Sandor LifeSciences
Biosciences Research Services

Protein Characterization

The bioengineered protein or peptide molecule should be highly analytically similar to the innovator molecule and not exhibit clinically meaningful differences to reduce further clinical costs. To ensure this, product quality modulations with parallel experimental design with multivariate analysis evolved into a field of utmost importance to assure release of safe and efficient products. Proteomics platforms known to be extensively used in the biomarker discovery phase is today fast replacing traditional techniques for efficient and cost effective characterization of bioengineered proteins and peptide.

Sample preparation is one of the most crucial processes in proteomics research. The results of the experiment depend on the condition of the starting material. Therefore, the proper experimental model and careful sample preparation is vital to obtain significant and trustworthy results, particularly in comparative proteomics. We have standardized protocols for extraction and purification of proteins or metabolites from a wide range of biological, nutraceutical sources. These extracted proteins and metabolites are used for further downstream applications.

Sources

  • Single step separation and extraction of proteins and metabolites from various tissues of the plant
  • Phenolic extraction of plant proteins
  • Removal of excipients and concentration of the proteins
  • Solubilization and quantification of proteins
  • Proteins are solubilized using different buffers for extracting hydrophilic and hydrophobic proteins.
  • Methods for protein extraction from cellular organelles
  • Solubilization and quantification of proteins
  • Proteins are extracted in a series of steps using Sequential Extraction Buffers
  • Samples are treated with different organic solvents for segregation of high and low abundant proteins.

The purifying proteins with fast protein liquid chromatography (FPLC) delivers quantities of the biologically active state target at satisfactory purity. Samples are separated based on the size, affinity, charge and hydrophobicity depending on the selected column.

AKTA pure protein purification systems is a user friendly chromatography system used for fast purification of proteins, peptides, and nucleic acids ranging from microgram levels to tens of grams of target product. AKTA pure and UNICORN™ software designed to work together with columns and chromatography media to meet several purification challenges.

In this approach substances are separated on the basis of their varying strengths of hydrophobic interactions with hydrophobic groups attached to an uncharged matrix. This technique is usually performed in the presence of moderately high concentrations of salts in the adsorption buffer. The choice of ligand, type and concentration of salt and pH are empirical values, which must be determined by screening experiments for each separation. Table below in house columns, which can take sample loads of 6mg/ml:

S.NO COLUMN NAME LIGAND
1 Phenyl Sepharose High Performance Phenyl
2 Phenyl Sepharose 6 Fast Flow (low sub) Phenyl
3 Phenyl Sepharose 6 Fast Flow (high sub) Phenyl
4 Butyl Sepharose High Butyl
5 Butyl Sepharose 4 Fast Flow Butyl
6 Butyl-S Sepharose 6 Fast Flow Butyl-S
7 Octyl Sepharose 4 Fast Flow Octyl

Based on the binding of charged sample molecules to oppositely charged groups attached to an insoluble matrix. Substances are bound to ion exchangers when they carry a net charge opposite to that of the ion exchanger. This binding is electrostatic and reversible. Table below in house columns for the purification use:

S.NO COLUMN NAME MATRIX SAMPLE LOAD
1 HiTrap SP FF SP Sepharose 70 mg/ml
2 HiTrap CM FF CM Sepharose 50 mg/ ml
3 HiTrap Q FF Q Sepharose 120 mg / ml
4 HiTrap DEAE FF DEAE Sepharose 110 mg / ml
5 HiTrap ANX FF (high sub) ANX Sepharose 43 mg / ml
6 HiTrap Q XL Q Sepharose >130 mg/ ml
7 HiTrap SP XL SP Sepharose >160 mg/ ml

Separates molecules on the basis of differences in size as they pass through porous beads packed in a column. SEC media consist of spherical particles with pores of different sizes where molecules small enough to enter the pores are retarded as compared to larger molecules pass through the interstitial spaces. Samples are eluted isocratically (single buffer, no gradient). SEC can be applied to separate a wide range of molecules including proteins as well as enzymes according to their difference in size.

S.NO COLUMN NAME MATRIX SAMPLE LOAD
1 Sephacryl S-200 HR Sephacryl 200-5 kDa

Accurate mass determination is the key for identification of wide range of samples from small organic molecules, intact glycoprotein and unambiguous proteins/peptides. Mass spectrometry offers accurate measurement over a range of molecular weight with minimum sample consumption. We offer both MALDI and ESI QTOF exact mass analysis.

Many proteins and peptides have isoforms which have to be characterized. We offer 2DE, DIGE (can see gel based proteomics) and IMS (Ion Mobility Mass spectrometry) technologies for isoform separation and characterization.

Chemical treatment of proteins/peptides releases its amino acids which can be quantitated by RP HPLC separation and comparison to standard amino acids. The molar ratios of the AA present in a protein are constant and need to be determined for all biosimilars. This method can also determine the in process results in free amino acids.

Peptide mapping identifies protein on the basis of unique peptides produced by each individual protein. Chemical or enzymatic treatment of protein results in formation of peptide fragments followed by separation (RP HPLC) and identification of the fragments in a reproducible way. It is a powerful test that is capable of identifying single amino acid changes also.

Protein purity is prime importance for efficacy and stability. The purity of the protein/peptide can be determined by both 2DE and RP HPLC technologies which we use. We use SE-HPLC for protein aggregation studies.

We offer method development and validation for characterization of impurities in the bioproducts. We do a combination of HPLC, LCMSMS technologies to identify and characterize the product impurities.

Protein folds into primary, secondary, tertiary and quaternary structures for activity and stability. The secondary and tertiary structures are determined by CD spectroscopy and florescence spectroscopy.

In order to characterize the complete protein molecule, from N- to C-terminal, we digest the intact pure protein by at least three proteases depending on the sequencing gaps obtained by trypsin digestion to give 100% sequence coverage. The raw data is processed by MassLynx 4.1 WATERS. The individual peptides MSMS spectra were matched to the database sequence for protein identification on PLGS software, WATERS. Bioinforamtic tools are used to stitch the complete sequence from the data obtained.

  • Protein Glycosylation
  • Phosphorylation
  • Acetylation
  • Sulphation
  • Methonine oxidation etc

We offer complete characterization of PTMs in proteins with information on site of modification, isolation and sequencing of modified peptides. We do complete free and bound glycan structure analysis by LCMSMS and GCMS.

We use SPR technology to determine the kinetic and binding constants for the following interactions:

  • protein-protein
  • protein-drug
  • protein-DNA/RNA
  • DNA-RNA binding
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