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Vaccine products represent one of the most successful public health measure to this day. This has been reflected during the current COVID-19 pandemic where more than 4.87 billion people have received at least one vaccine dose. In Latin America, Mexico occupies the second position in terms of the number of vaccinated people with 83.97 million people receiving at least a single dose. As in other countries, regulatory approval in Mexico is one of the key aspects that influences the public access to vaccines. This creates an active interplay between regulatory authorities establishing a regulatory framework to assure the quality, safety and efficacy of the vaccines, and applicants fulfilling this information. Mexico is a member of the International Council for Harmonisation (ICH) and it has adopted the Common Technical Document (CTD) for providing this information. This is particularly useful for vaccines developed abroad where it is expected to speed the evaluation of the new product. The Secretariat of Health of Mexico (SALUD) has published guidelines and laws or regulations related to GMP, labeling, stability, clinical trials, biocomparability and pharmacovigilance for drug products including vaccines which are classified as biological products. SALUD has also established guidelines and international homologating agreements to facilitate the application process for vaccine approval. Nevertheless, technical and scientific information and administrative processes for vaccine approval might be relatively concealed. Therefore, we aim to enable researchers and manufacturers in Mexico and overseas to better understand these requirements. To our knowledge, this is the most up-to-date and comprehensive attempt to present this information, also including information for COVID-19 vaccines. Here we describe the current requirements and processes by COFEPRIS, the national regulatory agency, for vaccine licensing and for emergency use authorization for COVID-19 vaccines in Mexico.

BACKGROUND Major depressive disorder (MDD) is a global health issue that affects 350 million people of all ages. Although between 2% and 5.6% of affected individuals are adolescents, research on young patients is limited. The inflammatory response contributes to the onset of depression, and in adult MDD patients, symptom severity has been linked to chemokine levels. AIM To determine the differences in circulatory levels of chemokines in healthy volunteers (HVs) and adolescents with MDD, and assess the changes induced by fluoxetine consume. METHODS The 22 adolescents with MDD were monitored during the first 8 wk of clinical follow-up and clinical psychiatric evaluation was done using the Hamilton depresión rating scale (HDRS). The serum levels of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, interleukin (IL)-8, interferon gamma-induced protein (IP)-10, and eotaxin were measured in patients and HVs. RESULTS In all cases, significant differences were detected in circulating chemokine levels between patients before treatment and HVs (P < 0.0001). All chemokines decreased at 4 wk, but only MCP-1 and IL-8 significantly differed (P < 0.05) between 0 wk and 4 wk. In the patients, all chemokines rose to their initial concentrations by 8 wk vs 0 wk, but only IP-10 did so significantly (P < 0.05). All patients experienced a significant decrease in HDRS scores at 4 wk (P < 0.0001) and 8 wk (P < 0.0001) compared with 0 wk. CONCLUSION Despite the consumption of fluoxetine, patients had significantly higher chemokine levels, even after considering the improvement in HDRS score. The high levels of eotaxin, IP-10, and IL-8 partially explain certain aspects that are affected in MDD such as cognition, memory, and learning.

Background The deterioration of the skin accentuates over time, affecting its aesthetic appearance. This is characterized by the weakening of the mechanisms involved in the regeneration and repair of the dermal matrix. Consequently, the skin losses elasticity and smoothness resulting in the formation of wrinkles. The alternatives for facial rejuvenation include surgery, injection of botulinum toxin, and the application of masks. Topic products are less invasive, can be self‐applied, and have an increased benefit/risk relationship. Aim We developed a liquid formulation containing collagen hydrolyzed and evaluated the product by cutting‐edge technology in order to define proper its quality attributes. Methods We employed nuclear magnetic resonance (NMR), size‐exclusion chromatography (SEC), and mass spectrometry (MS). Additionally, we analyzed its cosmetical effect in five volunteers and we demonstrate the product safety. Results Our results demonstrate the following: (a) a stable secondary structure identity associated to the known triple helix arrangement in liquid and solid states; (b) a typical conformational flexibility depending on its hydration state; (c) thermal stability confirmed by liquid‐ and solid‐state nuclear magnetic resonance schemes; and (d) a molecular mass distribution of peptides between 0.5 and 19.5 kDa. The product faded wrinkles in the forehead, an effect that remained after removing the mask. The formula was non‐irritating and hypoallergenic. Conclusion We characterized, using state‐of‐the‐art methodologies, the quality attributes that are critical for the safety and beneficial effect of a new collagen‐containing formula.

The expression of heterologous proteins in Escherichia coli is strongly affected by the type of carbon source used. In this work, the expression of a synthetic codon-optimized gene of human interleukin-2 in E. coli BL21-SI, carrying plasmid pET12a-hIL2 is presented. Glucose, fructose or Agave syrup from Agave salmiana, were used as carbon sources for production of recombinant human IL-2 (rhIL-2) in 1.5-L bioreactor aerobic cultures using mineral medium. Codon optimization of the native hIL-2 gene eliminated the presence of 35 rare codons for E. coli, and improved the codon usage up to 76% compared with the native gene sequence. Cultures using 10 g/L glucose showed the lowest production of rhIL-2, and in contrast, cultures using fructose improved the production of rhIL-2 1.9-times. The utilization of fructose from Agave syrup enhanced the rhIL-2 production 3.9-times, reaching 103.42±6.61 mgIL-2/L. The specific rhIL-2 production rate (5.52±0.33 mgIL-2/gDCW·h) using Agave syrup was also the highest. These results indicate that Agave syrup stimulates the production of rhIL-2 and it is an inexpensive alternative carbon source. This research abilities the potential use the Agaves to produce alternative and valuable biotechnological products instead the alcoholic beverages.

A complex mixture of peptides plays a key role in the regulation of the immune system; different sources as raw materials mainly from animals and vegetables have been reported to provide these extracts. The batch-to-batch product consistency depends on in-process controls established. However, when an immunomodulator is a customized product obtained from the same volunteer who will receive the product to personalize the treatment, the criteria to establish the consistency between volunteers are different. In this sense, it is expected to have the same molecular weight range although the profile of peptide abundance is different. Here, we characterized the peptide profile of three extracts of an immunomodulator obtained from the urine of different volunteers suffering from three different diseases (i.e., allergic rhinitis, rheumatoid arthritis, and chronic rhinopharyngitis), using size exclusion chromatography (SEC) and mass spectrometry (MS). The peptides contained in the immunomodulators were stable after six months, stored in a refrigerator. Our results showed a chromatographic profile with the same range of low molecular weight (less than 17 kDa) in all analyzed samples by SEC; these results were also confirmed by MS showing an exact mass spectrum from 3 to 13 kDa. The fact that the peptide profiles were conserved during a six-month period at refrigeration conditions (2 to 8°C) maintaining the quality and stability of the immunomodulator supports the notion that it might be an alternative in the treatment of chronic hypersensibility disorders.

Identity is a critical quality attribute that must be determined before releasing batches of medicinal and dietary products. However, the identities of peptide-derived products composed of a large number of diverse molecules is challenging since most analytical techniques cannot analyze multiple molecules simultaneously. Here, we proposed the determination of the weight-average molecular weight (

Therapeutic monoclonal antibodies (mAbs) are complex bioengineered proteins that require to be routinely characterized with robust and reliable bioassays. Infliximab was the first anti-TNFα mAb approved for use in humans and its use has revolutionized the treatment TNF-mediated inflammatory disorders. The mechanism of action (MOA) of infliximab involves its binding to soluble (s) and membrane (m) TNFα. Here, we describe two simple in vitro bioassays for the assessment of key activities of infliximab. First, a bioassay for TNFα neutralization, which evaluates the Fab binding to sTNFα and the consequent reduction in the activation of TNFα receptors and TNFα-induced expression of adhesion molecules on endothelial cells. A second bioassay evaluates the triggering of Complement-Dependent Cytotoxicity (CDC) in cells expressing mTNFα, which requires the interaction of infliximab-Fc with proteins of the complement system. In both cases, the biological responses are measured by flow cytometry, which is accessible for most laboratories. The methods reported here can be easily adapted to other therapeutic mAbs with similar MOA.

Therapeutic monoclonal antibodies (mAbs) are complex bioengineered proteins that require to be routinely characterized with robust and reliable bioassays. Infliximab was the first anti-TNFα mAb approved for use in humans and its use has revolutionized the treatment TNF-mediated inflammatory disorders. The mechanism of action (MOA) of infliximab involves its binding to soluble (s) and membrane (m) TNFα. Here, we describe two simple in vitro bioassays for the assessment of key activities of infliximab. First, a bioassay for TNFα neutralization, which evaluates the Fab binding to sTNFα and the consequent reduction in the activation of TNFα receptors and TNFα-induced expression of adhesion molecules on endothelial cells. A second bioassay evaluates the triggering of Complement-Dependent Cytotoxicity (CDC) in cells expressing mTNFα, which requires the interaction of infliximab-Fc with proteins of the complement system. In both cases, the biological responses are measured by flow cytometry, which is accessible for most laboratories. The methods reported here can be easily adapted to other therapeutic mAbs with similar MOA.

PEGylated protein purification with the required quality attributes has represented a bioengineering challenge and Affinity Monolith Chromatography (AMC) has never been exploited for this goal. This work reports the generation of a heparin-modified affinity monolith disk by reductive alkylation with raised ligand density for its use as chromatographic support in the separation of lysozyme PEGylation reactions (LPRs) with three different PEG sizes (1, 20 and 40 kDa). For immobilized heparin determination a modified toluidine colorimetric assay adapted to microplate format was proposed. The heparin modified-disk was able to differentiate positional isomers of 20 kDa mono-PEGylated lysozyme at neutral pH using a salt linear gradient. Identity of PEG-conjugates was verified by SDS-PAGE and positional isomers were partially characterized by peptide mapping mass spectrometry. 20 kDa mono-PEGylated lysozyme conjugate purity (99.69 ± 0.05%) was comparable with traditional chromatographic methods while productivity (0.0964 ± 0.0001 mg/mL*min) was increased up to 6.1 times compared to that obtained in heparin packed-bed affinity chromatography procedures. The proposed AMC method represents a reliable, efficient, easy-handling, fast and single-step operation for the analysis or preparative isolation of PEGylated proteins containing a heparin binding domain.

Collagen hydrolysates are dietary supplements used for nutritional and medical purposes. They are complex mixtures of low-molecular-weight peptides obtained from the enzymatic hydrolysis of collagen, which provide intrinsic batch-to-batch heterogeneity. In consequence, the quality of these products, which is related to the reproducibility of their mass distribution pattern, should be addressed. Here, we propose an analytical approach to determine the peptide pattern as a quality attribute of Colagenart®, a product containing collagen hydrolysate. In addition, we evaluated the safety by measuring the viability of two cell lines exposed to the product. The consistency of peptide distribution was determined using Size Exclusion Chromatography (SEC), Mass Spectrometry coupled to a reversed phase UPLC system (MS-RP-UPLC), and Shaped-pulse off-resonance water-presaturation proton nuclear magnetic resonance spectrometry [1 Hwater_presat NMR]. The mass distribution pattern determined by SEC was in a range from 1.35 to 17 kDa, and from 2 to 14 kDa by MS-RP-UPLC. [1 Hwater_presat NMR] showed the detailed spin-systems of the collagen hydrolysates components by global assignment of backbone Hα and NH, as well as side-chain proton resonances. Additionally, short-range intraresidue connectivity pathways of identified spin-regions were obtained by a 2D homonuclear shift correlation Shaped-pulse solvent suppression COSY scheme. Safety analysis of Colagenart® was evaluated in CaCo-2 and HepG2 cells at 2.5 and 25 μg/mL and no negative effects were observed. The results demonstrated batch-to-batch reproducibility, which evinces the utility of this approach to establish the consistency of the quality attributes of collagen hydrolysates. PRACTICAL APPLICATION: We propose state-of-the art analytical methodologies (SEC, MS, and NMR) to evaluate peptide profile and composition of collagen hydrolysates as quality attributes. These methodologies are suitable to be implemented for quality control purposes.

The development of biotherapeutics requires continuous improvement in analytical methodologies for the assessment of their quality attributes. A subset of biotherapeutics is designed to interact with specific antigens that are exposed on the membranes of target cells or circulating in a soluble form, and effector functions are achieved via recognition of their Fc region by effector cells that induce mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, ADCC induction is a critical quality attribute (CQA) that must be evaluated to ensure biotherapeutic efficacy. Induction of ADCC can be evaluated by employing effector cells from different sources, such as peripheral blood mononuclear cells (PBMC) and genetically modified cell lines (e.g., transfected NKs or Jurkat cells), and different approaches can be used for detection and results interpretation depending on the type of effector cells used. In this regard, validation of the assays is relevant to ensure the reliability of the results according to the intended purpose. Herein, we show the standardization and validation of ADCC assays to test the potency of three biotherapeutic proteins using primary NK cells obtained from fresh blood as effector cells and detecting cell death by flow cytometry. The advantage of using primary NKs instead of modified cells is that the response is closer to that occurring in vivo since cytotoxicity is evaluated in a direct manner. Our results indicate that in all cases, the assays exhibited a characteristic sigmoidal dose/response curve complying with accurate, precise and specific parameters. Thereby, the validated ADCC assay is an appropriate alternative to evaluate the biological activities of these type of biotherapeutics.

“Transferon Oral” is a peptide-derived product with immunomodulatory properties obtained from the lysis and dialysis of human buffy coat. Its active pharmaceutical ingredient, generically known as Dialyzable Leucocyte Extract, is a mixture of peptide populations with reproducible proportions among batches. “Transferon Oral” modulates IFN-γ, TNF-α, and IL-6 and increases the survival rate in a herpes infection murine model when oropharyngeally (ORO) administered, which correlate with clinical observations where “Transferon Oral” is used as a therapeutic auxiliary in inflammatory diseases. Notwithstanding, how a peptide-derived product elicits systemic modulation of cytokines when ORO administered remains unclear. To shed light on the pharmacology of “Transferon Oral” its peptide components must be known. Ten “Transferon Oral” batches were sequenced by mass spectrometry and the intact peptides were identified. The most abundant peptides were the monomeric human Ubiquitin (Ub), a globular low-molecular mass protein, and an Ub variant which lacks the two-terminal Gly (Ub-GG). Recombinant Ub prevented murine death when ORO administered in a herpes infection murine model. Besides, the percentage of survival increased in groups treated with Transferon Oral+Ub and decreased in groups treated with Ub-depleted “Transferon Oral” respect to the group treated with “Transferon Oral” only. Our findings indicate that the biological properties of “Transferon Oral” are partially associated to the Ub content. They suggest that Ub may activate its extracellular receptor (CXCR-4) in the stomach eliciting systemic immunomodulatory effects via vagus nerve. This is the first report that identifies an active component of “Transferon Oral” with the potential for the development of oral peptide immunomodulators.

The present work describes the development and validation of a first report including several non-invasive NMR schemes to identify parameters as local chemical environments, homo- and heteronuclear site-specific spin correlations, diffusion coefficient-dependent polydispersity indexes and quantification of identified peptide entities that composes a commercial human Dialyzable Leucocyte Extract (DLE), Transferon, an oral liquid formulation of low-molecular-weight peptides. The above parameters were useful indicators to verify reproducibility, consistency and homogeneity among the DLE batches manufactured at Good Manufacturing Practice (GMP) facilities and for batch-releasing purposes in a quality control laboratory. The results showed that peptide identity of the DLE is represented with both high reproducible one-dimensional proton spectra and diffusion coefficient distributions that predicts in turn a weight-average molecular weight of around 6.7-7.4 kDa and a mean polydispersity index of 1.13. The obtained NMR peptide fingerprint of the analyzed DLE allowed to i) confirm its structural homogeneity by line-shape analysis, ii) identify and quantify its peptide content within the total solution with qNMR methods iii) to confirm the robustness of the technique as a feasible alternative for routine analysis of Natural or non-Natural Complex Drugs, such as DLEs.

Therapeutic antibodies are recombinant proteins used in the treatment of cancer. There is a new generation of monoclonal antibodies with activity against cancer cells, known as antibody-drug conjugates. These molecules are made up of three elements: a monoclonal antibody, a highly potent cytotoxic drug, and a chemical linker that binds them together. The antibody recognizes tumor antigens, thereby allowing targeted delivery of the cytotoxic agent to cancer cells. After recognizing its antigen, the antibody-drug conjugate is endocytosed by the target cells, where the protein fraction is degraded into lysosomes, releasing the cytotoxic drug. This article reviews antibody-drug conjugates general characteristics and describes the clinical evidence of efficacy and safety of the first four approved by regulatory agencies in the United States and Europe.Los anticuerpos terapéuticos son proteínas recombinantes empleadas en el tratamiento del cáncer. Existe una nueva ­generación de anticuerpos monoclonales con actividad contra las células cancerosas, conocidos como anticuerpos conjugados a fármacos. Estas moléculas están integradas por tres elementos: un anticuerpo monoclonal, un fármaco citotóxico con alta potencia y un enlazador químico que los une. El anticuerpo reconoce antígenos tumorales, por lo que permite la entrega dirigida del agente citotóxico hacia las células cancerosas. Tras el reconocimiento de su antígeno, el anticuerpo conjugado a fármaco es endocitado por las células blanco, donde se induce la degradación lisosomal de la fracción proteica y se libera el fármaco citotóxico. En el presente artículo se revisan las características generales de los anticuerpos conjugados a fármacos y se describe la evidencia clínica de la eficacia y seguridad de los primeros cuatro aprobados por las agencias reguladoras de Estados Unidos y Europa.

espanolLos anticuerpos terapeuticos son proteinas recombinantes empleadas en el tratamiento del cancer. Existe una nueva generacion de anticuerpos monoclonales con actividad contra las celulas cancerosas, conocidos como anticuerpos conjugados a farmacos. Estas moleculas estan integradas por tres elementos: un anticuerpo monoclonal, un farmaco citotoxico con alta potencia y un enlazador quimico que los une. El anticuerpo reconoce antigenos tumorales, por lo que permite la entrega dirigida del agente citotoxico hacia las celulas cancerosas. Tras el reconocimiento de su antigeno, el anticuerpo conjugado a farmaco es endocitado por las celulas blanco, donde se induce la degradacion lisosomal de la fraccion proteica y se libera el farmaco citotoxico. En el presente articulo se revisan las carac EnglishTherapeutic antibodies are recombinant proteins used in the treatment of cancer. There is a new generation of monoclonal antibodies with activity against cancer cells, known as antibody-drug conjugates. These molecules are made up of three elements: a monoclonal antibody, a highly potent cytotoxic drug, and a chemical linker that binds them together. The antibody recognizes tumor antigens, thereby allowing targeted delivery of the cytotoxic agent to cancer cells. After recognizing its antigen, the antibody-drug conjugate is endocytosed by the target cells, where the protein fraction is degraded into lysosomes, releasing the cytotoxic drug. This article reviews antibody-drug conjugates general characteristics and describes the clinical evidence of efficacy and safety of the first four approved by regulatory agencies in the United States and Europe.

Prostate cancer (PCa) is the second most frequently diagnosed cancer in men worldwide. Dialyzed Leukocyte Extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that improve clinical responses in various diseases. Here, we analyzed the effects of TransferonTM, a commercial DLE with characterized active pharmaceutical ingredient and proven batch-to-batch reproducibility, in preclinical models of PCa. We employed v-Src-transformed murine prostate epithelial (PEC-Src) cells, which recapitulate the transcriptional profiles in human PCa, can be grown in immunocompetent mice, and consistently form bone and brain metastases. In vitro, TransferonTM did not induce cytotoxicity nor alterations in migration /invasion of PEC-Src cells. In vivo, TransferonTM reduced metastatic dissemination after intracardiac injection of PEC-Src and inhibited tumor growth of subcutaneous isotransplants. The antineoplastic effect of TransferonTM correlated with changes in tumor infiltration, increased serum concentrations of IL-12 and CXCL1, and reduced levels of VEGF. Our results suggest that the antineoplastic effect produced by TransferonTM is due to its immunomodulatory activity and not by a direct effect on cancer cells, and indicate that TransferonTM could be beneficial as adjuvant therapy in PCa patients.

Complex pharmaceuticals are in demand of competent analytical methods able to analyze charge heterogeneity as a critical quality attribute (CQA), in compliance with current regulatory expectations. A notorious example is glatiramer acetate (GA), a complex polypeptide mixture useful for the treatment of relapsing-remitting multiple sclerosis. This pharmaceutical challenges the current state of analytical technology in terms of the capacity to study their constituent species. Thus, a strong cation exchange methodology was designed under the lifecycle approach to support the establishment of GA identity, trough the evaluation of its chromatographic profile, which acts as a charge heterogeneity fingerprint. In this regard, a maximum relative margin of error of 5% for relative retention time and symmetry factor were proposed for the analytical target profile. The methodology met the proposed requirements after precision and specificity tests results, the former comprised of sensitivity and selectivity. Subsequently, method validation was conducted and showed that the method is able to differentiate between intact GA and heterogeneity profiles coming from stressed, fractioned or process-modified samples. In summary, these results provide evidence that the method is adequate to assess charge heterogeneity as a CQA of this complex pharmaceutical.

This work describes a strategy to optimize a downstream processing of a recombinant human growth hormone (rhGH) by incorporating a quality by design approach toward meeting higher quality specifications. The optimized process minimized the presence of impurities and degradation by-products during manufacturing by the establishment of in-process controls. Capillary zone electrophoresis, reverse phase, and size-exclusion chromatographies were used as analytical techniques to establish new critical process parameters for the solubilization, capture, and intermediate purification steps aiming to maintain rhGH quality by complying with pharmacopeial specifications. The results indicated that the implemented improvements in the process allowed the optimization of the specific recovery and purification of rhGH without compromising its quality. In addition, this optimization facilitated the stringent removal of the remaining impurities in further polishing stages, as demonstrated by the analysis of the obtained active pharmaceutical ingredient.

Analytical methods have been considered the “eyes” for development, characterization and batch releasing of biotherapeutics over the past 40 years. One of the most powerful analytical platform for biotherapeutic analysis is mass spectrometry coupled to liquid chromatography (LC-MS). Due to its wide flexibility and instrumental configurations, LC-MS can determine different physicochemical attributes of proteins, e.g. molecular mass, primary sequence, and posttranslational modifications. Intact molecular mass analysis of therapeutic proteins is essential to confirm their identity. Analytical methods must be validated to support drug quality information during its approval process. Although there are international guidelines that provide general information on validation of analytical methods, practical examples about the design, selection of validation attributes and acceptance criteria of identity LC-MS methods are scarce. Here, according to the recommendations of Q2R1 ICH guideline, we showcase the validation of an LC-MS-TOF method to identity rituximab by determining its intact and deglycosylated molecular mass profiles. The proposed method specifically identified the m/z profile and deconvoluted mass profile of rituximab from deglycosylated rituximab and from excipient blank (specificity) with a maximum error of 76.63 ppm (accuracy) and a maximum Relative Standard Deviation (RSD) of 0.00315% (precision). Besides, the system suitability test, which was based on the expected mass value of the mass calibrator, confirmed the reliability of the analytical results. In summary, validation showed that the proposed method is suitable for identifying rituximab based on its glycosylated (intact) and deglycosylated mass profile.

Background Glatiramer acetate (GA) is a drug for Multiple Sclerosis (MS) treatment. However, its administration induces anti-drug antibodies (ADA). This research evaluated the sex differences in humoral response against GA in RR-MS patients Methods We analyzed 69 RR-MS patients, 43 treated with GA and 26 treated with IFN-β. In all cases, the serum concentration of IgG antibodies was determined by UPLC, whereas the levels of IgG subclasses (1–4) of anti-GA antibodies and the concentration of IL-6 were detected by Multiplex and IL-10, and IFN-γ were detected by ELISA. Results The total concentration of IgG antibodies in patients did not differ between treatments, whereas the IgG levels of ADA were higher in male and female patients treated with GA (P ≤ 0.0001). The subclasses of IgG anti-GA antibodies were as follows: IgG4>>IgG3>IgG1>IgG2. Statistical analysis showed differences in the IgG2 (P ≤ 0.01) and IgG4 (P ≤ 0.0001) subclasses by sex in RR-MS patients. Levels of IgG1 subclass in male patients correlated positively with the circulatory levels of IL-6 (rs = 0.587, P ≤ 0.04) and IFN-γ (rs = 0.721, P ≤ 0.001), while IgG2 subclass levels in female patients correlated with serum levels of IFN-γ (rs = 0.628, P ≤ 0.0006). Statistical analysis did not detect correlations between the levels of IgG (1–4) subclasses of anti-GA antibodies and the evaluated clinical parameters. Conclusion This study showed differences in the levels of IgG2 and IgG4 subclasses of ADA between male and female RR-MS patients. Further studies are necessary to take advantage of the clinical potential of this finding.

Disulfide bonds are post-translational modifications that provide conformational stability to proteins relevant to perform their biological functions. In this study, the quantification of free thiols by using Ellman’s reagent was validated to demonstrate, that the Filgrastim contained in the Filatil® exhibits the same amount of disulfide bonds than its reference product under both native and denaturing-reducing conditions. The validation exercise demonstrated to be suitable for its intended use. The comparability exercise for this attribute showed no significant differences (p=1.000; α , 0.05) in free cysteines content among the two evaluated products. These results provide evidence of similitude for this attribute, which along with an extended comparability exercise for other physicochemical characteristics, contribute to the demonstration of similarity among the physicochemical and functional properties set as critical quality attributes for Filgrastim according to the regulatory guidelines.

Biotherapeutic products which are derived from living organisms using recombinant DNA technology significantly contribute to the progress in the treatment of life-threatening and chronic diseases. The worldwide sale of biological drugs in 2016 was near US $263,700 million. In Latin America, where monoclonal antibodies market was worth US $7000 million, being Mexico the second largest market. Approval is one of the key aspects which influences the market of medicinal products, thus it is responsibility of the regulatory authority to establish a regulatory framework that ensure safety and efficacy of the products, and it is responsibility of the applicants to provide a high quality dossier in accordance with the registration requirements of the country. The applicants submitting registration requests in Mexico need to be aware of the requirements. Similar to many other countries, Mexico has adopted the Common Technical Document (CTD) structure for organizing dossier of the medicinal product for submission into main modules (i.e., quality, non-clinical, and clinical). This facilitates the submission process of medicinal products following a logical sequence aligned to the International Council on Harmonisation (ICH) guidelines. Moreover, this structure improves the transparency and clarity of the dossier in process of evaluation of medicinal products. In Mexico, the Ministry of Health has published a regulation, NOM-257-SSA1-2014, which established the general requirements to be followed by applicants to complete the registration of biotherapeutics. This regulation stipulates that the evaluation process is supported by a regulatory framework involving Good Manufacturing Practices, labeling, stability, clinical trials, biocomparability studies, pharmacovigilance, and a technical evaluation performed by a multidisciplinary team of experts in biotherapeutics development. Additionally, the Mexican regulatory agency, COFEPRIS, has published specific guidelines to facilitate the application process. Despite the availability of this information, the scope is limited to regulatory and administrative purposes, rather than technical-scientific supporting knowledge. The aim of this article is to provide concise information to improve and promote communication between industry and regulatory agencies. Herein, we describe the current process of COFEPRIS in regulating biotherapeutics in Mexico. This process explains the basis for the organization and structure of the technical-scientific information of biotherapeutics required for registration application.

Physicochemical and structural properties of proteins used as active pharmaceutical ingredients of biopharmaceuticals are determinant to carry out their biological activity. In this regard, the assays intended to evaluate functionality of biopharmaceuticals provide confirmatory evidence that they contain the appropriate physicochemical properties and structural conformation. The validation of the methodologies used for the assessment of critical quality attributes of biopharmaceuticals is a key requirement for manufacturing under GMP environments. Herein we present the development and validation of a flow cytometry-based methodology for the evaluation of adalimumab’s affinity towards membrane-bound TNFα (mTNFα) on recombinant CHO cells. This in vitro methodology measures the interaction between an in-solution antibody and its target molecule onto the cell surface through a fluorescent signal. The characteristics evaluated during the validation exercise showed that this methodology is suitable for its intended purpose. The assay demonstrated to be accurate (r 2 = 0.92, slope = 1.20), precise (%CV ≤ 18.31) and specific (curve fitting, r 2 = 0.986–0.997) to evaluate binding of adalimumab to mTNFα. The results obtained here provide evidence that detection by flow cytometry is a viable alternative for bioassays used in the pharmaceutical industry. In addition, this methodology could be standardized for the evaluation of other biomolecules acting through the same mechanism of action.

Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.

The function of proteins, such as the catalytic enzyme activity, depends on the interaction of their active sites with their specific substrates and the environment conditions that affect the stability of those sites. This study presents a structure-to-function characterization of the folding process of a recombinant 6×-His tag leucine aminopeptidase (rLAP) based on a platform of analytical techniques. The results demonstrated an increase up to 31 U/mg in the activity of the enzyme after folding as revealed by circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and free thiol analysis. Collectively, these techniques revealed a larger number of covalent and non-covalent bonds within the protein seen as an increase in the chemical and thermal stability, while exhibited a lower level of non-bonded cysteines after the protein was folded. Mass spectrometry analysis showed the maintenance of the distribution of the enzyme isoforms related to N-terminal histidine residues after folding, which confirmed that the enzymatic activity of rLAP depends on its three-dimensional structure rather than N-terminal self-processing activity. In summary, the studied attributes allow a better understanding of the structure-to-function relationship of rLAP, that permit a more proficient manufacturing of the enzyme that would improve the bioprocesses in which is employed.

Glatiramer Acetate (GA) is an immunomodulatory medicine approved for the treatment of multiple sclerosis, whose mechanisms of action are yet to be fully elucidated. GA is comprised of a complex mixture of polypeptides with different amino acid sequences and structures. The lack of sensible information about physicochemical characteristics of GA has contributed to its comprehensiveness complexity. Consequently, an unambiguous determination of distinctive attributes that define GA is of highest relevance towards dissecting its identity. Herein we conducted a study of characteristic GA heterogeneities throughout its manufacturing process (process signatures), revealing a strong impact of critical process parameters (CPPs) on the reactivity of amino acid precursors; reaction initiation and polymerization velocities; and peptide solubility, susceptibility to hydrolysis, and size-exclusion properties. Further, distinctive GA heterogeneities were correlated to defined immunological and toxicological profiles, revealing that GA possesses a unique repertoire of active constituents (epitopes) responsible of its immunological responses, whose modification lead to altered profiles. This novel approach established CPPs influence on intact GA peptide mixture, whose physicochemical identity cannot longer rely on reduced properties (based on complete or partial GA degradation), providing advanced knowledge on GA structural and functional relationships to ensure a consistent manufacturing of safe and effective products.

This protocol shows the measurement of the apoptotic activity neutralization of TNFα in a mouse fibroblast cell model (WEHI 164) using an anti-TNFα mAb. In addition, this protocol can be used to evaluate other anti-TNFα molecules, such as fusion proteins. The cellular model employed here is sensitive to TNFα-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Prostate cancer (PCa) is the second most frequently diagnosed cancer in men worldwide. Dialyzed Leukocyte Extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that improve clinical responses in various diseases. Here, we analyzed the effects of Transferon

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (

In order to demonstrate physicochemical comparability of a recombinant protein medicament as a drug substance (Active Pharmaceutical Ingredient or API), such as Rituximab, one must first choose suitable orthogonal analytical methods that selectively provide information regarding the identity and heterogeneity of the molecule. The application of these methods in the investigation of process-derived variability of the reference molecule can yield valuable information to establish comparability criteria. This could become the basis to determine the critical physicochemical attributes that should be verified by relevant in-process controls and batch release criteria. In this paper we show the determination and analysis of relevant attributes from two different proposed biosimilars. We show differences in Reditux proposed Rituximab biosimilar, mainly in charge heterogeneity patterns, whereas no major differences were found for Kikuzubam as related to the Mabthera reference batches. The detected differences are...

Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase ( r LAP) to explain the gain of activity observed when incubated at 37 °C after its production. Unfolding transitions of r LAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8 ± 0.4 and 3.7 ± 0.4 kcal mol −1 , respectively. Thermal stability conformation of r LAP is 2.6 ± 0.1 and 6.1 kcal mol −1 for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with l -leucine- p -nitroanilide (L- p NA) as substrate. The catalytic efficiency was 3.87 ± 0.10 min −1 μM −1 at 37 °C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and r LAP; however r LAP is selective and specific to remove N-terminal groups from amino acids.

Infliximab is therapeutic monoclonal antibody (mAb) against TNF-α employed in the treatment of immunoinflammatory diseases. The development of biosimilar mAbs is a global strategy to increase drug accessibility and reduce therapy-associated costs. Herein we compared key physicochemical characteristics and biological activities produced by infliximab and infliximab-Probiomed in order to identify functionally relevant differences between the mAbs. Binding of infliximab-Probiomed to TNF-α was specific and had kinetics comparable to that of the reference product. Both mAbs had highly similar neutralizing efficacy in HUVEC cell cultures stimulated with TNF-α. In vitro induction of CDC and ADCC were also similar between the evaluated products. In vivo comparability was assessed using a transgenic mouse model of arthritis that expresses human TNF-α in a 13-week multiple-administration study. Infliximab and infliximab-Probiomed showed comparable efficacy, safety, and pharmacokinetic profiles. Our results indicate that infliximab-Probiomed has highly similar activities to infliximab in preclinical models, warranting a clinical evaluation of its biosimilarity.

Highlights • The HEK-Blue IFN-α/β system was validated to comply international guidelines. • This system complied in accuracy, precision, specificity and, system suitability. • The validated system showed robustness to evaluate different commercial type I IFN. • This bioassay could be a complementary method to evaluate type I IFN activity. • The system met the validation criteria to be used in pharmaceutical environments., The biotherapeutic type I interferons (IFN-I) are indicated to treat several diseases. These products are regulated to guarantee safety and efficacy through critical quality attributes. For this purpose, the development of robust assays is required, followed by its validation to demonstrate their suitability for its intended purpose. Despite there are some commercial kits to evaluate IFN-I signaling, these are focused on measuring in vitro biological response instead of their validation, which is a pharmaceutical industry requirement. The aim of this work was to validate the HEK-Blue IFN-α/β system evaluating the biological activity of IFN-α/β under good laboratory practices, according to international standards. Our results demonstrated that HEK-Blue IFN-α/β system comply with accuracy (r2>0.95) precision (CV

Analytical platforms that characterize charge heterogeneity in therapeutic proteins, such as mAbs, are important tools that can be used to define quality attributes. CZE separates protein moieties close to their native state and is a valuable physicochemical analytical method that can be used in parallel with other orthogonal methods for characterization and comparability. In this study, custom conditions for the analysis of charge heterogeneity of two mAbs were developed with regard to critical parameters in the BGE, running conditions, and sample treatment. The method application was tested for up to four mAbs and one mAb fragment. The electropherograms showed specific profiles and contrasting levels of basic and acidic isoforms with respect to the main isoform. Issues that surround this method, such as peak tailing and capillary lifetime, are summarized. Using this method, the identities of rituximab and trastuzumab were confirmed, based on the correspondence between the biosimilars and reference products, noninterference of the sample matrix, and the ability to separate spiked samples of related mAbs. The RSD of the isoform content and migration time for the method repeatability were less than 2 and 1%, respectively.

Leucine aminopeptidase from Vibrio proteolyticus (LAP) is used to remove N-terminal methionine from recombinant therapeutic proteins in biotechnology processes. The native enzyme is efficient for this purpose, but it contains proteins and endotoxins from V. proteolyticus that need to be removed in the manufacturing process and quantified as contaminants of the active pharmaceutical ingredient (API). In this study, a synthetic gene was designed to encode a mature and active 32 kDa LAP and thus avoid the post-translational processing of the 54 kDa pro-protein. A 6×-histidine tag was fused to the N-terminus to facilitate purification of the recombinant LAP (rLAP) by immobilized-metal ion-affinity chromatography (IMAC). An active enzyme with purity above 95% was obtained; it is able to remove the N-terminal methionine from recombinant human methionine-interferon alpha-2b (met-rhIFN α-2b), giving a primary structure identical to the human one. Therefore, rLAP can be used as a biologically active therapeutic protein. In addition, a kinetic characterization of rLAP was performed. The kinetic parameters V max , k m and k cat were 93.62 μM h −1 , 27.26 μM and 3.07 h −1 , respectively. Thus, this work provides an rLAP that can be used in industrial processes for N-terminal methionine excision (NME) from such recombinant products as interferon α-2b, human growth hormone and granulocyte macrophage-colony stimulating factor.

The presence of endocrine disruptors bisphenol-A, bisphenol-A-dimethacrylate, bisphenol-A-diglycidyl-ether, phthalic-acid, dibutyl-phthalate, diethyl-phthalate and dioctyl-phthalate was determined in vegetable cans, baby bottles and microwaveable containers from the Mexican market. Gas-Chromatography-Mass-Spectrometry was used for the identification and High-Performance-Liquid-Chromatography with UV/Visible light and fluorescence detectors was used for the quantification. Endocrine disruptors were found in all samples. PA and DOP were the substances most commonly found, and maximum concentrations were 9.549 and 0.664 μg/kg, respectively from a jalapeno peppers can. Bisphenol A, phthalic-acid, bisphenol-A-dimethacrylate, bisphenol-A-diglycidyl-ether, dioctyl-phtalate and dibutyl-phthalate were found in baby bottles and microwaveable containers.

Interferon-alpha 2b (IFN-alpha 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-alpha 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-alpha 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r(2)) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain.

The production of human interferon gamma (hIFN-γ) using a synthetic gene in Escherichia coli BL21-SI was optimized by response surface methodology (RSM) and a Box-Behnken design. The process variables studied were temperature, bio-mass concentration at induction time and the NaCl concentration as inducer. According to the Box-Behnken design, a second order response function was developed. The optimal expression conditions were a temperature of 32.6°C, induction biomass of 0.31 g/L and 0.3 M NaCl in minimal medium. The model prediction for the maximum hIFN-γ production was 77.3 mg/L, which corresponded satisfactorily with the experimental data. The hIFN-γ concentration attained under optimized conditions was 13-times higher than that obtained using the non-optimized conditions. We conclude that RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Production of periplasmic human interferon-gamma (hINF-gamma) and human interleukin-2 (hIL-2) by the Tat translocation pathway in Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-gamma was processed and the protein was transported to periplasm. Up to 30.1% of hINF-gamma was found in the periplasmic soluble fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction.

UV spectrophotometric measurement is a widely accepted and standardized routine analysis for quantitation of highly purified proteins; however, the reliability of the results strictly depends on the accuracy of the employed extinction coefficients. In this work, an experimental estimation of the differential refractive index (dn/dc), based on dry weight measurements, was performed in order to determine accurate extinction coefficients for four biotherapeutic proteins and one synthetic copolymer after separation in a size-exclusion ultra-performance liquid chromatograph coupled to an ultraviolet, multiangle light scattering and refractive index (SE-UPLC-UV-MALS-RI) multidetection system. The results showed small deviations with respect to theoretical values, calculated from the specific amino acid sequences, for all the studied immunoglobulins. Nevertheless, for proteins like etanercept and glatiramer acetate, several considerations, such as glycan content, partial specific volume, polarizability, and higher order structure, should be considered to properly calculate theoretical extinction coefficient values. Herein, these values were assessed with simple approximations. The precision of the experimentally obtained extinction coefficients, and its convergence towards the theoretical values, makes them useful for characterization and comparability exercises. Also, these values provide insight into the absorbance and scattering properties of the evaluated proteins. Overall, this methodology is capable of providing accurate extinction coefficients useful for development studies.

Comparability between a biosimilar and its reference product requires the evaluation of critical quality attributes that may impact on its pharmacological response. Herein we present a physicochemical characterization of a biosimilar trastuzumab focused on the attributes related to the pharmacokinetic response. Capillary isoelectrofocusing (cIEF) and cation exchange chromatography (CEX) were used to evaluate charge heterogeneity; glycosylation profiles were assessed through hydrophilic interaction liquid chromatography (HILIC); aggregates content was evaluated through size exclusion chromatography (SEC) while binding affinity to FcRn was evaluated using isothermal titration calorimetry (ITC). The biosimilar trastuzumab and its reference product exhibited a high degree of similarity for the evaluated attributes. In regard to the pharmacokinetic parameters, randomized, double blind, and two-arm parallel and prospective study was employed after the administration of a single intravenous dose in healthy volunteers. No significant differences were found between the pharmacokinetic profiles of both products. Our results confirm that similarity of the critical quality attributes between a biosimilar product, obtained from a different manufacturing process, and the reference product resulted in comparable pharmacokinetic profiles, diminishing the uncertainty related to the biosimilar’s safety and efficacy.

Worldwide, the most highly consumed meat is of porcine origin. The production and distribution of swine meat are affected by diverse health matters, such as influenza and diarrhea, which cause head losses and require the use of antibiotics and other drugs in hog farms. To stimulate newborn piglet immune responses and increase resistance to infections, we developed a spray-drying technique to produce dried swine dialyzable spleen extract (sDSE), an immunomodulator. Based on the size-exclusion ultra performance liquid chromatography quantitative analysis, it was possible to recover up to 58% of the product after the drying process. The biological activity of orally administered dried sDSE increased mouse survival and induced cytokine production in a herpes infection model.

Cheese whey (CW) is the major subproduct from cheese manufacturing and it is considered as a waste pollutant since its high content of lactose. In this work a fermentation process for the production of penicillin acylase (PA) by a recombinant Escherichia coli and using CW as unique carbon source and inducer was developed. A design factorial 3(2) was used to evaluate the influence of independent variables (dissolved oxygen and CW concentration) on the ability of E. coli W3110/pPA102 to produce PA. Maximum specific PA activity of 781 U g(-1) was attained at 5 g L(-1) of CW and 3% dissolved oxygen. The results showed that CW can be used successfully as unique carbon source and inducer for the production of recombinant proteins using constructions driven by the lac promoter and this way reducing the discharges of that pollutant to the environment.

Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.

In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

Analytical platforms that characterize charge heterogeneity in therapeutic proteins, such as mAbs, are important tools that can be used to define quality attributes. CZE separates protein moieties close to their native state and is a valuable physicochemical analytical method that can be used in parallel with other orthogonal methods for characterization and comparability. In this study, custom conditions for the analysis of charge heterogeneity of two mAbs were developed with regard to critical parameters in the BGE, running conditions, and sample treatment. The method application was tested for up to four mAbs and one mAb fragment. The electropherograms showed specific profiles and contrasting levels of basic and acidic isoforms with respect to the main isoform. Issues that surround this method, such as peak tailing and capillary lifetime, are summarized. Using this method, the identities of rituximab and trastuzumab were confirmed, based on the correspondence between the biosimilars and reference products, noninterference of the sample matrix, and the ability to separate spiked samples of related mAbs. The RSD of the isoform content and migration time for the method repeatability were less than 2 and 1%, respectively.

A synthetic human interferon gamma (hIFN-gamma) gene was fused to SP1 and SP3, two Sec-dependent artificial signal peptides to transport the hIFN-gamma to the periplasm of Escherichia coli BL21-SI. The processing efficiency of both SP1-hIFN-gamma and SP3-hIFN-gamma was dependent on the culture medium as well as the post-induction temperature. Both precursors were processed completely when cells were cultured using minimal medium and a post-induction temperature of 32.5 degrees C, and only the processed hIFN-gamma was detected. The SP3 signal peptide was more efficient than SP1 for the secretion of hIFN-gamma. Sixty percent of the total hIFN-gamma was secreted to the periplasm using the SP3 signal peptide and a post-induction temperature of 20 degrees C. Using Tris-sucrose-dithiothreitol (TSD) hypertonic buffer, the periplasmic soluble hINF-gamma was recovered with a purity of 85%.

A human interferon beta (hINF-beta) synthetic gene was optimized and expressed in Escherichia coli BL21-SI using a vector with the T7 promoter. To determine the best culture conditions such as culture medium, temperature, cell density and inducer concentration, we used the response surface methodology and a Box-Behnken design to get the highest hINF-beta production. The maximum hINF-beta production of 61 mg l(-1) was attained using minimum medium and the following predicted optimal conditions: temperature of 32.5 degrees C, cell density of 0.64, and inducer concentration of 0.30 M NaCl. This is the first report showing the successful performance of the BL21-SI system in a minimum medium. The response surface methodology is effective for the optimization of recombinant protein production using synthetic genes.