Currently, Laboratorios Beta has created the Biotechnology R&D unit conformed by the molecular biology and mammalian cell culture laboratory, a microbiology fermentation area and the downstream laboratory. A pilot plant for biotechnological processes was built in 2005 to carry out the scaling up procedure of the upstream and downstream processes. 

 

A. Our activities in the biotechnology R&D laboratories

 

Molecular Biology:

The molecular biology laboratory is dedicated to the development of genetically modified microorganisms and mammalian cells in order to obtain the highest expression level of the desired recombinant protein.

 

 

 A.1 The yeast expression system platform

In order to maximize the productivity of different goal proteins and taking into account the methylotrophic yeast expression system benefits, we have developed the following technological platform:

 

Cloning of the selected genes into different vectors and transformation of yeast;

Screening of recombinant yeast to obtain the highest expression strains; 

Expression of heterologous genes under highly induced promoters or metabolic regulated promoters or a combination of both;

Genetic modification of strains with chaperones (PDI) in order to improve the folding and secretion pathway of heterologous proteins;

Generation of the Master and Working Cell Bank.

 A.2 The mammalian cells expression system 

Those proteins with complex glycosidic residues in their structures must be expressed in mammalian cells cultures. We have optimised the expression of recombinant glycoproteins in CHO/dhfr^- cells. Stable transfected CHO cells with the capacity to express glycoproteins of human therapeutic interest are optimised in our R&D laboratories.

 

 

A.3 The Fermentation of yeast    

Optimise the fermentation process to derive the highest yield possible. At the laboratory scale this includes:

Selection of high level expression clones trough induction in Erlenmeyer’s cultures

fermentation in a 2-litre bioreactor to establish the optimal culture conditions (medium, pH. Tº, pO₂, etc)

scaling-up modelling to a 20-litre fermentor.

 

The product recovered as well as the contaminants and/or impurities generated are identified, characterized and quantified by SDS-PAGE, Western Blot, and HPLC.

 

A.4 Downstream Processing

 

The following steps are carried out in our R&D area:

 

capture of the recombinant protein from the supernatant,

 intermediate purification and polishing,

chemical or enzymatic modifications of proteins,

crystallization,

in process control,                                                                                                

QC analysis of the drug substance and drug product*,

HPLC characterization of the product and related impurities.  

 

* The quality control include the following assays

 

A.5 Host contaminants in the final product

 

Host DNA and protein quantification by PCR and ELISA, respectively.

Biological Assay

Viability cell culture assay

Measurement of biological activity using cultures of different mammalian cell lines or Balb/c mice.

 

 

B. Our activity at the Pilot Plant

Upstream and Downstream Scaling Up

 

B.1 Scaling-up to the Pilot Plant in order to assess the manufacturing reproducibility. This includes:

fermentation processing in a 300-litre bioreactor,

comparative study of the main variables behaviour between the different scales,

standardization of a quality control procedure of the fermentation,

separation of cells using continuous centrifugation and cross flow filtration.

B.2 The Purification Room is equipped with a 6mm Bioprocess and different size chromatographic columns in which the following activities are carried out:

ion exchange chromatography, gel filtration or hydrophobic interaction chromatography for the capture, intermediate and polishing steps;

 crystallization   

 

 

 C. Biotechnology pipeline

rh-Insulin and rh insulin-analogues for diabetes mellitus,

rh-insulin and rh-growth factors for cell culture medium

Growth factors for the treatment of wound healing diseases,

TNF antagonists in CHO cells,

Proteases

 

D. Patents

 

“Expression of a human insulin precursor in Pichia pastoris” USP 7,091,032”

“Cepa de Levaduras Metilotróficas Recombinantes Productoras de un Precursor de Insulina Humana, Construcciones de ADN y Métodos para Obtener La Cepa”. AR025646B1; IMP 267651

“Procedimientos para Incrementar el Rendimiento de Proteínas Recombinantes Producidas en Levaduras Metilotróficas”. AR040162B1.

 

Patents Pending Approval

Cepa de Levaduras Metilotróficas Recombinantes productoras del Homodímero BB de PDGF Humano, Construcciones de ADN y Método para obtener dicha cepa. P 0101105599.

Un método Para Producir un Precursor Análogo de Insulina Humana y Cepa de Levadura Metilotrófica que lo Produce. AR 041517 A1.

Cepa de Levadura Transformada productora de Insulina Humana y Análogos de Insulina Humana, vectores y Métodos para la Producción de Heterodímeros y Métodos para la Obtención de Insulina Humana o sus Análogos. P 040101283

“Un procedimiento par aobtener insulina aspártica que utiliza una cepa de levaduras de Pichia pastoris” P-090102686