Recombinant Protein Lot Consistency

Recombinant Human TGF-beta 1: An Analysis of Lot-to-lot Consistency

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Intro & Set-Up
Transforming Growth Factor-beta 1 (TGF-β1) is a widely used multifunctional cytokine that regulates immune function, cell differentiation, proliferation, migration, and fibrosis 1-2.  In a mechanism to prevent immune system over-activation, TGF-β 1 inhibits cell proliferation induced by IL-4 3.  We used the industry standard cell-based assay established on this inhibition mechanism, to test the lot-to-lot consistency in reported biological activity and the potency of Recombinant Human TGF-β 1 (rhTGF-β1) manufactured by Shenandoah Biotechnology (catalog number 100-39). 

Results
The biological activity of rhTGF-β1 was measured by the dose dependent inhibition of IL-4-induced HT-2 cell proliferation. Three lots of rhTGF-β1, A, B and C, were tested simultaneously.  The results showed similar Effective Dose (ED50) values (Figure 1) and virtually overlapping dose response curves.  Each lot’s ED50 value fell below Shenandoah’s acceptance criteria for this product (ED50 ≤ 0.5 ng/mL) and was within the ED50 ranges of other suppliers in the industry.  The data shows that rhTGF-β1 lots produced by Shenandoah over the course of time show consistent measurements of biological activity in the HT-2 assay. 


Figure 1:  rhTGF- β1 Lots A, B and C were simultaneously tested in an inhibition HT-2 cell growth assay. 


The ED50 values of nine other production lots of rhTGF-β1 manufactured and tested over a period of three years (designated as Lots A-I) were also compiled to determine the consistency of production and activity testing over time.  The average ED50 was calculated as 0.147 ng/mL ± 0.072 ng/mL (Table 1). The biological activity data over time demonstrated a high degree of consistency between lots of rhTGF-β1 produced by Shenandoah Biotechnology.

In addition, Shenandoah Biotechnology’s rhTGF-β1 was calibrated against the reference standard material for rhTGF-β1 (NIBSC code 89/514). The activity of Shenandoah Biotechnology’s rhTGF-β1 showed a greater activity of 21,510 RU/μg compared to the reference standard of 18,750 RU/μg (Table 2). These data showed that in addition to consistency, rhTGF- β1 from Shenandoah Biotechnology was more potent than the reference standard.  By calibrating Shenandoah’s rhTGF-β1 to the reference standard, customers can also compare product performance across suppliers.

 

Conclusions  
Lot-to-lot consistency is a valuable asset to consider when selecting recombinant proteins for biological applications.  Lot-specific biological activity of Shenandoah Biotechnology’s rhTGF-β1 was measured both in parallel and sequentially.  All results showed consistent ED50 values.  Further, rhTGF-β1 was more potent than the reference standard.  Shenandoah’s strict adherence to established production protocols and acceptance criteria for biological activity ensures that each lot of proteins produced by Shenandoah Biotechnology is consistent and reliable.

Shenandoah’s high standards can be extended to other quality assurance specifications, including purity and endotoxin standards.  Every specification test is reported on lot-specific Certificate of Analyses (COAs).  COAs are available prior to purchase in a digital library and also physically accompany each product when shipped. 

Summary:

  • Lot-to-lot consistency is important for functional experiments.
  • Shenandoah’s rhTGF-β1 is consistent when tested side-by-side and over time.
  • When calibrated, Shenandoah’s rhTGF-β1 is more potent than the NIBSC reference standard.
  • Strict high-quality standards allow for lot-specific activity to be reported for each lot of Shenandoah’s rhTGF-β1.

 

References                                                                                                                                                          
1. Stockis J, Dedobbeleer O, Lucas S. Role of GARP in the activation of latent TGF-β1. Mol Biosystems. 2017 Sep 26;13(10):1925-1935 

  1. Dünker N, Krieglstein K. Targeted mutations of transforming growth factor-beta genes reveal important roles in mouse development and adult homeostasis. Eur J Biochem. 2000 Dec; 267(24):6982-8.
  2. Toyota N, Hashimoto Y, Matsuo S, Iizuka H. Transforming growth factor beta 1 inhibits IL-3- and IL-4-dependent mouse connective tissue-type mast cell proliferation. Arch Dermatol Res. 1995;287(2):198-201.