Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition.     

Prey preference and egg production of the carabid beetleAgonum dorsale

In order to elucidate whether the aphidRhopalosiphum padi is low quality food for the carabid beetleAgonum dorsale, as it has previously been found to be for cereal spiders, we performed a series of experiments using fruit fliesDrosophila melanogaster as a standard alternative prey (‘control prey’): 1) Prey preference, 2) Aphid consumption for satiated and starved beetles, 3) Egg production on different diets and 4) Preference experiments with fruit flies coated with a taste of different prey types. Other alternative prey types used were earthworms and sciarid midges, all potential prey in the field.A. dorsale has a low preference for aphids compared to fruit flies. Apparently there is an upper limit to the consumption of aphids independent of hunger and much below the beetles' total food demand. Egg production on a pure diet of aphids is lower than on a pure diet of fruit flies; it is extremely low on a pure diet of earthworms; the highest fecundity is found on a mixed diet. As fruit flies coated with a taste of aphids or sciarid midges are less prefered than flies covered with a taste of fruit flies there may be a chemical factor, acting through taste, involved in determining prey preference.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

The neuroprotective effect of acute moderate alcohol consumption on caspase-3 mediated neuroapoptosis in traumatic brain injury: The role of lysosomal cathepsin L and nitric oxide

Our aim in this study was to investigate the effect of moderate acute alcohol administration on cysteine protease mediated neuronal apoptosis and nitric oxide production in the traumatic brain injury. A total of 29 adult Sprague–Dawley male rats weighing 250–300 g were used. The rats were allocated into four groups. The first group was the control (sham-operated) group in which only a craniotomy was performed, the others were alcohol, trauma and trauma + alcohol groups. Caspase-3 enzyme activity in the trauma group increased significantly in comparison with the control group. The alcohol given group showed a decreased caspase-3 enzyme activity compared to the trauma group. The level of caspase-3 enzyme activity in the alcohol + trauma group decreased in comparison to the trauma group. SF/FEL ratio of cathepsin-L enzyme activity in the trauma group was significantly higher than in the control group. Our results indicate that moderate alcohol consumption may have protective effects on apoptotic cell death after traumatic brain injury. Protective effects of moderate ethanol consumption might be related to inhibition of lysosomal protease release and nitric oxide production.     

Pharmacological undertreatment of coronary risk factors in patients with psoriasis: observational study of the Danish nationwide registries

Background

Patients with psoriasis have increased prevalence of coronary risk factors and limited recent results have suggested that these risk factors are undertreated in patients with psoriasis. This may contribute to the increased risk of cardiovascular diseases observed in patients with psoriasis.

Objective

To examine the pharmacological treatment of coronary risk factors in patients with severe psoriasis treated with biologic agents in a real-world setting.

Methods and Findings

Medical history of patients with severe psoriasis treated with biologic agents in the time period 2007–09 was retrieved from a Danish nationwide registry (DERMBIO). Individual-level linkage of nationwide administrative registries of hospitalizations, concomitant medications, and socioeconomic status was performed to gain insights into the use of pharmacological treatment. A total of 693 patients (mean age 46.1±12.7 years, 65.7% male) with severe psoriasis treated with biologic agents were identified. Hypertension, hypercholesterolemia, and diabetes mellitus were identified in 16.6%, 9.2%, and 6.7% of cases, respectively. Patients with severe psoriasis were significantly less likely to receive cardiovascular pharmacotherapy compared to age, sex, and coronary risk factor matched controls. In psoriatic patients with hypertension 27.7% received no antihypertensive pharmacotherapy. Patients with dyslipidemia received cholesterol-lowering medications in 55.8% of cases and patients with diabetes mellitus received angiotensin converting enzyme inhibitors/angiotensin II receptor blockers and cholesterol-lowering medications in 42.1% and 23.7% of cases, respectively. Similar results were found for the subset of patients with >1 coronary risk factor and for high risk patients with established atherosclerotic disease.

Conclusion

This nationwide study of patients with severe psoriasis demonstrated substantial undertreatment of coronary risk factors. Increased focus on identifying cardiovascular risk factors and initiation of preventive cardiovascular pharmacotherapy in patients with psoriasis is warranted.     

A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

Pseudomonas aeruginosa produces extracellular DNA which functions as a cell-to-cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular beta-galactosidase released from lacZ-containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI, pqsA, pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber-grown wild-type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria and the cap-forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm.     

The Interaction of Equine Lysozyme:Oleic Acid Complexes with Lipid Membranes Suggests a Cargo Off-Loading Mechanism

The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to α-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of α-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human α-lactalbumin made lethal to tumor cells. Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and “soft” lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with bovine serum albumin, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA, which shifts towards free OA as ELOA is progressively diluted, indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA towards a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein.     

环境噪声对临安和阜阳两地白头鹎鸣声频率的影响

为了解环境噪声对白头鹎(Pycnonotus sinensi)鸣声频率的影响,在浙江临安和安徽阜阳两地分别对白头鹎在高低噪声水平环境中的鸣声做了研究。把用数码录音机记录到的鸣声输入计算机,利用计算机声谱分析系统进行分析,再对每一鸣声的每个音节的主频进行Mann—Whitney U检验。结果显示：高噪声水平区和低噪声水平区相比,在两地白头鹎每一鸣声的各音节主频中,最低和最高主频以及第I、Ⅱ音节的主频都有显著提高;在阜阳第Ⅲ音节的主频也有显著提高。这表明白头鹎可以通过提高声音频率来避免环境噪声对鸣声的影响,从而保证噪声环境中声信息的有效传递;而噪声对各音节的不同影响也表明,同一鸣句的各音节,对于噪声通讯中的信息识别,具有不同的地位。