HGF Human, CHO

What is human HGF and why is it predominantly produced in CHO cells?

Human Hepatocyte Growth Factor is a potent multi-functional protein that affects morphogenesis, cell migration, organ regeneration, and tumor invasion in various tissues. It functions as a strong mitogen for hepatocytes and primary epithelial cells, and synergizes with interleukin-3 and GM-CSF to stimulate colony formation of hematopoietic progenitor cells .

CHO cells are the preferred expression system for producing recombinant human HGF (rhHGF) because:

• They provide proper post-translational modifications essential for biological activity

• They can secrete high quantities of functional rhHGF

• They have demonstrated capacity for overexpression in serum-free medium culture conditions, achieving yields exceeding 13 mg/l in a 5-day batch culture using a 7.5l bioreactor

• They produce rhHGF that exhibits properties similar to native human HGF, making it suitable for both research and potential therapeutic applications

What are the structural characteristics of CHO-derived rhHGF?

CHO-derived rhHGF typically exists as a heterodimeric protein with the following characteristics:

• Molecular weight: Approximately 80-105 kDa, consisting of:

  • α-chain of 463 amino acids

  • β-chain of 234 amino acids

• The rhHGF protein from CHO cells is found as a mixture of:

  • Inactive pro-HGF (single-chain precursor)

  • Active heterodimeric form (processed)

• The CHO-derived rhHGF has a higher molecular weight than its counterpart expressed in insect cells, suggesting different glycosylation patterns

• The inactive pro-HGF rapidly converts to the active heterodimeric form in the presence of serum, indicating this conversion would likely occur when injected into the human body

How should researchers properly store and reconstitute rhHGF?

Proper storage and reconstitution are critical for maintaining the biological activity of rhHGF:

Storage conditions:

• Lyophilized rhHGF is stable at room temperature for up to 3 weeks but should ideally be stored desiccated below -18°C for long-term stability

• Upon reconstitution, rhHGF should be stored at 4°C if used within 2-7 days

• For extended periods, store reconstituted rhHGF below -18°C

• Avoid repeated freeze-thaw cycles as they can degrade the protein

Reconstitution protocol:

• Reconstitute lyophilized rhHGF in sterile 18MΩ-cm H₂O to a concentration not less than 100 μg/ml

• For long-term storage of reconstituted rhHGF, it is recommended to add a carrier protein (0.1% Human Serum Albumin or Bovine Serum Albumin)

• The reconstituted solution can be further diluted into other aqueous buffers as needed for specific applications

What purification methods effectively isolate rhHGF from CHO cell culture supernatants?

Purification of rhHGF from CHO cell culture supernatants typically involves a multi-step chromatographic approach:

Standard purification protocol:

• Harvest culture supernatant from CHO cells after a 5-day batch culture in serum-free medium

• Apply a two-step chromatographic procedure:

  • Primary capture step: Often using affinity or ion exchange chromatography

  • Polishing step: Size exclusion or hydrophobic interaction chromatography

• This protocol typically results in approximately 35% recovery rate of purified rhHGF

Purity assessment:

• Reversed-phase HPLC (RP-HPLC) analysis

• SDS-PAGE under reducing conditions

• Protein quantitation via:

  • UV spectroscopy at 280 nm using an absorbency value of 1.83 as the extinction coefficient for a 0.1% solution

  • Comparative RP-HPLC using a calibrated solution of HGF as reference standard

High-purity rhHGF preparations generally exceed 97% purity as determined by these analytical methods .

How is the biological activity of rhHGF accurately assessed?

The biological activity of rhHGF can be evaluated through several functional assays:

Cell proliferation assays:

• Dose-dependent proliferation of monkey 4MBr-5 indicator cells, with an ED50 typically in the range of 20-40 ng/ml

• Specific activity measurements calibrated against WHO Hepatocyte Growth Factor reference standard (NIBSC code: 96/556)

Cell scattering activity:

• Assessment of the morphological changes and cell motility in epithelial cell lines

• The half-maximal effective concentration (EC50) for purified rhHGF has been reported as 36.3 ng/ml in cell scattering activity assays

Receptor binding and activation:

• Binding affinity to HGF receptor (c-Met) using surface plasmon resonance (SPR):

  • Affinity constant (KD) of 0.486 nM when measured by Biacore 8K

  • Affinity constant of 3.29 nM when measured by BLI assay (ForteBio Octet Red96e)

• c-Met tyrosine phosphorylation in liver tissues following administration

Downstream functional assessments:

• Stimulation of IL-11 secretion by Saos-2 cells, with a specific activity of >6.00 × 10⁵ IU/mg

What is the pharmacokinetic profile of rhHGF in experimental models?

The pharmacokinetics of rhHGF varies based on administration route and the physiological state of the experimental model:

In normal rats (intravenous administration of 0.1 mg/kg):

• Peak serum concentration: 89.7 ± 20.6 ng/ml at 5 minutes post-injection

• Elimination half-life: 2.4 minutes

• Primary tissue distribution: Liver

• Induces c-Met tyrosine phosphorylation in liver tissues

Route-dependent differences:

• Portal vein administration results in lower serum HGF levels but increased hepatic distribution compared to systemic intravenous injection

Disease state modifications:

• Rats with 70% partial hepatectomy or liver cirrhosis show:

  • Increased serum levels of human HGF

  • Prolonged elimination half-life compared to normal rats

These pharmacokinetic characteristics suggest that despite its short half-life, bolus injection of rhHGF may induce therapeutic effects in patients with liver disease, though dosing should be adjusted according to the degree of liver injury .

How do glycosylation patterns of CHO-derived rhHGF differ from other expression systems?

The glycosylation pattern of rhHGF varies significantly between expression systems and affects its biological properties:

CHO-derived rhHGF vs. insect cell-derived rhHGF:

• CHO-derived rhHGF has a higher molecular weight than its counterpart expressed in insect cells

• This difference is attributed to more complex glycosylation patterns in mammalian CHO cells

• The CHO-derived protein better mimics the glycosylation found in native human HGF

Functional implications of glycosylation differences:

• Glycosylation affects protein stability, half-life, and receptor binding

• CHO-derived rhHGF exhibits a mixture of inactive pro-HGF and active heterodimeric forms

• The conversion of inactive pro-HGF to the active form occurs rapidly in the presence of serum, suggesting this process would occur when injected into the human body

Researchers should consider these glycosylation differences when designing experiments, as they may impact experimental outcomes depending on the specific application and required biological activity.

What strategies can optimize rhHGF expression and recovery in CHO cell systems?

Several strategies have been developed to optimize rhHGF production in CHO cells:

Cell line engineering:

• Establishment of CHO cells overexpressing rhHGF through genetic modifications

• Selection of high-producing clones with stable expression characteristics

Culture optimization:

• Use of serum-free medium to simplify downstream purification

• Implementation of fed-batch or perfusion culture modes to extend culture duration and increase yields

• Current optimized systems can achieve over 13 mg/l of rhHGF protein in a 5-day batch culture using a 7.5l bioreactor (5l working volume)

Recovery and purification enhancements:

• Development of efficient two-step chromatographic procedures

• Current methods achieve approximately 35% recovery rate of purified rhHGF

• Process modifications to minimize protein degradation during purification

Quality considerations:

• Implementation of analytical methods to ensure consistent glycosylation patterns

• Monitoring of pro-HGF to active HGF conversion during production and storage

• Assessment of biological activity throughout the production process

These optimization strategies are critical for producing sufficient quantities of biologically active rhHGF for both research applications and potential therapeutic development.

How does controlled release of HGF affect cellular responses and extracellular vesicle production?

Recent research has investigated the effects of controlled HGF release on cellular responses, particularly regarding extracellular vesicle secretion:

Controlled release systems:

• HGF can be incorporated into controlled release systems such as microbeads within the size range of 50–200 μm

• These systems are made with ultrapurified low-viscosity high-guluronic acid (UP-LVG) materials

Effects on extracellular vesicle secretion:

• Administration of HGF to urine-derived stem cells (USCs) via controlled release significantly enhances the levels of small extracellular vesicle (sEV) secretion during 7 days of culture

• This enhancement is superior to bolus administration of an equivalent amount of HGF

Mechanism and implications:

• Controlled release maintains a more consistent concentration of HGF in the cellular microenvironment

• This steady-state concentration likely provides sustained signaling through the c-Met receptor

• The increased production of sEVs may have implications for paracrine signaling and regenerative medicine applications

These findings suggest that the method of HGF delivery (bolus vs. controlled release) significantly impacts cellular responses, which may be important for both research applications and therapeutic development strategies.

What are the key differences between single-chain and heterodimeric forms of rhHGF?

Understanding the different forms of rhHGF is essential for experimental design and interpretation:

Structural differences:

• Pro-HGF (single-chain): The inactive precursor form of HGF (~90 kDa)

• Heterodimeric HGF: The biologically active form consisting of an α-chain and β-chain linked by disulfide bonds

Conversion process:

• Conversion from pro-HGF to heterodimeric HGF occurs through proteolytic cleavage

• This conversion happens rapidly in the presence of serum due to serum proteases

• When working with purified rhHGF, researchers should be aware that preparations may contain mixtures of both forms

Experimental implications:

• For in vitro experiments lacking serum, the proportion of active heterodimeric HGF may need to be considered

• For in vivo applications, conversion is expected to occur naturally after administration

• Analytical methods such as SDS-PAGE under reducing and non-reducing conditions can help determine the proportion of each form

What controls and standards should be included in rhHGF bioactivity assays?

Rigorous controls and standards are essential for reliable bioactivity assessment:

Reference standards:

• WHO Hepatocyte Growth Factor (precursor) (Human rDNA derived) (NIBSC code: 96/556) is the recommended international standard

• Commercial rhHGF preparations with established activity for internal standardization

Essential controls for bioactivity assays:

• Positive control: Validated active rhHGF preparation

• Negative control: Buffer-only or heat-inactivated rhHGF

• Dose-response curve: Serial dilutions to establish ED50 values

• Specificity control: Anti-HGF neutralizing antibodies to confirm specificity of observed effects

Reporting standards:

• Activity should be reported in International Units (IU) where possible

• Specific activity (IU/mg) should be calculated and reported

• The assay system used should be clearly described, including cell type, detection method, and incubation conditions

• Comparison to the WHO standard enhances reproducibility and comparability between studies

How should researchers address batch-to-batch variability in rhHGF preparations?

Batch-to-batch variability can significantly impact experimental outcomes:

Sources of variability:

• Cell culture conditions (passage number, media composition, harvest time)

• Purification process variations

• Pro-HGF to active HGF ratio differences

• Glycosylation pattern variations

Mitigation strategies:

• Implement robust quality control testing for each batch:

  • Protein concentration determination by multiple methods

  • Purity assessment by RP-HPLC and SDS-PAGE

  • Bioactivity testing in standardized assays

  • Glycosylation analysis where appropriate

Experimental design considerations:

• Use the same batch for all experiments within a study when possible

• Include internal standards in each experiment

• Validate critical findings with multiple batches

• Report batch information in publications to enhance reproducibility

By implementing these measures, researchers can minimize the impact of batch-to-batch variability on experimental outcomes and improve the reliability and reproducibility of their results.