CHOK1SV GS-KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

Rational optimization of a monoclonal antibody for simultaneous improvements in its solution properties and biological activity.

Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies. MAB1 and its variants were formulated in the same platform buffer and subjected to a battery of experiments to assess their solution behaviors, and biological activities. Five of the seven (71%) variants of MAB1 demonstrated improved biophysical attributes in multiple experimental testings. Contrary to the commonly expressed reservations about potential biological activity loss upon developability optimizations, the improvements in solution behavior of MAB1 also increased its biological activity up to ~180%. In particular, concentrate-ability and apparent solubility of V44KE59S improved to ~150% and ~160%, respectively. Its diffusion interaction parameter (kD) reduced to 28% and viscosity at ~100 mg/ml decreased to less than half of the corresponding values for MAB1. V44KE59S is also slightly more active and its transfections in CHO cells were more productive. It also degraded slower than MAB1 in three month long 25°C and 40°C formulation stability studies. These results open doors to an exciting realm of structure-based biologic drug design where developability and biological activity can be simultaneously optimized at the molecular engineering stages.

Strategic deployment of CHO expression platforms to deliver Pfizer's Monoclonal Antibody Portfolio.

Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1463-1467, 2017.

Health concerns of heterotrophic plate count (HPC) bacteria in dental equipment water lines.

There is an unsubstantiated concern as to the health relevance of HPC (heterotrophic plate count) bacteria in dental equipment waterlines. The American Dental Association (ADA) web site includes guidelines for controlling HPC populations and implies that HPC populations >500 CFU/mL as a "health" benchmark. The world-wide published literature including the United Nations fully examined this situation and concluded that HPC bacteria are not a health risk, but merely a general water quality parameter for all waters including dental water lines. This review provides documentation that the standard measurement of HPC bacteria in waters alone do not pose a health risk and the ADA already provides appropriate practices to minimize HPC bacteria in dental equipment water.

Rational design of viscosity reducing mutants of a monoclonal antibody: hydrophobic versus electrostatic inter-molecular interactions.

High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region. The disruption of an APR that lies at the interface of light and heavy chain variable domains, VH and VL, via L45K mutation destabilized MAB 1 and abolished antigen binding. However, mutation at the preceding residue (V44K), which also lies in the same APR, increased apparent solubility and reduced viscosity of MAB 1 without sacrificing antigen binding or thermal stability. Neutralizing the negatively charged surface patch (E59Y) also increased apparent solubility and reduced viscosity of MAB 1, but charge reversal at the same position (E59K/R) caused destabilization, decreased solubility and led to difficulties in sample manipulation that precluded their viscosity measurements at high concentrations. Both V44K and E59Y mutations showed similar increase in apparent solubility. However, the viscosity profile of E59Y was considerably better than that of the V44K, providing evidence that inter-molecular interactions in MAB 1 are electrostatically driven. In conclusion, neutralizing negatively charged surface patches may be more beneficial toward reducing viscosity of highly concentrated antibody solutions than charge reversal or aggregation prone motif disruption.

Drinking water microbial myths.

Accounts of drinking water-borne disease outbreaks have always captured the interest of the public, elected and health officials, and the media. During the twentieth century, the drinking water community and public health organizations have endeavored to craft regulations and guidelines on treatment and management practices that reduce risks from drinking water, specifically human pathogens. During this period there also evolved misunderstandings as to potential health risk associated with microorganisms that may be present in drinking waters. These misunderstanding or "myths" have led to confusion among the many stakeholders. The purpose of this article is to provide a scientific- and clinically-based discussion of these "myths" and recommendations for better ensuring the microbial safety of drinking water and valid public health decisions.

Molecular and functional characterization of cynomolgus monkey IgG subclasses.

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

Interchain disulfide bonding in human IgG2 antibodies probed by site-directed mutagenesis.

Human IgG2 exists as a mixture of disulfide-linked structural isoforms that can show different activities. To probe the contribution of specific cysteine residues to the formation of structural isoforms, we characterized a series of Cys-->Ser mutant IgG2 recombinant monoclonal antibodies, focused on the first C(H)1 cysteine and the first two hinge cysteines. These residues participate in the formation of structural isoforms that have been noted by nonreduced capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, and cation exchange chromatography. We show that single Cys-->Ser mutants can greatly reduce heterogeneous disulfide bonding in human IgG2 and maintain in vitro activity. The data demonstrate the feasibility of applying site-directed mutagenesis to reduce disulfide bond heterogeneity in human IgG2 while preserving the activity of this therapeutically important class of human antibodies.

Disulfide connectivity of human immunoglobulin G2 structural isoforms.

In this communication we present the detailed disulfide structure of IgG2 molecules. The consensus structural model of human IgGs represents the hinge region positioned as a flexible linker connecting structurally isolated Fc and Fab domains. IgG2 molecules are organized differently from that model and exhibit multiple structural isoforms composed of (heavy chain-light chain-hinge) covalent complexes. We describe the precise connection of all the disulfide bridges and show that the IgG2 C H1 and C-terminal C L cysteine residues are either linked to each other or to the two upper hinge cysteine residues specific to the IgG2 subclass. A defined arrangement of these disulfide bridges is unique to each isoform. Mutation of a single cysteine residue in the hinge region eliminates these natural complexes. These results show that IgG2 structure is significantly different from the conventionally accepted immunoglobulin structural model and may help to explain some of the unique biological activity attributed only to this subclass.

Identification of novel small molecule enhancers of protein production by cultured mammalian cells.

Small molecule additives to cell culture media (e.g., sodium butyrate) that are capable of enhancing the expression of recombinant proteins have significant utility in the production and manufacture of therapeutic polypeptides. To identify novel small molecule enhancers (SMEs) of recombinant protein expression in Chinese Hamster Ovary (CHO) cells, we screened two separate small molecule libraries for compounds capable of enhancing the expression of either a fluorescent reporter protein or a monoclonal antibody. Several compounds that increased recombinant protein expression were identified, and these compounds fell into three broad classes: (1) aromatic carboxylic acids, (2) hydroxamic acids, and (3) acetamides. We examined the impact of SME addition to CHO cell cultures expressing different classes of recombinant proteins including monoclonal antibodies (MAbs). For CHO cell pools or clones grown in production shake-flasks or bioreactors, recombinant protein titers up to 60% higher than control cultures were observed. Analysis of mRNA levels suggest that transcriptional activation plays a role in the expression enhancement seen for some SMEs, but other mechanisms may be involved for at least one compound. Finally, we tested many of the identified SMEs for their ability to increase MAb production by a hybridoma cell line. Hexanohydroxamic acid increased shake-flask MAb production by 40% relative to a control. Taken together, these data demonstrate the potential utility of the compounds in the production of therapeutically relevant proteins from diverse cell-based production systems.

Human IgG2 antibodies display disulfide-mediated structural isoforms.

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C(L)) and the heavy chain C(H)1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C(H)1-C(L)-hinge)(2) complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either kappa or lambda light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.

Issues for microbial regulation: Aeromonas as a model.

The decision by public health agencies to regulate specific microorganisms that may be found in drinking water can only be made if specific criteria find that a microorganism poses a health risk. These criteria should include: (1) there is a clinical history of an organism causing disease from the ingestion of drinking water; (2) there is epidemiological evidence that drinking water rather than food or other vectors is a major source of disease; (3) there is sufficient evidence that the target organism, if found in water, possesses virulence factors capable of causing disease in humans; (4) there is sufficient evidence that the target organism is not readily removed or inactivated by multi-barrier conventional water treatment process (e.g., coagulation-filtration-disinfection); (5) there is sufficient evidence that the target organism, if surviving conventional treatment, will be viable, virulent, and present in sufficient numbers to cause disease; (6) there are robust analytical methods for the target organism which have acceptable sensitivity, specificity, and reproducibility to measure accurately the presence of the target organism in treated water; and (7) the performance criteria of analytical method(s) for the target organism have been certified by the appropriate public health agency, and there is intra-laboratory field-test performance data to base this certification.

Virulence and risk from drinking water of heterotrophic plate count bacteria in human population groups.

Bacteria are natural inhabitants of all aqueous environments. The heterotrophic plate count is a means of assessing the concentration of these bacteria in foods, water, and water filtration systems. Methods vary, but are designed to enumerate bacteria that have evolved an environmental lifestyle. Most commonly, low nutrient, low ionic strength culture media are employed. The group of environmental bacteria enumerated depends on the media formulation and incubation conditions but are commonly known as heterotrophic plate count (HPC) bacteria; in Europe, this group is also referred to as autochthonous flora. While HPC inhabit an environmental niche, there has been concern that at some concentration they may be a human health risk. A review of the literature, including animal and human feeding studies, analysis of virulence factors, and outbreaks demonstrates that HPC bacteria as enumerated on HPC culture media have not been established as a human health threat at any concentration in drinking water or foods.

Heterotrophic plate count bacteria--what is their significance in drinking water?

While the literature documents the universal occurrence of heterotrophic plate count (HPC) bacteria in soils, foods, air, and all sources of water, there is a lingering question as to whether this group of organisms may signal an increased health risk when elevated populations are present in drinking water. This paper reviews the relevant literature on HPC bacteria in drinking water, the lack of clinical evidence that elevated populations or specific genera within the HPC flora pose an increased health risk to any segment of the population, and the appropriate uses of HPC data as a tool to monitor drinking water quality changes following treatment. It finds no evidence to support health-based regulations of HPC concentrations.

Arg343 in human surfactant protein D governs discrimination between glucose and N-acetylglucosamine ligands.

Surfactant protein D (SP-D), one of the members of the collectin family of C-type lectins, is an important component of pulmonary innate immunity. SP-D binds carbohydrates in a calcium-dependent manner, but the mechanisms governing its ligand recognition specificity are not well understood. SP-D binds glucose (Glc) stronger than N-acetylglucosamine (GlcNAc). Structural superimposition of hSP-D with mannose- binding protein C (MBP-C) complexed with GlcNAc reveals steric clashes between the ligand and the side chain of Arg343 in hSP-D. To test whether Arg343 contributes to Glc > GlcNAc recognition specificity, we constructed a computational model of Arg343-->Val (R343V) mutant hSP-D based on homology with MBP-C. Automated docking of alpha-Me-Glc and alpha-Me-GlcNAc into wild-type hSP-D and the R343V mutant of hSP-D suggests that Arg343 is critical in determining ligand-binding specificity by sterically prohibiting one binding orientation. To empirically test the docking predictions, an R343V mutant recombinant hSP-D was constructed. Inhibition analysis shows that the R343V mutant binds both Glc and GlcNAc with higher affinity than the wild-type protein and that the R343V mutant binds Glc and GlcNAc equally well. These data demonstrate that Arg343 is critical for hSP-D recognition specificity and plays a key role in defining ligand specificity differences between MBP and SP-D. Additionally, our results suggest that the number of binding orientations contributes to monosaccharide binding affinity.

Serum SP-D is a marker of lung injury in rats.

Pulmonary surfactant protein D (SP-D) is expressed in alveolar type II and bronchiolar epithelial cells and is secreted into alveoli and conducting airways. However, SP-D has also been measured in serum and is increased in patients with acute respiratory distress syndrome, pulmonary fibrosis, and alveolar proteinosis. To demonstrate that SP-D can be measured in rat serum, we instilled rats with keratinocyte growth factor, which produces type II cell hyperplasia and an increase in SP-D in bronchoalveolar lavage fluid (BALF). To evaluate serum SP-D as a biomarker of lung injury, we examined several injury models. In rats treated with 1 unit of bleomycin, serum SP-D was elevated on days 3, 7, 14, and 28 after instillation, and SP-D mRNA was increased in focal areas as detected by in situ hybridization. However, there was no increase in whole lung SP-D mRNA when the expression was normalized to whole lung 18S rRNA. After instillation of 2 units of bleomycin, the serum levels of SP-D were higher, and SP-D was also increased in BALF and lung homogenates. In another model of subacute injury, serum SP-D was increased in rats treated with paraquat plus oxygen. Finally to evaluate acute lung injury, we instilled rats with HCl; SP-D was increased at 4 h after instillation. Our data indicate that serum SP-D may be a useful indicator of lung injury and type II cell hyperplasia in rats.