A general evidence-based sequence variant control limit for recombinant therapeutic protein development.

Sequence variants (SVs) resulting from unintended amino acid substitutions in recombinant therapeutic proteins have increasingly gained attention from both regulatory agencies and the biopharmaceutical industry given their potential impact on efficacy and safety. With well-optimized production systems, such sequence variants usually exist at very low levels in the final protein products due to the high fidelity of DNA replication and protein biosynthesis process in mammalian expression systems such as Chinese hamster ovary cell lines. However, their levels can be significantly elevated in cases where the selected production cell line has unexpected DNA mutations or the manufacturing process is not fully optimized, for example, if depletion of certain amino acids occurs in the cell culture media in bioreactors. Therefore, it is important to design and implement an effective monitoring and control strategy to prevent or minimize the possible risks of SVs during the early stage of product and process development. However, there is no well-established guidance from the regulatory agencies or consensus across the industry to assess and manage SV risks. A question frequently asked is: What levels of SVs can be considered acceptable during product and process development, but also have no negative effects on drug safety and efficacy in patients? To address this critical question, we have taken a holistic approach and conducted a comprehensive sequence variant analysis. To guide biologic development, a general SV control limit of 0.1% at individual amino acid sites was proposed and properly justified based on extensive literature review, SV benchmark survey of approved therapeutic proteins, and accumulated experience on SV control practice at Regeneron.

Nutrient supplementation strategy improves cell concentration and longevity, monoclonal antibody production and lactate metabolism of Chinese hamster ovary cells.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 106 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry.

Effects of Succinic Acid Supplementation on Stable Cell Line Growth, Aggregation, and IgG and IgA Production.

BACKGROUND: Immunoglobulin (Ig) G is the most commonly used therapeutic antibodies. Recently, the interest in IgA antibodies to treat respiratory infectious diseases has been increasing. The reason for the inefficient use of IgA is recombinant antibody aggregation in cell culture, affecting the longevity and productivity of cell lines. Lactate is an important metabolite that affects the cultivation of stable cell lines producing monoclonal antibodies. METHODS: In the present study, we investigated whether different combinations of succinic acid and micro-additives affect lactate production, which correlates with productivity. The effect of succinic acid substitution on productivity of cells producing IgG/IgA was analyzed using the static culture method in a six-well plate. Lactate was measured in supernatant of cell culture indirectly by using the activity of Lactate Dehydrogenase (LDH).A low lactate level was observed in cultivation medium supplemented with succinic acid or asparagine combined with some inorganic salts. RESULTS: The results also demonstrated the effect of component supplementation on homogeneity, longevity, and productivity of cell culture. Supplementation of succinic acid eliminated cell aggregation and improved homogeneity of stable cell lines producing IgG and, especially, IgA. CONCLUSION: Overall, succinic acid supplementation to the culture medium has potential biotechnological applications in the production IgG and IgA.

Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

Real-time quantification and supplementation of bioreactor amino acids to prolong culture time and maintain antibody product quality.

Real-time monitoring of cell cultures in bioreactors can enable expedited responses necessary to correct potential batch failure perturbations which may normally go undiscovered until the completion of the batch and result in failure. Currently, analytical technologies are dedicated to real-time monitoring of bioreactor parameters such as pH, dissolved oxygen, and temperature, nutrients such as glucose and glutamine, or metabolites such as lactate. Despite the importance of amino acids as the building blocks of therapeutic protein products, other than glutamine their concentrations are not commonly measured. Here, we present a study into amino acid monitoring, supplementation strategies, and how these techniques may impact the cell growth profiles and product quality. We used preliminary bioreactor runs to establish baselines by determining initial amino acid consumption patterns, the results of which were used to select a pool of amino acids which gets depleted in the bioreactor. These amino acids were combined into blends which were supplemented into bioreactors during a subsequent run, the concentrations of which were monitored using a mass spectrometry based at-line method we developed to quickly assess amino acid concentrations from crude bioreactor media. We found that these blends could prolong culture life, reversing a viable cell density decrease that was leading to batch death. Additionally, we assessed how these strategies might impact protein product quality, such as the glycan profile. The amino acid consumption data were aligned with the final glycan profiles in principal component analysis to identify which amino acids are most closely associated with glycan outcomes.

Consequences of trace metal variability and supplementation on Chinese hamster ovary (CHO) cell culture performance: A review of key mechanisms and considerations.

Trace metals are supplied to chemically-defined media (CDM) for optimal Chinese hamster ovary (CHO) cell culture performance during the production of monoclonal antibodies and other therapeutic proteins. However, lot-to-lot and vendor-to-vendor variability in raw materials consequently leads to an imbalance of trace metals that are supplied to CDM. This imbalance can yield detrimental effects rooted in several primary mechanisms and pathways including oxidative stress, apoptosis, lactate accumulation, and unfavorable glycan synthesis. Recent research endeavors involve supplying zinc, copper, and manganese to CDM in excess to further maximize culture productivity and product quality. These treatments significantly impact critical quality attributes and furthermore highlight the degree to which trace metal availability can affect CHO cell culture performance. This review highlights the role of trace metal variability, supplementation, and interplay on key cellular mechanisms responsible for overall culture performance and the production and quality of therapeutic proteins.

Generation of Monoclonal Antibodies Against Natural Products.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Lateral Flow Assessment and Unanticipated Toxicity of Kratom.

The leaves of the Mitragynine speciosia tree (also known as Kratom) have long been chewed, smoked, or brewed into a tea by people in Southeastern Asian countries, such as Malaysia and Thailand. Just this past year, the plant Kratom gained popularity in the United States as a "legal opioid" and scheduling it as a drug of abuse is currently pending. The primary alkaloid found in Kratom is a µ-opioid receptor agonist, mitragynine, whose structure contains a promising scaffold for immunopharmacological use. Although Kratom is regarded as a safe opioid alternative, here we report the LD50 values determined for its two main psychoactive alkaloids, mitragynine and 7-hydroxymitragynine, as comparable to heroin in mice when administered intravenously. Given Kratom's recent emergence in the U.S., there is currently no diagnostic test available for law enforcement or health professionals, so we sought to design such an assay. Mitragynine was used as a starting point for hapten design, resulting in a hapten with an ether linker extending from the C9 position of the alkaloid. Bacterial flagellin (FliC) was chosen as a carrier protein for active immunization in mice, yielding 32 potential monoclonal antibodies (mAbs) for assay development. Antimitragynine mAbs in the range of micro- to nanomolar affinities were uncovered and their utility in producing a convenient lateral flow detection assay of human fluid samples was examined. Antibodies were screened for binding to mitragynine, 7-hydroxymitragynine, and performance in lateral flow assays. Two monoclonal antibodies were subcloned and further purified with 93 and 362 nM affinity to mitragynine. Test strip assays were optimized with a detection cut off of 0.5 µg/mL for mitragynine in buffer and urine (reflecting projected clinically relevant levels of drug in urine), which could be beneficial to law enforcement agencies and health professionals as the opioid epidemic in America continues to evolve.

Application of microRNA Targeted 3'UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools.

The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3'UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3'UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3'UTR targets of the let-7f miR are then cloned in the DHFR host 3'UTR to determine the impact on gene expression (HMGA2 3'UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3'UTR and its ability to bind let-7f. The same 3'UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.

Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis.

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017.

Generation and Application of Monoclonal Antibody Against Lycopene.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17-25, 2017.

Effect of copper variation in yeast hydrolysate on C-terminal lysine levels of a monoclonal antibody.

The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C-terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large-scale production of a murine cell line exhibited variation in the C-terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small-scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C-terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C-terminal lysine levels of the batches in manufacturing. Small-scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C-terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast-hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463-468, 2017.

Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117.

Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.

Molecular Selection, Modification and Development of Therapeutic Oligonucleotide Aptamers.

Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the detailed advantages and disadvantages of antibodies and aptamers in therapeutic applications and summarize recent progress in aptamer selection and modification approaches. We will present therapeutic oligonucleotide aptamers in preclinical studies for skeletal diseases and further discuss oligonucleotide aptamers in different stages of clinical evaluation for various disease therapies including macular degeneration, cancer, inflammation and coagulation to highlight the bright commercial future and potential challenges of therapeutic oligonucleotide aptamers.

Development of a low-serum medium for the production of monoclonal antibody against congenital adrenal hyperplasia by hybridoma culture.

Statistically designed experiments were used in developing a low-serum medium for the production of a diagnostic monoclonal antibody against congenital adrenal hyperplasia using hybridoma 192. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate, and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco's modified Eagle's medium (DMEM; containing 4 mM L-glutamine, 1% antibiotic-antimycotic agent) supplemented with 0.4% by volume fetal bovine serum (FBS), 311.8 mM ferric citrate, 17.3 nM sodium selenite, and 4.5 mM zinc sulfate (LSD) was found to support the growth of the hybridoma. Specific cell growth rate in the LSD (0.033 ± 0.001/h) was slightly lower than in the control medium (i.e., basal DMEM supplemented with 2% FBS; 0.0045 ± 0.003/h). Nevertheless, the specific MAb production rate for LSD was higher (0.057 ± 0.015 pg/cell · h versus 0.004 ± 0.002 pg/cell · h in LSD and control, respectively). The antibody produced in the LSD showed high specificity and no cross-reactivity with the other structural resemblance's steroid hormones, revealing no structural changes owing to the new medium formulation developed. The new medium formulation effectively reduced the medium cost by up to 64.6%.

The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals.

Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever.