RESUMO
The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.
Assuntos
Vetores Genéticos , Lentivirus , Lentivirus/genética , Ligantes , Reprodutibilidade dos Testes , Terapia Genética/métodosRESUMO
Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS2Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 - currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as 'C-tag', composed of the four amino acids, glutamic acid - proline - glutamic acid - alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions. Biochemical and immunological analysis showed that the C-tagged and hexa-histidine-tagged P. falciparum reticulocyte-binding protein homolog 5 proteins are comparable. The C-tag technology has the potential to form the basis of a current good manufacturing practice-compliant platform, which could greatly improve the speed and ease with which novel protein-based products progress to clinical testing.
Assuntos
Proteínas de Transporte/química , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/metabolismo , Animais , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , CoelhosRESUMO
Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process-related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VH H antibody fragments as "tunable" immunoaffinity ligands for separation of product-related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma-carboxylglutamic acid domain.
Assuntos
Cromatografia de Afinidade/métodos , Anticorpos/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Exotoxinas/isolamento & purificação , Protrombina/isolamento & purificaçãoRESUMO
Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.
Assuntos
Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Camelídeos Americanos/imunologia , Fragmentos de Imunoglobulinas/imunologia , Proteínas Recombinantes/imunologia , Infecções por Rotavirus/virologia , Rotavirus/imunologia , Animais , Anticorpos Neutralizantes/uso terapêutico , Western Blotting , Diarreia Infantil/virologia , Genótipo , Humanos , Imunização , Fragmentos de Imunoglobulinas/uso terapêutico , Lactente , Camundongos , Proteínas Recombinantes/uso terapêutico , Rotavirus/genética , Rotavirus/patogenicidade , Infecções por Rotavirus/tratamento farmacológicoRESUMO
AIMS: Using genetically engineered lactobacilli, producing high avidity llama VHH domains (referred to as anti-rotavirus proteins; ARPs), to test the effect of multimeric antibody fragments as prophylaxis and therapy against rotavirus infection. METHODS: Two ARPs, ARP1 and ARP3, shown to bind to different epitopes and act synergistically against rotavirus, were displayed on the surface of Lactobacillus paracasei as monovalent or bivalent proteins (mono- or bi-specific). RESULTS: Although a nonsignificant difference was observed between lactobacilli producing bispecific ARP3-ARP1 and monomeric ARPs, lactobacilli producing bispecific ARP3-ARP1 were superior at reducing the rate of diarrhea when used for prophylactic and therapeutic intervention in a mouse model of rotavirus infection in comparison to nontreated animals. CONCLUSION: Expression of bispecific antibodies in lactobacilli resulted in slight improvement of their efficacy. Furthermore, increasing the specificity would theoretically reduce the rate of appearance of viral escape mutants and would have a broader capacity to be effective against a range of viral serotypes.
Assuntos
Anticorpos Biespecíficos/biossíntese , Camelídeos Americanos/imunologia , Diarreia/prevenção & controle , Lactobacillus/metabolismo , Infecções por Rotavirus/prevenção & controle , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Camelídeos Americanos/genética , Diarreia/virologia , Imunização Passiva , Lactobacillus/genética , Camundongos , Camundongos Endogâmicos BALB C , Rotavirus/imunologia , Infecções por Rotavirus/virologiaRESUMO
Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.
Assuntos
Antibacterianos/farmacologia , Anticorpos Antibacterianos/farmacologia , Camelídeos Americanos/imunologia , Cárie Dentária/prevenção & controle , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Streptococcus mutans/efeitos dos fármacos , Animais , Camelídeos Americanos/fisiologia , Cárie Dentária/microbiologia , Glucose Oxidase/farmacologia , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/farmacologia , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/farmacologia , Imunotoxinas/toxicidade , Boca/microbiologia , Ratos , Ratos Sprague-Dawley , Streptococcus mutans/enzimologia , Streptococcus mutans/genéticaRESUMO
Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.