Protein Synthesis and Purification Testing Service

Protein Synthesis and Purification Testing Service – Comprehensive Quality Control and Characterisation for Recombinant Proteins, Antibodies and Enzymes

As an ISO/IEC 17025 accredited contract research laboratory, we offer specialised protein synthesis and purification testing services to Bulgarian and international pharmaceutical, biotechnology, academic, and industrial clients. Recombinant proteins, monoclonal antibodies, enzymes, and protein‑based therapeutics require rigorous characterisation to ensure identity, purity, potency, and stability. Our testing platform covers the entire workflow – from gene synthesis and expression to protein purification, folding, and final formulation analysis – using a combination of chromatographic, electrophoretic, spectroscopic, and functional assays. All methods are aligned with ICH Q6B (Specifications for Biotechnological Products), Ph. Eur. 2.7 (General Methods for Biological Products), USP <1055> (Biotechnology‑Derived Articles), and BDS (Bulgarian Institute for Standardisation) guidelines. Our reports are recognised by the Bulgarian Food Safety Agency (BFSA), the Bulgarian Drug Agency (BDA), and the European Medicines Agency (EMA) reference laboratories for product registration, batch release, and quality assurance.

Protein Samples and Constructs We Test

Our laboratory handles a wide variety of protein types and expression systems. Typical test articles include:

• Recombinant proteins – growth factors, cytokines, enzymes, hormones, and structural proteins
• Monoclonal antibodies (mAbs) – IgG, IgA, IgM, and antibody fragments (Fab, scFv, VHH)
• Fusion proteins – Fc‑fusions, albumin‑fusions, and multi‑domain constructs
• Viral proteins and vaccine antigens – for subunit and recombinant vaccine development
• Industrial enzymes – proteases, lipases, cellulases, amylases, and polymerases
• Protein conjugates – PEGylated proteins, antibody‑drug conjugates (ADCs), and reporter enzyme conjugates
• Biosimilar and biobetter candidates – for comparability and similarity assessments
• Intracellular and membrane proteins – for functional and structural studies

Gene Synthesis and Expression Testing – From Sequence to Protein

• Gene synthesis and codon optimisation – we design and synthesise codon‑optimised genes for optimal expression in the chosen host system (E. coli, yeast, insect cells, mammalian cells). The gene sequence is verified by full‑length Sanger sequencing.
• Expression vector construction and verification – we clone the gene into appropriate expression vectors (e.g., pET, pGEX, pPICZ, pcDNA3.1) and verify the plasmid construct by restriction digestion and sequencing.
• Small‑scale expression screening (2‑50 mL) – we perform expression screening in multiple host strains and induction conditions (temperature, inducer concentration, induction time) to identify the optimal conditions for soluble protein yield. The expression level is quantified by SDS‑PAGE and Western blot.
• Cell lysis and protein extraction – we evaluate different lysis methods (sonication, high‑pressure homogenisation, enzymatic lysis) and extraction buffers (pH, salt concentration, detergent composition) to maximise protein recovery.
• Insoluble protein solubilisation (inclusion bodies) – for proteins expressed as inclusion bodies, we test a range of solubilisation agents (urea, guanidine hydrochloride) and refolding buffers (oxidation/reduction systems, arginine, detergent) to obtain active, soluble protein.
• Folding and refolding optimisation – for proteins that require post‑expression folding, we optimise the refolding protocol (dilution, dialysis, or on‑column refolding) and monitor the recovery of native conformation using intrinsic fluorescence, circular dichroism (CD), and activity assays.

Protein Purification and Chromatographic Characterisation

• Affinity chromatography – yield and purity assessment – we test the efficiency of affinity purification using His‑tag, GST‑tag, Strep‑tag, and Protein A/G resins. We measure the binding capacity (mg protein/mL resin), the elution recovery (%), and the purity of the eluted protein (by SDS‑PAGE) to optimise the purification protocol.
• Ion‑exchange chromatography – charge variant analysis – using cation‑ or anion‑exchange columns (Mono Q, SP, DEAE), we separate protein charge variants (acidic, basic, and main isoforms). The percentage of each variant is reported, and the chromatographic profile is used for batch‑to‑batch consistency.
• Size‑exclusion chromatography (SEC) – aggregation and oligomerisation – we perform analytical SEC (HPLC) to determine the native molecular weight and to quantify high‑molecular‑weight (HMW) aggregates, low‑molecular‑weight (LMW) fragments, and the monomer content. For therapeutic proteins, a monomer content ≥ 95 % is typically required.
• Hydrophobic interaction chromatography (HIC) – hydrophobicity analysis – for assessing the surface hydrophobicity of the protein, we use HIC (e.g., butyl‑, phenyl‑, or octyl‑Sepharose) to separate variants with different hydrophobic character. This is particularly important for antibody‑drug conjugates and proteins prone to aggregation.
• Reversed‑phase high‑performance liquid chromatography (RP‑HPLC) – for purity and sequence verification – we use C4 or C18 RP‑HPLC columns to separate protein variants based on differences in hydrophobicity, and to quantify the percentage purity and detect any truncated forms or oxidation products. For monoclonal antibodies, the intact mAb peak is typically ≥ 95 %.
• Multidimensional purification – process development – for complex samples, we combine two or more purification steps (e.g., affinity + ion‑exchange + SEC) and evaluate the overall yield and purity improvement at each stage.

Purity and Identity – Electrophoretic and Immunochemical Methods

• SDS‑PAGE (reducing and non‑reducing) – molecular weight and purity assessment – we run gradient SDS‑PAGE gels to determine the apparent molecular weight, check for degradation products, and assess the percentage purity (by densitometry). For monoclonal antibodies, the reduced profile shows the heavy and light chains; the non‑reduced profile shows the intact antibody.
• Native PAGE – charge and native conformation – using native (non‑denaturing) PAGE, we assess the protein’s charge, native size, and any aggregation or oligomerisation.
• Western blot – identity confirmation and degradation – we perform Western blot using specific antibodies (e.g., anti‑His, anti‑GST, target‑specific antibodies) to confirm the identity of the expressed protein and to detect any degradation products or truncated forms.
• Isoelectric focusing (IEF) – charge heterogeneity – we determine the isoelectric point (pI) of the protein and identify any charge variants (post‑translational modifications, deamidation, sialylation). The pI is compared to the theoretical pI calculated from the amino acid sequence.
• Two‑dimensional gel electrophoresis (2D‑PAGE) – comprehensive purity and isoform analysis – for complex samples, we perform 2D‑PAGE (IEF in the first dimension, SDS‑PAGE in the second) to resolve and quantify isoforms, degradation products, and co‑purifying contaminants.

Mass Spectrometry (MS) – Molecular Weight, Sequence and Post‑Translational Modifications

• Intact mass determination (ESI‑TOF or Q‑TOF) – molecular weight confirmation – we measure the intact molecular mass of the protein (with an accuracy of ≤ 10 ppm) to verify that the mass matches the theoretical mass of the desired protein, and to detect any modifications (e.g., glycosylation, oxidation, truncation).
• Peptide mapping (LC‑MS/MS) – sequence coverage and modification identification – we digest the protein with a specific protease (trypsin, Lys‑C, Glu‑C) and analyse the peptide mixture by LC‑MS/MS. The sequence coverage and the identification of any post‑translational modifications (phosphorylation, glycosylation, acetylation, deamidation) are reported.
• Glycosylation analysis – N‑linked and O‑linked glycan profiling – for glycoproteins (including mAbs), we release the glycans (by PNGase F for N‑glycans or chemical cleavage for O‑glycans) and analyse them by HPLC‑FLR (2‑AB labelling) or by MALDI‑TOF MS. The glycan profile (including high‑mannose, complex, and hybrid glycans) is quantified, and the distribution is reported as a percentage of each glycan species.
• N‑terminal and C‑terminal sequencing (Edman degradation) – for sequence verification – we perform N‑terminal sequencing for 5‑20 cycles (Edman chemistry) to confirm the first few amino acids; for C‑terminal analysis, we perform C‑terminal peptide identification by MS after digestion with carboxypeptidase.
• Disulphide bond mapping – for cysteine‑containing proteins – we digest the protein under non‑reducing conditions and analyse the resulting disulphide‑linked peptides by LC‑MS/MS to confirm the disulphide connectivity pattern. The correct disulphide bonding is essential for the stability and activity of many proteins.

Biological Activity and Potency – Functional Assays

• Enzyme activity assays – specific activity determination (U/mg) – for enzyme products, we develop and validate a kinetic assay (e.g., continuous spectrophotometric, fluorometric, or end‑point assay) to measure the specific activity. The initial rate is determined under optimised conditions, and the specific activity (units per mg protein) is calculated from a standard curve.
• Cell‑based potency assays (for therapeutic proteins and antibodies) – we perform cell‑based bioassays (e.g., proliferation assay, cytotoxicity assay, cytokine release assay) to measure the biological activity of the protein relative to a reference standard. The half‑maximal effective concentration (EC₅₀) or the half‑maximal inhibitory concentration (IC₅₀) is determined using a dose‑response curve.
• Binding affinity (ELISA, SPR, or ITC) – for antibodies and receptors – we measure the binding affinity (Kd) of the protein to its target (antigen, receptor, ligand) using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or ELISA. The association rate (kon) and dissociation rate (koff) are determined, and the Kd (in nM or pM) is reported.
• Neutralisation assays – for viral or bacterial neutralisation – for vaccine antigens or neutralizing antibodies, we perform a neutralisation assay (e.g., plaque reduction neutralisation test – PRNT, or a cell‑based virus neutralisation test – VNT) to measure the neutralising titre (e.g., the dilution that inhibits 50 % of the viral plaque formation).

Stability and Formulation – Assessing Shelf Life and Storage Conditions

• Accelerated stability testing – ICH Q1A / Q5C – we store the formulated protein at elevated temperatures (e.g., 25 °C, 37 °C, 40 °C) and test it at defined time points (0, 1, 3, 6 months) for purity (SEC, SDS‑PAGE), activity (enzyme or binding assay), and aggregate/fragment content. The degradation kinetics are analysed, and the shelf life is estimated using Arrhenius modeling.
• Freeze‑thaw stability – for frozen formulations – we subject the protein to 3‑5 freeze‑thaw cycles (‑20 °C to 25 °C or ‑80 °C to 25 °C) and assess the purity and activity after each cycle to determine the acceptable number of freeze‑thaw cycles.
• Aggregation propensity – thermal stress (DSC) – we perform differential scanning calorimetry (DSC) to measure the melting temperature (Tm) of the protein. A high Tm indicates a stable, well‑folded protein; a low Tm suggests a tendency to aggregate or unfold under thermal stress.
• Formulation development – excipient screening – we test a panel of excipients (sugars, amino acids, surfactants, antioxidants) to identify formulations that minimise aggregation and maintain activity during storage. The effects of pH, buffer type, and salt concentration are also evaluated.
• Forced degradation studies – to develop stability‑indicating methods – we subject the protein to stress conditions (heat, light, oxidation, pH extremes) and analyse the degradation products to identify the primary degradation pathways.

Quality Control and Reference Standards

To ensure the reliability and reproducibility of our protein tests, we follow a strict quality control framework.

• Reference standards – we use certified reference standards (where available) from national pharmacopoeias (e.g., Ph. Eur., USP) or in‑house characterised reference materials for calibration, system suitability, and identity verification.
• System suitability testing – we perform system suitability testing (e.g., resolution, peak symmetry, column performance) for each chromatographic method and use control charts to monitor the performance of each analytical instrument.
• Proficiency testing – we participate in external proficiency testing schemes (e.g., FAPAS, BIPEA) and inter‑laboratory comparisons for key protein assays (e.g., ELISA, SDS‑PAGE, SEC) to verify the accuracy and reliability of our results.
• Positive and negative controls – in each assay, we include positive controls (active protein) and negative controls (blank or inactive protein) to ensure that the method works correctly and that the sample is not contaminated.

Report Acceptance & Compliance with Bulgarian and European Regulatory Standards

All protein synthesis and purification tests are performed under our ISO/IEC 17025 accreditation and in compliance with ICH Q6B and Good Laboratory Practice (GLP) principles, where applicable. Our final test reports include a complete description of the protein (construct, expression system, purification history), the analytical methods used (chromatography, electrophoresis, MS, activity assays), the raw and processed data (chromatograms, gel images, MS spectra, dose‑response curves), the calculated parameters (purity percentage, specific activity, EC₅₀/IC₅₀, glycan profile), statistical summaries (mean, standard deviation, confidence intervals), and a clear conclusion on the identity, purity, activity, and stability of the protein. These reports are accepted by the Bulgarian Food Safety Agency (BFSA), the Bulgarian Drug Agency (BDA), the European Medicines Agency (EMA), and Bulgarian and international pharmaceutical and biotech companies for quality assurance, batch release, and regulatory filing. Bilingual (Bulgarian/English) versions are available to facilitate submissions to national and European authorities.