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Collagen accounts for approximately 25% of total protein in the human body, making it the most abundant structural protein. It is widely distributed in the skin, bones, tendons, blood vessels, and other tissues, playing a critical role in maintaining structural integrity and physiological function. To date, 28 collagen types (Type I–XXVIII) have been identified, among which Type I and Type III represent approximately 95% of total collagen content.
Native collagen consists of three α-chains, each composed of approximately 1,014 amino acids, forming a characteristic triple-helix structure with a molecular weight of about 300 kDa. This unique conformation underpins its diverse biological functions: enhancing skin elasticity and hydration, promoting wound healing, strengthening joints and bones, improving hair and nail quality, and even supporting visual health.
As illustrated above, collagen-based products can be broadly categorized into three types: Collagen, Gelatin, and Collagen Peptides.
The global collagen market continues to expand steadily. According to Grand View Research, the market size reached USD 17.258 billion in 2022 and is projected to grow to USD 22.622 billion by 2027 (CAGR 5.42%). Among application sectors, healthcare is the primary growth driver (expected to reach USD 11.117 billion by 2027, accounting for approximately 50%), followed by cosmetics and food & beverages. Different application fields correspond to distinct regulatory frameworks and quality standards, and with ongoing technological advancements, the boundaries of collagen applications continue to expand.
Currently, collagen production mainly follows two technical routes: animal-derived extraction and recombinant expression. Recombinant collagen is classified into three categories:
- Recombinant human collagen (with a complete triple-helix structure)
- Recombinant humanized collagen (containing partial human-derived sequences)
- Recombinant collagen-like proteins (with lower sequence homology)
Common expression systems include Escherichia coli, yeast, and mammalian cells (such as CHO cells). Although process details vary depending on the source and expression platform, the overall manufacturing workflow typically includes:
Fermentation/Extraction → Clarification → Purification (removal of impurities, endotoxins, viruses, etc.) → Buffer exchange/Concentration → Sterile filtration, along with other critical processing steps.
Clarification
During the clarification stage after extraction or fermentation, as well as after precipitation or enzymatic digestion in purification, the solution often has high turbidity and contains cells, debris, precipitates, and other impurities. Proper clarification is needed to ensure smooth downstream processing.
For convenience and effective clarification, depth filtration is usually the preferred choice. Considering the complexity of the solution and different process requirements, Cobetter offers a range of depth filtration solutions with varying precision and characteristics, allowing flexible adaptation to different process steps.
Case Study 1 (CHO-Expressed Collagen Project)：
CHO cell fermentation broth, after enzymatic digestion and pH adjustment, left to settle overnight. The resulting supernatant initial turbidity>300 NTU.
For certain process steps where consumable reuse or high solids content is a concern, such as yeast fermentation, microfiltration hollow fibers or microfiltration TFF cassettes can be used for clarification.
Case study 2:
Recombinant humanized collagen expressed in a yeast system. After centrifugation, the feed had a turbidity of 147 NTU. Choose 0.2 μm, 1 mm ID hollow fiber (HFEMIN0201030P) for testing.
Results: the feed solution was concentrated around 6 times and diafiltered 6 times, maintaining a high flux of ~30 LMH. Post-filtration turbidity dropped to 3.41 NTU, indicating excellent clarification. No target protein was detected in the retentate, demonstrating negligible adsorption or retention of the product.
Purification
Traditional chromatography resins are costly and prone to shortened lifespan when handling high-viscosity materials. Moreover, conventional operations often rely on large-scale chromatography systems, placing higher demands on equipment investment, facility space, and layout.
Consequently, the industry is gradually shifting toward membrane chromatography technology. With higher loading capacity, faster flow rates, and stronger fouling resistance, membrane chromatography can significantly reduce consumable usage, shorten process time, and eliminate cumbersome steps such as column packing, unpacking, and storage. It offers greater operational flexibility and a smaller footprint.
Case Study 3:
In the purification process of recombinant type I and III collagen, the anion exchange step compared traditional resins with Cobetter’s Pultrix™ XQ membrane chromatography filter. The membrane chromatography filter achieved a loading capacity of 60 g/L—four times that of traditional resins (15 g/L)—while removing over 95% of host cell proteins (HCPs). In terms of yield, type I collagen achieved 90% with membrane chromatography compared to only ~60% with traditional resins.Type III collagen yield was also increased by 5%, as shown below.
Based on customer process requirements, Cobetter offers membrane chromatography products with multiple mechanisms, including anion exchange (AEX), cation exchange (CEX), and hydrophobic interaction (HIC). Among them, AEX membrane chromatography is also widely applied in critical steps such as endotoxin removal.
Concentration / Buffer Exchange
Conventional buffer exchange and concentration can be performed using ultrafiltration cassettes or hollow fibers. However, for high-viscosity materials such as animal-derived collagen, viscosity increases significantly after concentration, posing a serious challenge for sterile filtration. It is recommended to use sterile hollow fiber filters: perform sterile filtration at a lower concentration first, then transfer via aseptic connection to a sterile hollow fiber system for concentration to the target level. This approach effectively improves process efficiency and product recovery.
Case Study 4:
For an animal-derived collagen product with a molecular weight above 300 kDa and an initial concentration of 1.0 mg/mL, the goal was to concentrate it to 5–10 mg/mL. Cobetter ultrafiltration cassette UFCLA0100001P (RC membrane, standard coarse screen, 100 kDa) was tested. The process involved dialysis four times for impurity removal, followed by concentration five times. The average flux during dialysis was 137.44 LMH, and during concentration 114.26 LMH.
Case Study 5:
For a natural collagen product with a target molecular weight of 300 kDa, tangential flow filtration was applied to remove small-molecule impurities. Performance was compared between a 100K TFF cassette (standard coarse screen) and a hollow fiber (0.5 mm fiber ID). During dialysis and buffer exchange, pressure and flow performance were recorded as shown below.
As indicated in the table, both the cassette and hollow fiber demonstrated high process flux during buffer exchange and impurity removal. After cleaning, water flux recovery exceeded 95%, meeting customer requirements for completing the process in a short time. Both filters can be reused, effectively reducing production costs.
Viral Removal
The selection and loading capacity of virus removal filters are closely related to the molecular weight of the target protein:
- For recombinant collagen with a molecular weight below 200 kDa, PES membrane virus filters (such as ViruClear™ VF or ViruClear™ VF Plus) are generally recommended.
- For collagen above 200 kDa or full-length sequences, the higher molecular weight and increased viscosity may lead to rapid fouling and limited loading when using PES membranes. In such cases, RC membrane virus filters (such as ViruClear™ RCH) are recommended, offering higher loading capacity and lower flux decay.
Currently, RCH has been successfully implemented by customers in viral clearance processes at a production scale of 6,000 L.
Case Study 6:
In another customer case, the original process utilized an other brand's virus filtration solution, achieving a recovery of only 30%. Through optimization of both prefiltration and viral filtration steps with Cobetter filters, overall recovery was improved to approximately 90%.
Sterile Filtration
In a yeast-based expression system for recombinant humanized collagen, the impact of protein concentration on sterile filtration performance was evaluated. Cobetter’s PVDF filter (LHPVND0022) was used to filter solutions at 15 g/L (left figure) and 40 g/L (right figure), respectively. The results are summarized below.
In this case, the sterile filtration loading capacity of the concentrated solution was relatively low (approximately 45 L/m²). In contrast, for unconcentrated or lower-concentration solutions, the loading capacity increased by nearly 3 times (approximately 130 L/m²).
Therefore, as discussed earlier, it is advisable to consider performing sterile filtration prior to concentration during process design, followed by aseptic concentration using sterile hollow fiber filter to reach the target concentration. Final process configuration should be evaluated comprehensively based on cost, risk, and operational flexibility. In addition, a combined filtration strategy may also be considered. Appropriate prefiltration can significantly improve the loading capacity of the sterilizing-grade filter.
This article presents Cobetter’s end-to-end collagen processing solutions, spanning from upstream to downstream operations. By integrating representative case studies with analysis of key technical challenges, we aim to provide practical insights for process optimization and support the advancement of collagen manufacturing technologies.
It should be noted that, due to the diverse sources of collagen, significant differences in molecular weight, and varying process routes, real-world applications often encounter distinct technical bottlenecks.
