How Is CJC-1295 Made?

Overview of Peptide Manufacturing

CJC-1295 is manufactured using solid-phase peptide synthesis (SPPS), the same sophisticated chemical process used for other therapeutic peptides. However, CJC-1295's synthesis is particularly complex due to the need to attach the Drug Affinity Complex (DAC) modification after the peptide backbone is assembled. The manufacturing process requires specialized equipment, expertise, and quality control to produce a peptide suitable for human use, though the quality of CJC-1295 available through research chemical suppliers varies enormously.

Unlike FDA-approved medications manufactured under strict Good Manufacturing Practice (GMP) regulations, most CJC-1295 is produced by contract manufacturers, often in China or other countries with less stringent oversight. This creates significant quality variability—some suppliers provide relatively pure, well-characterized products, while others sell material of questionable quality. Understanding the manufacturing process helps users appreciate the complexity involved and the potential for quality issues.

Solid-Phase Peptide Synthesis (SPPS)

The foundation of CJC-1295 manufacturing is solid-phase peptide synthesis, a method that builds the peptide chain one amino acid at a time while the growing chain is attached to an insoluble solid support (typically a resin bead). This approach, developed by Bruce Merrifield in the 1960s (earning him the Nobel Prize in Chemistry in 1984), revolutionized peptide manufacturing and made therapeutic peptides commercially viable.

The SPPS Process

SPPS begins with a solid resin bead to which the first amino acid (the C-terminal amino acid of the final peptide) is chemically attached. The amino acid's amino group is protected by a temporary protecting group (typically Fmoc or Boc) to prevent unwanted reactions. The synthesis then proceeds through repeated cycles of deprotection and coupling.

In the deprotection step, the protecting group is removed from the amino terminus of the growing peptide chain, typically using a base like piperidine (for Fmoc chemistry) or acid like trifluoroacetic acid (for Boc chemistry). This exposes the free amino group, making it reactive for the next coupling step.

In the coupling step, the next amino acid (with its amino group protected and its carboxyl group activated) is added to the reaction vessel. The activated carboxyl group reacts with the free amino group on the resin-bound peptide, forming a new peptide bond. Coupling reagents like HBTU, HATU, or DIC are used to activate the carboxyl group and facilitate the reaction.

For CJC-1295's approximately 29-amino acid sequence, this deprotection-coupling cycle repeats 28 times. Each cycle must proceed with extremely high efficiency—even 99% efficiency per step would result in only about 75% of chains having the correct sequence after 29 steps. Modern SPPS achieves >99.5% efficiency per coupling through optimized conditions, but this still requires careful monitoring and quality control.

Challenges in CJC-1295 Synthesis

Several factors make CJC-1295 synthesis challenging. First, the 29-amino acid length approaches the practical limits of standard SPPS—longer peptides accumulate more synthesis errors and deletion sequences (peptides missing one or more amino acids). Second, certain amino acid sequences in CJC-1295 may be prone to aggregation or incomplete coupling, requiring special handling or modified conditions.

Third, the peptide must be synthesized with a specific lysine residue available for DAC attachment while other reactive groups are protected. This requires careful selection of protecting group strategies to ensure the correct lysine is accessible while others are blocked. Fourth, the synthesis must produce peptide of sufficient purity for the DAC modification step—impurities can complicate the modification reaction and reduce final product quality.

To address these challenges, manufacturers may use advanced SPPS techniques including microwave-assisted synthesis (which accelerates reactions and improves coupling efficiency), pseudoproline dipeptides (which disrupt aggregation-prone sequences), double coupling (repeating coupling steps to ensure completeness), and optimized coupling reagents and conditions for difficult sequences. The exact methods used vary by manufacturer and are often proprietary.

DAC Modification

After the peptide backbone is synthesized, the Drug Affinity Complex (DAC) modification must be attached. This is the critical step that distinguishes CJC-1295 from other GHRH analogs and enables its extended half-life. The DAC modification involves attaching a maleimidoproprionic acid (MPA) derivative to a specific lysine residue in the peptide sequence.

The Modification Process

The DAC attachment typically occurs while the peptide is still on the solid support, though it can also be performed in solution after cleavage. The process involves several steps. First, the protecting group on the target lysine's side chain (typically an Mtt or Alloc group that can be selectively removed) is removed while other protecting groups remain in place. This exposes the lysine's amino group for modification.

Next, the MPA derivative (containing the maleimide group that will bind to albumin) is coupled to the lysine's amino group. This coupling uses similar chemistry to peptide bond formation but attaches the MPA group via an amide linkage to the lysine side chain. The reaction must be highly selective to avoid modifying other reactive groups in the peptide.

After DAC attachment, the completed peptide is cleaved from the solid support and all remaining protecting groups are removed. This cleavage step typically uses strong acid (trifluoroacetic acid with scavengers) that simultaneously removes protecting groups and releases the peptide from the resin. The crude peptide is then precipitated, washed, and dried.

Quality Control of DAC Modification

Verifying correct DAC attachment is critical for quality control. Mass spectrometry can confirm the molecular weight increase from the DAC group. HPLC can separate DAC-modified from unmodified peptide. Functional assays can verify albumin binding capacity. However, not all manufacturers perform these verification steps, and some "CJC-1295" products may actually be unmodified peptide or incorrectly modified material.

Purification

After synthesis and DAC modification, crude CJC-1295 contains the desired product along with numerous impurities: deletion sequences (peptides missing one or more amino acids), truncated sequences, peptides with incomplete deprotection, unmodified or incorrectly modified peptides, aggregates, and residual reagents. Purification to research-grade quality (typically >95% purity) requires sophisticated chromatographic techniques.

High-Performance Liquid Chromatography (HPLC)

The primary purification method is preparative reversed-phase HPLC, which separates peptides based on hydrophobicity. Crude CJC-1295 is dissolved and injected onto a large HPLC column packed with hydrophobic stationary phase (typically C18 or C8 silica). A gradient of increasing organic solvent (typically acetonitrile with trifluoroacetic acid) elutes peptides in order of increasing hydrophobicity.

CJC-1295, with its DAC modification, is more hydrophobic than unmodified peptide and elutes later in the gradient. The HPLC separation must resolve CJC-1295 from closely related impurities that differ by only one or two amino acids or have incorrect DAC attachment. This requires careful optimization of column type, gradient profile, temperature, and flow rate.

Multiple HPLC runs may be needed to achieve research-grade purity. Fractions containing pure CJC-1295 are collected, pooled, and the organic solvent is removed by lyophilization (freeze-drying). The purified peptide is typically a white to off-white powder.

Additional Purification Steps

Depending on the impurity profile, additional purification steps may be employed. Ion exchange chromatography can remove peptides with different charge properties. Size exclusion chromatography can remove aggregates and fragments. However, each additional step reduces yield, so manufacturers must balance purity requirements with economic considerations.

For research chemical CJC-1295, purification rigor varies widely. Some suppliers perform multiple purification steps and achieve >98% purity. Others perform minimal purification, resulting in products with significant impurities. Third-party testing (when available) can verify purity, but many products lack such testing.

Quality Control and Analytical Testing

Proper quality control involves extensive analytical testing to verify identity, purity, potency, and safety. However, the level of quality control for research chemical CJC-1295 varies dramatically between suppliers.

Identity and Structure Verification

Mass spectrometry determines the molecular weight with high precision, confirming the correct amino acid sequence and DAC modification. The expected molecular weight for CJC-1295 with DAC is approximately 3,647 Da (exact value depends on the specific formulation). Peptide mapping (enzymatic digestion followed by HPLC-MS analysis) can confirm the sequence and identify the DAC attachment site.

Amino acid analysis quantifies each amino acid after acid hydrolysis, verifying the composition. However, this technique is expensive and not routinely performed for research chemicals. Nuclear magnetic resonance (NMR) spectroscopy can provide detailed structural information but is rarely used for routine quality control.

Purity Assessment

Analytical HPLC quantifies CJC-1295 purity and identifies impurities. A typical purity specification is >95% by HPLC, with individual impurities below defined limits. However, HPLC purity doesn't capture all quality aspects—peptides can have correct HPLC purity but contain incorrect sequences, improper DAC modification, or other defects not detected by HPLC.

Capillary electrophoresis provides an orthogonal purity assessment using different separation principles. Size exclusion chromatography quantifies aggregates and fragments. These multiple analytical methods provide more comprehensive purity characterization but are often not performed for research chemicals.

Potency Testing

Biological potency assays confirm that CJC-1295 retains its ability to activate GHRH receptors and bind to albumin. Cell-based assays measure receptor binding and activation using cells expressing human GHRH receptors. Albumin binding assays verify the DAC modification's functionality. These assays are important for confirming biological activity but are expensive and rarely performed for research chemicals.

Safety Testing

Safety testing for pharmaceutical peptides includes endotoxin testing (using Limulus amebocyte lysate assay), sterility testing, heavy metals testing, and residual solvent analysis. These tests protect against contamination that could cause adverse effects. However, research chemical CJC-1295 often lacks comprehensive safety testing, creating potential risks for users.

Manufacturing Quality Tiers

CJC-1295 manufacturing exists on a quality spectrum from pharmaceutical-grade (which doesn't exist for CJC-1295 since it's not approved) to low-quality research chemicals.

ConjuChem's Original Manufacturing

ConjuChem's clinical trial material was manufactured under GMP conditions with rigorous quality control. This represented the highest quality CJC-1295 ever produced, with comprehensive testing, validated manufacturing processes, and regulatory oversight. However, this material is no longer available since ConjuChem discontinued development.

High-Quality Research Chemical Suppliers

Some research chemical suppliers provide relatively high-quality CJC-1295 with third-party testing, certificates of analysis, and reasonable purity (>95%). These suppliers typically work with reputable contract manufacturers and perform at least basic quality control. However, even "high-quality" research chemicals lack pharmaceutical-grade quality assurance and regulatory oversight.

Mid-Tier Suppliers

Many suppliers provide CJC-1295 of moderate quality—reasonably pure but with limited testing and quality control. Purity may be 90-95%, with some impurities and batch-to-batch variability. These products may be adequate for some users but carry more risk than higher-quality options.

Low-Quality Suppliers

Some suppliers provide low-quality CJC-1295 with significant impurities, incorrect dosing, mislabeling, or even wrong compounds. These products pose serious risks including ineffectiveness, unexpected side effects, or contamination-related adverse events. Unfortunately, distinguishing low-quality from higher-quality products can be difficult without testing.

Modified GRF 1-29 Manufacturing

Modified GRF 1-29 (CJC-1295 without DAC) is manufactured similarly to CJC-1295 but without the DAC modification step. This simplifies manufacturing and reduces cost, as the complex DAC attachment chemistry is eliminated. The peptide is synthesized using standard SPPS, cleaved from the resin, and purified by HPLC.

The simpler manufacturing process for Modified GRF 1-29 may result in more consistent quality compared to CJC-1295 with DAC, as there's one less complex step where errors can occur. However, quality still varies widely between suppliers, and the same quality control considerations apply.

Storage and Stability

Proper storage is critical for maintaining CJC-1295 quality. The lyophilized (freeze-dried) powder should be stored at -20°C (freezer) or 2-8°C (refrigerator) in a sealed container protected from light and moisture. Under proper storage, lyophilized CJC-1295 can remain stable for 1-2 years or longer.

After reconstitution with bacteriostatic water, CJC-1295 should be stored refrigerated (2-8°C) and used within 2-4 weeks. The exact stability depends on formulation, pH, and storage conditions. Some degradation is inevitable over time, reducing potency. Reconstituted peptide should never be frozen, as freeze-thaw cycles can cause aggregation and loss of activity.

Improper storage (room temperature, exposure to light, contamination) can rapidly degrade CJC-1295. Users should follow storage guidelines carefully and be suspicious of suppliers who don't provide proper storage instructions or ship without appropriate temperature control.

Quality Verification for Users

Users have limited ability to verify CJC-1295 quality without access to analytical equipment. However, some strategies can help minimize risk:

Supplier Selection

Choose suppliers with good reputations, third-party testing, certificates of analysis, proper storage and shipping, clear product information, and responsive customer service. Be skeptical of unusually cheap products or suppliers making unrealistic claims.

Visual Inspection

Lyophilized CJC-1295 should be a white to off-white powder forming a solid cake in the vial. Discoloration, unusual texture, or visible contamination suggests quality problems. Reconstituted peptide should be clear and colorless. Cloudiness, particles, or discoloration indicate degradation or contamination.

Third-Party Testing

Some users send products to independent laboratories for testing. This can verify identity, purity, and absence of contamination but is expensive ($200-500 per test). For users purchasing large quantities or concerned about quality, testing may be worthwhile.

Biological Response

Monitoring biological responses (IGF-1 levels, subjective effects) can provide indirect evidence of quality. Lack of expected IGF-1 increase or effects suggests low potency or wrong compound. However, individual variability makes this an imperfect quality indicator.

Compounding Pharmacy CJC-1295

Some compounding pharmacies provide CJC-1295 by prescription. These pharmacies are regulated and must follow USP (United States Pharmacopeia) standards for compounding. This provides more quality assurance than research chemical suppliers, though compounded CJC-1295 still lacks FDA approval and the rigorous testing of approved medications.

Compounding pharmacy CJC-1295 is typically more expensive than research chemicals but offers advantages including regulatory oversight, professional preparation, proper labeling, and medical supervision. For users who can access and afford it, compounding pharmacy CJC-1295 may be a safer option than research chemicals.