cleavage and Global Deprotection | TFA/water/TIS (95:2.5:2.5) or equivalent scavenger cocktail | 2-3 hours | Complete peptide release and side-chain deprotection |
Manufacturing facilities utilize automated peptide synthesizers equipped with microwave irradiation capability to accelerate coupling reactions and improve synthesis efficiency. Microwave-assisted SPPS reduces coupling times from 30-60 minutes to 5-10 minutes while maintaining or improving coupling yields, enabling economical production of multi-kilogram peptide batches. Recent advances in sustainable SPPS methodologies, including ultrasound-assisted synthesis, have demonstrated 83-88% reduction in solvent consumption per coupling cycle, representing significant environmental and economic benefits for commercial manufacturing operations[3].
Critical process parameters (CPPs) for TB-500 synthesis include amino acid equivalents (typically 3-5 fold excess), coupling reagent selection and concentration, reaction temperature, coupling time, deprotection reagent concentration, and washing efficiency. These parameters must be optimized during process development and maintained within validated ranges during commercial production to ensure consistent coupling yields exceeding 99% per cycle. For a seven-residue peptide like TB-500, achieving 99.5% average coupling yield results in crude peptide purity of approximately 96.5%, providing suitable starting material for downstream purification operations.
3. Crude Peptide Cleavage and Precipitation
Following completion of the synthesis sequence and N-terminal acetylation, the protected peptide-resin must undergo simultaneous cleavage from the solid support and removal of side-chain protecting groups. This operation employs trifluoroacetic acid (TFA)-based cleavage cocktails containing scavenger reagents to prevent side-chain modification during the deprotection process. The standard cleavage cocktail for TB-500 comprises TFA (95%), water (2.5%), and triisopropylsilane (TIS, 2.5%), providing effective scavenging of carbocations generated during Trt (trityl) and tBu (tert-butyl) protecting group removal.
The cleavage operation proceeds at ambient temperature for 2-3 hours with periodic mixing to ensure homogeneous reagent contact with the peptide-resin. Following cleavage completion, the peptide solution is separated from the resin by filtration, and the crude peptide is precipitated by addition to cold diethyl ether (typically 10-15 volumes relative to the TFA solution). The precipitated peptide is collected by centrifugation or filtration, washed multiple times with fresh cold ether to remove TFA and organic soluble impurities, and dried under nitrogen or vacuum to yield crude TB-500 as an off-white to tan solid.
Quality control testing of crude TB-500 includes analytical HPLC to determine crude purity (typically 70-90% for optimized synthesis), mass spectrometry to confirm molecular weight and detect synthesis errors, and amino acid analysis to verify sequence composition. Crude peptide batches meeting minimum acceptance criteria (typically >70% purity by HPLC) proceed to purification operations, while batches falling below specifications require investigation and potential reprocessing or rejection.
4. Reversed-Phase HPLC Purification Strategy
Purification of crude TB-500 employs preparative reversed-phase high-performance liquid chromatography (RP-HPLC), the industry standard for peptide purification at scales ranging from grams to multi-kilogram production. RP-HPLC provides excellent resolution of the target peptide from synthesis-related impurities including deletion sequences (missing one or more amino acids), truncation products, incomplete deprotection products, and amino acid substitution impurities. The purification process achieves final product purity exceeding 98% by HPLC analysis, meeting pharmaceutical quality standards for peptide active pharmaceutical ingredients (APIs).
| Purification Parameter | Specification | Rationale |
|---|---|---|
| Column Type | C18 reversed-phase silica, 10-20 μm particle size | Optimal retention and selectivity for hydrophobic peptides |
| Column Dimensions | 5-30 cm diameter × 20-50 cm length (scale-dependent) | Adequate loading capacity and resolution for target batch size |
| Mobile Phase A | Water + 0.1% TFA (v/v) | Protonates basic residues, improves peak shape |
| Mobile Phase B | Acetonitrile + 0.1% TFA (v/v) | Organic modifier for gradient elution |
| Gradient Profile | 15-45% B over 30-60 minutes (method-dependent) | Optimized separation of target from impurities |
| Flow Rate | 50-500 mL/min (scale-dependent) | Maintains resolution while maximizing throughput |
| Detection Wavelength | 214-220 nm | Peptide bond absorbance maximum |
| Column Loading | 5-25 g crude peptide per liter column volume | Balance between throughput and resolution |
| Fraction Collection Criteria | Peak fractions meeting >95% purity threshold | Ensures final product meets purity specifications |
The preparative HPLC purification workflow begins with dissolution of crude TB-500 in dilute aqueous TFA or acetic acid solution, clarification by filtration (0.45 μm or 0.22 μm), and injection onto the preparative column equilibrated in starting mobile phase conditions. The peptide mixture is separated using a shallow acetonitrile gradient optimized during method development to maximize resolution between TB-500 and related impurities. UV detection at 214-220 nm monitors peptide elution, and automated fraction collectors capture eluent corresponding to the target peak[4].
Quality control analysis of collected fractions by analytical HPLC determines purity and guides pooling decisions. Fractions meeting purity specifications (typically >95-98%) are combined to form the purified peptide pool, while fractions with lower purity may be reprocessed or rejected depending on the specific impurity profile. Multi-cycle purification strategies enable efficient processing of large crude peptide batches, with typical recovery yields of 40-60% based on crude peptide input mass.
Alternative purification technologies including counter-current chromatography and simulated moving bed chromatography offer potential advantages for high-volume production, but RP-HPLC remains the dominant purification platform for TB-500 manufacturing due to its proven reliability, scalability, and regulatory acceptance. Facilities producing pharmaceutical-grade TB-500 must validate purification processes to demonstrate consistent achievement of purity specifications, removal of process-related impurities, and compliance with residual solvent limits.
5. Desalting and Lyophilization Operations
Purified TB-500 fractions contain substantial quantities of TFA and acetonitrile from the HPLC mobile phase, which must be removed prior to final product formulation. The desalting operation employs one of several approaches depending on batch scale and facility capabilities: rotary evaporation to remove acetonitrile followed by lyophilization, direct lyophilization of the purified fraction pool, or buffer exchange chromatography followed by lyophilization. The most common approach for commercial manufacturing involves concentration of the purified pool by rotary evaporation under vacuum to remove acetonitrile, followed by lyophilization to remove water and volatile acids.
Lyophilization (freeze-drying) converts the aqueous peptide solution into a stable solid form suitable for long-term storage and distribution. The lyophilization cycle comprises three sequential phases: freezing, primary drying (ice sublimation), and secondary drying (removal of bound water). Proper cycle design ensures complete water removal while maintaining peptide stability and achieving acceptable reconstitution properties[5].
| Lyophilization Phase | Parameters | Duration | Critical Quality Attributes |
|---|---|---|---|
| Freezing | Cool from +5°C to -40°C at 1°C/min; hold at -40°C | 2-4 hours | Complete ice crystallization, uniform temperature |
| Primary Drying | -40°C to -10°C, vacuum 50-200 mTorr | 24-48 hours | Ice sublimation, residual moisture reduction to <5% |
| Secondary Drying | -10°C to +25°C, vacuum 20-100 mTorr | 8-24 hours | Removal of bound water to achieve <2% final moisture |
| Post-Drying | Nitrogen backfill to atmospheric pressure, sealing | 1-2 hours | Maintain low oxygen environment, prevent moisture uptake |
Critical process parameters for TB-500 lyophilization include freezing rate, shelf temperature profile, chamber pressure, and total cycle duration. These parameters must be optimized during process development to achieve target residual moisture content (typically <2% w/w) while maintaining peptide chemical stability and physical appearance. Residual moisture content represents a critical quality attribute impacting long-term stability, with higher moisture levels increasing rates of hydrolysis, oxidation, and aggregation reactions.
Quality control testing of lyophilized TB-500 includes residual moisture determination by Karl Fischer titration or thermogravimetric analysis, appearance inspection for cake integrity and color, reconstitution time testing, and purity analysis by HPLC to confirm the lyophilization process did not induce degradation. Modern pharmaceutical facilities employ quality by design (QbD) approaches to lyophilization process development, utilizing mathematical modeling and design of experiments methodology to define a robust design space ensuring consistent product quality[6].
6. Analytical Testing and Quality Control
Comprehensive analytical testing of TB-500 batches ensures product identity, purity, potency, and compliance with established specifications. The analytical control strategy encompasses a suite of orthogonal methods providing complementary information about chemical structure, purity profile, physical properties, and microbiological quality. All analytical methods must be validated according to ICH Q2(R2) guidelines, demonstrating specificity, accuracy, precision, linearity, range, detection limit, quantitation limit, and robustness as appropriate for the intended application[7].
| Test | Method | Specification | Frequency |
|---|---|---|---|
| Identity (Sequence) | LC-MS/MS peptide mapping or amino acid analysis | Conforms to reference standard | Each batch |
| Identity (Molecular Weight) | ESI-MS or MALDI-TOF MS | 963.13 ± 1.0 Da (acetate salt) | Each batch |
| Purity (HPLC) | RP-HPLC, C18 column, UV 220 nm | ≥98.0% main peak area | Each batch |
| Related Substances | RP-HPLC with gradient method | Single impurity ≤1.0%; Total impurities ≤2.0% | Each batch |
| Water Content | Karl Fischer titration | ≤5.0% w/w | Each batch |
| Acetate Content | Ion chromatography or 1H-NMR | 3-8% w/w (for acetate salt form) | Each batch |
| TFA Content | 19F-NMR or ion chromatography | ≤0.5% w/w | Each batch |
| Peptide Content | Amino acid analysis with internal standard | 85-100% on anhydrous, salt-free basis | Each batch |
| Bacterial Endotoxins | LAL (Limulus Amebocyte Lysate) assay | ≤5 EU/mg peptide | Each batch |
| Bioburden | Plate count method (bacteria and fungi) | ≤100 CFU/g | Each batch |
| Sterility (if aseptic) | USP <71> Direct Inoculation Method | No growth in 14 days | Each batch (sterile products) |
High-performance liquid chromatography with UV detection represents the primary purity assay for TB-500, utilizing a validated analytical method with specificity for the target peptide and resolution of potential impurities. The analytical HPLC method typically employs a C18 column with acetonitrile/water gradient containing 0.1% TFA, detection at 214-220 nm where the peptide bond exhibits maximum absorbance, and integration of peak areas to calculate purity as percentage of main peak relative to total peak area. Method validation must demonstrate ability to separate TB-500 from known impurities including deletion sequences, oxidation products, and amino acid substitution variants[8].
Mass spectrometry provides orthogonal identity confirmation and molecular weight determination with high accuracy and precision. Electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generates molecular ion peaks corresponding to the protonated peptide [M+H]+, allowing verification of correct molecular weight within ±1 Da tolerance. Advanced MS/MS fragmentation analysis enables complete sequence confirmation through identification of b-ions and y-ions corresponding to peptide bond cleavage products.
Peptide content determination by quantitative amino acid analysis provides an absolute measurement of peptide quantity on an anhydrous, salt-free basis. The method involves acid hydrolysis of the peptide sample (typically 6M HCl at 110°C for 20-24 hours under nitrogen), derivatization of liberated amino acids, chromatographic separation, and quantitation against calibration standards. Results are expressed as percentage peptide content, accounting for water content, counter-ion content, and residual solvents determined by orthogonal methods.
7. Batch Manufacturing Specifications
Commercial TB-500 batches must meet comprehensive specifications encompassing chemical, physical, and microbiological attributes. These specifications are established during product development based on batch analysis data, stability study results, literature precedent, and regulatory guidance. Specifications define acceptable ranges for each quality attribute, and batches failing to meet any specification parameter are rejected and cannot be released for distribution. Manufacturing facilities implement statistical process control to monitor batch quality trends and identify process drift requiring corrective action.
| Attribute | Target Specification | Test Method |
|---|---|---|
| Appearance | White to off-white lyophilized powder | Visual inspection |
| Solubility | Clear, colorless solution at 5 mg/mL in water | Visual inspection after reconstitution |
| Identity (Retention Time) | RRT 0.98-1.02 relative to reference standard | RP-HPLC |
| Identity (Mass) | 963.13 ± 1.0 Da (acetate salt) | ESI-MS or MALDI-TOF MS |
| Purity by HPLC | ≥98.0% (main peak area percentage) | RP-HPLC, UV 220 nm |
| Single Largest Impurity | ≤1.0% | RP-HPLC |
| Total Impurities | ≤2.0% | RP-HPLC (sum of all peaks excluding main peak) |
| Water Content | ≤5.0% w/w | Karl Fischer titration |
| Acetate Content | 3.0-8.0% w/w | Ion chromatography or 1H-NMR |
| TFA Content | ≤0.5% w/w | 19F-NMR or ion chromatography |
| Peptide Content (net) | 85.0-100.0% (anhydrous, salt-free basis) | Amino acid analysis |
| Bacterial Endotoxins | ≤5.0 EU/mg | LAL Gel-Clot or Kinetic Chromogenic |
| Bioburden | ≤100 CFU/g (total aerobic count) | USP <61> <62> Plate Count |
| Heavy Metals | ≤10 ppm | ICP-MS or USP <231> colorimetric |
Batch size for commercial TB-500 production typically ranges from 10 grams to 100 kilograms depending on market demand and manufacturing scale. Small-scale batches (10-100 g) are produced using laboratory or pilot-scale synthesizers and purification equipment, while large-scale batches (1-100 kg) require industrial-scale synthesis vessels and preparative chromatography systems. Regardless of batch size, manufacturing processes must be validated to demonstrate consistent achievement of quality specifications and process performance metrics.
Process validation follows a lifecycle approach encompassing process design (Stage 1), process qualification (Stage 2), and continued process verification (Stage 3). Stage 2 qualification includes execution of at least three consecutive conforming batches under routine manufacturing conditions, demonstrating process capability and reproducibility. Statistical analysis of validation batch data confirms that critical quality attributes fall within specification limits with adequate process capability indices (Cpk ≥ 1.33 preferred).
8. Stability Studies and Shelf-Life Determination
Stability testing of TB-500 follows ICH Q1A(R2) guidelines for new drug substances, establishing shelf-life and storage conditions based on systematic evaluation of chemical and physical stability under defined environmental conditions. The stability program includes long-term stability studies at recommended storage conditions, accelerated stability studies to assess degradation kinetics, and stress testing to identify degradation pathways and validate stability-indicating analytical methods[9].
| Study Type | Storage Conditions | Duration | Testing Frequency |
|---|---|---|---|
| Long-Term Stability | -20°C ± 5°C (recommended storage) | 36 months minimum | 0, 3, 6, 9, 12, 18, 24, 36 months |
| Long-Term Stability (Alternative) | 2-8°C (refrigerated) | 24 months minimum | 0, 3, 6, 9, 12, 18, 24 months |
| Accelerated Stability | 25°C ± 2°C / 60% ± 5% RH | 6 months | 0, 3, 6 months |
| Intermediate Stability | 30°C ± 2°C / 65% ± 5% RH | 6 months (if needed) | 0, 3, 6 months |
| Stress Testing (Temperature) | 40°C, 50°C, 60°C | 1-4 weeks per condition | Initial and final timepoints |
| Stress Testing (pH) | pH 2, 4, 7, 10 at 25°C | 1-4 weeks | Initial and final timepoints |
| Stress Testing (Oxidation) | 0.3% H₂O₂ at 25°C | 24-48 hours | Initial and final timepoints |
| Photostability | ICH Option 2 light exposure | Until target exposure reached | Post-exposure analysis |
Stability sample testing includes the full battery of release tests plus additional assessments specifically designed to detect degradation. Key stability-indicating parameters for TB-500 include HPLC purity (monitoring increase in degradation products), mass spectrometry (detecting oxidation, deamidation, or fragmentation), visual appearance (detecting discoloration or aggregation), and reconstitution characteristics (detecting changes in solubility). Stability batches represent commercial manufacturing processes and are packaged in the proposed commercial container-closure system to ensure data relevance.
Degradation pathways identified during stress testing of TB-500 typically include oxidation of methionine residues (if present in related peptides, though TB-500 lacks methionine), deamidation of glutamine residues to glutamic acid, hydrolysis of peptide bonds (particularly Asp-Pro and Asp-Gly sequences), and aggregation through intermolecular disulfide bond formation or hydrophobic interactions. Understanding these degradation mechanisms guides formulation development, including selection of pH, excipients, and container-closure systems that minimize degradation rates.
Shelf-life determination follows ICH Q1E guidelines for evaluation of stability data. For lyophilized TB-500 stored at -20°C, typical shelf-life assignments range from 24 to 36 months based on long-term stability data demonstrating maintenance of specifications throughout the proposed shelf-life period. Extrapolation beyond observed data may be justified when stability data shows no significant degradation trend, but extrapolation should not exceed 12 months beyond real-time data and should not exceed twice the period covered by long-term data[10].
9. Storage and Handling Requirements
Proper storage and handling of TB-500 ensures maintenance of product quality throughout the distribution chain and during end-user handling. Storage requirements are established based on stability data and must account for both the lyophilized powder and reconstituted solution forms. Manufacturing facilities, distributors, and end users must implement appropriate storage conditions, monitoring systems, and handling procedures to prevent product degradation, contamination, or quality excursions.
| Product Form | Storage Conditions | Stability Period | Special Handling |
|---|---|---|---|
| Lyophilized Powder (Unopened) | -20°C ± 5°C in sealed container | 24-36 months (per CoA) | Protect from light and moisture; store in original container |
| Lyophilized Powder (Unopened Alternative) | 2-8°C (refrigerated) | 12-24 months (per CoA) | Protect from light and moisture; do not freeze-thaw repeatedly |
| Lyophilized Powder (Short-term) | Room temperature (20-25°C) | Up to 30 days | Keep sealed; return to -20°C for long-term storage |
| Reconstituted Solution (Sterile Water) | 2-8°C (refrigerated) | 7-14 days | Use aseptic technique; store in sterile container; discard if cloudy |
| Reconstituted Solution (Bacteriostatic Water) | 2-8°C (refrigerated) | 14-28 days | Use aseptic technique; store in sterile container |
| Shipping (Ambient) | Insulated packaging with temperature monitoring | Transit time ≤5 days | Avoid direct sunlight; temperature loggers included |
| Shipping (Refrigerated) | 2-8°C with gel packs or dry ice | Per shipping validation | Qualified shipping containers; temperature monitoring |
Container-closure systems for lyophilized TB-500 typically consist of Type I borosilicate glass vials with rubber stoppers and aluminum crimp seals, providing excellent moisture barrier properties and chemical compatibility. Vials are often backfilled with nitrogen or argon during lyophilization to minimize oxygen exposure and reduce oxidation potential during storage. Package integrity testing validates the container-closure system's ability to maintain sterility and exclude moisture throughout the shelf-life period.
Temperature excursions during storage or shipping require assessment to determine product acceptability. Short-term temperature excursions (e.g., 2-8 hours at room temperature) typically do not compromise product quality if the material is returned to proper storage conditions promptly. Extended temperature excursions or exposure to temperatures exceeding 25°C require formal evaluation using stability data or additional testing to confirm continued compliance with specifications. Documentation of temperature excursions and disposition decisions must be maintained as part of the quality record.
Reconstitution of lyophilized TB-500 should be performed using aseptic technique with appropriate diluents including sterile water for injection, bacteriostatic water for injection (containing 0.9% benzyl alcohol), or sterile saline solution. The lyophilized cake typically dissolves within 1-3 minutes with gentle swirling, forming a clear to slightly opalescent solution. Vigorous shaking should be avoided to minimize aggregation and foaming. Reconstituted solutions should be inspected visually for particulate matter and discoloration prior to use, with any solution showing turbidity, precipitation, or color change being discarded.
10. Certificate of Analysis (CoA) Documentation
The Certificate of Analysis represents the official quality documentation accompanying each TB-500 batch, certifying that the batch has been manufactured according to established procedures, tested using validated analytical methods, and found to meet all specifications. The CoA provides critical information for quality assurance, regulatory compliance, and traceability, enabling customers to verify product identity, purity, and quality before use. Manufacturing facilities must implement robust document control procedures ensuring CoA accuracy, completeness, and proper authorization[11].
| CoA Section | Required Information | Example Data |
|---|---|---|
| Product Identification | Product name, catalog number, CAS number, molecular formula | TB-500 (Thymosin Beta-4 Fragment 17-23), Cat# TB500-5MG, CAS 77591-33-4 |
| Batch Information | Batch/lot number, manufacture date, retest/expiry date | Batch: TB500-2024-001, Mfg: 15-Jan-2024, Exp: 15-Jan-2027 |
| Physical Description | Appearance, net weight per vial, number of vials | White lyophilized powder, 5.2 mg net peptide per vial, 100 vials |
| Identity Tests | HPLC retention time, mass spectrometry molecular weight | RT: 18.5 min (conforms), MW: 963.1 Da (conforms) |
| Purity by HPLC | Main peak area percentage, chromatogram reference | 98.7% (Spec: ≥98.0%), Chromatogram: CHR-2024-001 |
| Related Substances | Individual impurity percentages, total impurities | Largest impurity: 0.6%, Total impurities: 1.3% (Spec: ≤2.0%) |
| Water Content | Percentage by Karl Fischer titration | 3.2% (Spec: ≤5.0%) |
| Peptide Content | Net peptide content by amino acid analysis | 92.5% (Spec: 85.0-100.0%, anhydrous, salt-free basis) |
| Endotoxins | LAL assay result in EU/mg | <0.5 EU/mg (Spec: ≤5.0 EU/mg) |
| Microbiological Quality | Total aerobic count, sterility (if applicable) | <10 CFU/g (Spec: ≤100 CFU/g); Sterile: Pass |
| Storage Conditions | Recommended storage temperature and conditions | Store at -20°C ± 5°C; protect from light and moisture |
| Approval | QC manager name, signature, date of approval | Approved by: J. Smith, QC Manager, Date: 22-Jan-2024 |
Certificate of Analysis format and content should comply with industry standards and regulatory expectations, including clear presentation of test results, specifications, and conformance statements. Electronic CoAs in PDF format have become standard practice, enabling efficient distribution and archiving while maintaining document integrity through digital signatures and version control. Some manufacturers provide QR codes or batch-specific URLs enabling customers to verify CoA authenticity and access additional batch documentation.
Retention of CoA records follows pharmaceutical documentation requirements, typically requiring retention for a minimum of one year beyond the product expiration date or as specified by applicable regulations. Electronic document management systems facilitate CoA retrieval, version control, and audit trail maintenance, supporting regulatory inspections and customer inquiries. Facilities subject to FDA oversight must maintain CoA records in compliance with 21 CFR Part 11 for electronic records and electronic signatures.
Conclusion
TB-500 manufacturing requires integration of advanced peptide synthesis technology, high-resolution purification methods, comprehensive analytical testing, and stringent quality management systems to consistently produce pharmaceutical-grade material meeting established specifications. This manufacturing profile has outlined the critical process operations, quality control strategies, and documentation requirements necessary for commercial TB-500 production compliant with current Good Manufacturing Practices and applicable regulatory standards.
Manufacturing facilities must validate all critical processes including SPPS synthesis, HPLC purification, and lyophilization to demonstrate process capability and reproducibility. Analytical methods must be validated to ensure reliable detection of the target peptide and related impurities throughout manufacturing and stability testing. Stability programs following ICH guidelines establish shelf-life and storage conditions based on systematic evaluation of chemical and physical stability parameters.
The complexity of peptide manufacturing demands highly trained personnel, specialized equipment, and rigorous quality oversight to maintain product quality and regulatory compliance. As therapeutic peptide markets continue expanding, manufacturing organizations must implement continuous improvement initiatives, adopt emerging technologies, and maintain current knowledge of regulatory expectations to remain competitive and deliver high-quality peptide products to the market. The principles and practices described in this manufacturing profile provide a comprehensive framework for TB-500 production suitable for research, clinical development, and commercial distribution applications.
References
- Bachem. "Solid Phase Peptide Synthesis (SPPS) Explained." https://www.bachem.com/articles/peptides/solid-phase-peptide-synthesis-explained/
- Behrendt R, White P, Offer J. "Automated solid-phase peptide synthesis to obtain therapeutic peptides." PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC4077397/
- Science Direct. "Sustainable Ultrasound-Assisted Solid-Phase peptide synthesis (SUS-SPPS): Less Waste, more efficiency." https://www.sciencedirect.com/science/article/pii/S1350417725000367
- PubMed. "Synthesis and characterization of the N-terminal acetylated 17-23 fragment of thymosin beta 4 identified in TB-500." https://pubmed.ncbi.nlm.nih.gov/22962027/
- PMC. "Recommended Best Practices for Lyophilization Validation 2021 Part II: Process Qualification and Continued Process Verification." https://pmc.ncbi.nlm.nih.gov/articles/PMC8575750/
- PMC. "Recommended Best Practices for Lyophilization Validation—2021 Part I: Process Design and Modeling." https://pmc.ncbi.nlm.nih.gov/articles/PMC8373746/
- International Conference on Harmonisation. "ICH Quality Guidelines." https://www.ich.org/page/quality-guidelines
- PubMed. "Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry." https://pubmed.ncbi.nlm.nih.gov/23084823/
- ICH. "Q1A(R2) Stability Testing Guidelines." https://database.ich.org/sites/default/files/Q1A(R2)%20Guideline.pdf
- International Conference on Harmonisation. "ICH Quality Guidelines - Stability Testing." https://www.ich.org/page/quality-guidelines
- PekCura Labs. "TB-500 - Certificates of Analysis." https://pekcuralabs.com/tb-500-certificates-of-analysis/