b型流感嗜血杆菌结合疫苗冻干剂型的研究

对b型流感嗜血杆菌结合疫苗冻干剂型进行了研制。选用2.5%的甘氨酸作为稳定剂对疫苗进行冻干,对冻干疫苗的抗原性、免疫原性和安全性等理化特性和免疫学特性进行了检定,并与同类疫苗进行比较。各项检定结果及系统观察表明:冻干疫苗安全性良好,抗原性及免疫原性没有发生改变。与同类疫苗相比,针对HibCPS抗原的IgG抗体及针对TT抗原的抗体水平具有相同的变化趋势。冻干疫苗稳定性较冻干前明显变好,效期可延长至3年;不仅有利于保存和运输,而且方便与其他疫苗联合使用。     

冻干甲型肝炎减毒活疫苗的研制

研制冻干甲肝减毒活疫苗,提高疫苗的稳定性,便于保存及运输,保证疫苗的接种效果.采用CA-9冻干保护剂,按13.5的比例加入疫苗,制备冻干疫苗.冻干甲肝减毒活疫苗具有与液体疫苗相同的安全性及免疫原性.在2～8℃保存的有效期较液体疫苗的3～5个月提高到18个月.在室温和37℃保存的稳定性也明显提高.冻干甲肝减毒活疫苗的稳定性良好,无需低温保存、冷链运输,便于甲肝疫苗的大规模推广应用.     

冻干风疹活疫苗生产工艺研究

文报告了冻干风疹减毒活疫苗生产工艺及疫苗研制结果。在对日本化学血清疗法研究所疫苗生产株—松叶株全面检定的基础上,通过对原代兔肾细胞培养条件及病毒培养条件的试验优化,建立了疫苗生产工艺并制备出了冻干风疹活疫苗制剂,稳定性试验表明其安全有效、质量稳定可靠,而且其生产工艺切实可行、投入产出率高。     

冻干水痘减毒活疫苗转瓶生产工艺的建立

应用旋转培养的方法,建立冻干水痘减毒活疫苗的生产工艺。选择长成致密单层的2BS细胞,接种带状疱疹病毒Oka株,待细胞病变达75％以上时,收获病毒液,经超声破碎、离心、澄清,冻干后,按常规检定,疫苗各项检定符合《WHO水痘活疫苗规程》及《冻干水痘减毒活疫苗制造及检定试行规程》要求。与克氏瓶相比,应用旋转培养,不但提高了疫苗单产,降低了牛血清蛋白残留量,而且疫苗质量也保持稳定。     

容量滴定法和库伦滴定法测定冻干疫苗水分含量的比较

目的在适宜的温度和湿度条件下,评价容量滴定法和库伦滴定法测定冻干疫苗水分是否具有差异性。方法确定冻干疫苗水分测定的适宜温度和湿度条件,在此条件下,分别从水分含量区间、冻干疫苗类型和进样量三个主要影响方面,对容量滴定法(volumetric titration)和库伦滴定法(coulometric titration)进行比较。结果冻干疫苗水分测定的适宜条件为:温度25℃、湿度45%。冻干疫苗水分的质量分数为1.0%～2.0%时,容量滴定法和库伦滴定法的检测结果差异有统计学意义(P0.05);水分的质量分数在2.0%～3.0%时,两种滴定法的检测结果差异无统计学意义(P0.05)。对于不同类型冻干疫苗和不同进样量的冻干疫苗,两种滴定法检测结果差异均无统计学意义(P均0.05)。结论在适宜的温度和湿度条件下,库伦滴定法更适用于低水分含量冻干疫苗的水分测定;其他情况下两种滴定法无明显差异。     

病毒活疫苗冻干保护剂筛选研究

为提高冻干病毒性活疫苗的成品滴度和稳定性及提高疫苗生产的出品率,本研究通过对多种保护剂成分如明胶、山梨醇、蔗糖、乳糖、右旋糖苷、精氨酸等及其配比的大量反复筛选试验,已初步选定了数种可供选择并进一步优化完善的冻干保护剂配方。与现行冻干保护剂相比,其疫苗出品率在相同投入下可提高三倍。     

冷冻干燥对重组酵母乙型肝炎疫苗效力(抗原性)的影响

重组酵母乙型肝炎疫苗(酵母乙肝疫苗)加入保护剂后冷冻干燥,观察是否影响疫苗抗原性和稳定性。按5％和1％的比例,在酵母乙肝疫苗原液中分别加入蔗糖和明胶,低温冷冻干燥,制备3批冻干疫苗,分别进行体外相对效力和小鼠ED50测定。加保护剂冻干后酵母乙肝疫苗体外相对效力和小鼠ED50值较冻干前改变不大,而4℃放置和热加速后的稳定性均优于液体疫苗。因此现有酵母乙肝疫苗加入保护剂冻干后,显著提高了疫苗的稳定性,为进一步制备稳定的疫苗参考品打下了初步基础。     

冻干腮腺炎活疫苗细胞培养生产工艺研究

报告了冻干腮腺炎活疫苗细胞培养生产工艺研究结果,通过对鸡胚疫苗株的适应培养及对细胞培养生产工艺的试验优化,建立了连续细胞培养多次收获疫苗生产工艺并制备出了冻干细胞培养腮腺炎活疫苗制剂。本生产工艺切实可行,生产成本低、投入产出率高,所用原材料规范、质量易于控制,具有明显的技术优势。生产的冻干疫苗制剂质量稳定可靠,符合中国生物制品规程要求,有利于预防腮腺炎的规模化推广使用     

麻疹疫苗生产连续培养多次收获工艺研究

本研究对连续培养多次收获工艺用于麻疹疫苗生产的可行性、疫苗维持液保护剂、冻干保护剂及冻干过程等进行了大量反复试验,结果表明在现行条件下,本生产工艺具有重复性好、成本低、投入产出率高、易于质量控制等优势。对本工艺生产疫苗进行全面检定,表明成品滴度和稳定性试验指标等均高于９０版《生物制品规程》要求,并在试验基础上制定出了生产工艺流程。     

水痘疫苗冻干保护剂的改进

明胶是水痘减毒活疫苗中常用的冻干赋形剂组分,是源于动物组织的大分子蛋白质。由于明胶可能引起疫苗接种后的不良反应,且与某些民族的宗教信仰相冲突,近年来WHO建议减少和避免明胶在病毒疫苗中的使用[1]。我国目前在疫苗冻干保护剂方面的研究与国外还有相当大的差距,明胶作为病毒性疫苗的冻干保护剂组分仍然广泛使用,但目前国内还没有相应的药用辅料标准,因此必须尽快建立相应的质量标准;同时还应对疫苗中明胶含量、分布情况、以及对病毒的稳定性研究和替代物等方面进行全面的研究,以便将来在疫苗生产中取消明胶的添加。本文主要对常规使用的冻干保护剂1和不含明胶的冻干保护剂2进行对比,以便筛选出更适合的水痘疫苗保护剂。     

动态浊度法检测冻干甲型肝炎减毒活疫苗细菌内毒素含量的研究

探讨动态浊度法检测冻干甲型肝炎减毒活疫苗细菌内毒素含量的可行性。参照《中国药典》2015年版通则1143细菌内毒素检测法中动态浊度法,对甲肝疫苗进行标准曲线可靠性试验、干扰初筛试验、干扰验证试验及内毒素含量的测定,同时与经凝胶法检定合格的同批疫苗进行比较。标准曲线可靠性试验结果符合规定。干扰初筛试验疫苗稀释160、320及640倍,回收率在50%~200%之间,均无干扰,符合要求。干扰验证试验结果进一步表明:疫苗稀释160倍对试验无干扰作用。采用动态浊度法检测的10批甲肝疫苗细菌内毒素含量均小于该疫苗的限值L=20 EU/m L,且与经凝胶法检定的同批疫苗结果一致。采用动态浊度法检测甲肝疫苗细菌内毒素含量是可行的,值得推广应用。     

Near‐infrared spectroscopic evaluation of lyophilized viral vaccine formulations

This article examines the applicability of near‐infrared spectroscopy (NIRS) to evaluate the virus state in a freeze‐dried live, attenuated vaccine formulation. Therefore, this formulation was freeze‐dried using different virus volumes and after applying different pre‐freeze‐drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze‐dried product was measured directly after freeze‐drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300–4000 cm^?1 region containing the amide A/II band which might reflect information on the coated proteins of freeze‐dried live, attenuated viruses; and (ii) the C–H vibration overtone regions (10,000–7500 and 6340–5500 cm^?1) which might supply information on the lipid layer surrounding the freeze‐dried live, attenuated viruses. The different pre‐freeze‐drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS‐DA) were developed and evaluated, allowing classification of the freeze‐dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1573–1586, 2013     

The Preservation of Plant Pathogenic Bacteria

Some 160 cultures were preserved by freeze drying, under mineral oil and in soil. After storage for 5 years all freeze dried cultures were viable; most cultures of xanthomonads were viable under oil and in soil; pseudomonads survived well in soil but only moderately well under oil; soft-rotting Erwinia spp. survived poorly but storage under oil was better than in soil; other Erwinia spp. and most Corynebacterium spp. survived well in soil and under oil. The mean half lives in years ( h ) calculated for freeze dried cultures of groups of closely related bacteria were: Erwinia 'chrysanthemi group', 0·40; Erwinia 'carotovora group', 0·51; Pseudomonas 'syringae group', 0·50; Xanthomonas spp., 0·84 years. Estimated half lives for Corynebacterium spp. ranged from 1·8 to 6·5 years. There was no evidence that bacteria which had been in culture for more than 3 years before being freeze dried had a longer storage life than those freeze dried within 3 years of isolation. Cultures of the Pseudomonas 'syringae group'had a longer storage life when freeze dried by Greaves'method ( h = 0·73) than when freeze dried by Annear's method ( h =0·50). There appeared to be no general correlation between half life in storage and either the proportion of cells surviving the freeze drying process or the viable cell count immediately after freeze drying. Most of the variation in the results could be attributed to variation in viable cell count between different ampoules of the same batch of a culture.     

FTIR spectroscopy for the detection and evaluation of live attenuated viruses in freeze dried vaccine formulations

This article examines the applicability of Fourier Transform Infrared (FTIR) spectroscopy to detect the applied virus medium volume (i.e., during sample filling), to evaluate the virus state and to distinguish between different vaccine doses in a freeze dried live, attenuated vaccine formulation. Therefore, different formulations were freeze dried after preparing them with different virus medium volumes (i.e., 30, 100, and 400 µl) or after applying different pre‐freeze‐drying sample treatments (resulting in different virus states); i.e., (i) as done for the commercial formulation; (ii) samples without virus medium (placebo); (iii) samples with virus medium but free from antigen; (iv) concentrated samples obtained via a centrifugal filter device; and (v) samples stressed by 96h exposure to room temperature; or by using different doses (placebo, 25‐dose vials, 50‐dose‐vials and 125‐dose vials). Each freeze‐dried product was measured directly after freeze‐drying with FTIR spectroscopy. The collected spectra were analyzed using principal component analysis (PCA) and evaluated at three spectral regions, which might provide information on the coated proteins of freeze dried live, attenuated viruses: (i) 1700–1600 cm^?1 (amide I band), 1600–1500 cm^?1 (amide II band) and 1200–1350 cm^?1 (amide III band). The latter spectral band does not overlap with water signals and is hence not influenced by residual moisture in the samples. It was proven that FTIR could distinguish between the freeze‐dried samples prepared using different virus medium volumes, containing different doses and using different pre‐freeze‐drying sample treatments in the amide III region. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1107–1118, 2015     

Evaluation of hydrogen peroxide efficacy against AZD1222 chimpanzee adenovirus strain in the recombinant COVID-19 vaccine for application in cleaning validation in a pharmaceutical manufacturing industry

This study aimed to evaluate the performance of hydrogen peroxide vapour (HPV) to inactivate the chimpanzee adenovirus AZD1222 vaccine strain used in the production of recombinant COVID-19 vaccine for application in cleaning validation in pharmaceutical industries production areas. Two matrixes were tested: formulated recombinant COVID-19 vaccine (FCV) and active pharmaceutical ingredient (API). The samples were dried on stainless steel and exposed to HPV in an isolator. One biological indicator with population >10⁶ Geobacillus stearothermophilus spores was used to validate the HPV decontamination cycle as standard. HPV exposure resulted in complete virus inactivation in FVC (≥5·03 log₁₀) and API (≥6·40 log₁₀), showing HPV efficacy for reducing chimpanzee adenovirus AZD1222 vaccine strain. However, the optimum concentration and contact time will vary depending on the type of application. Future decontamination studies scaling up the process to the recombinant COVID-19 vaccine manufacturing areas are necessary to evaluate if the HPV will have the same or better virucidal effectivity in each specific production area. In conclusion, HPV showed efficacy for reducing AZD1222 chimpanzee adenovirus strain and can be a good choice for pharmaceutical industries facilities disinfection during recombinant COVID-19 vaccine production.     

Rheological properties of suspensions containing cross-linked starch nanoparticles prepared by spray and vacuum freeze drying methods

The rheological behavior of suspensions containing vacuum freeze dried and spray dried starch nanoparticles was investigated to explore the effect of these two drying methods in producing starch nanoparticles which were synthesized using high pressure homogenization and mini-emulsion cross-linking technique. Suspensions containing 10% (w/w) spray dried and vacuum freeze dried nanoparticles were prepared. The continuous shear viscosity tests, temperature sweep tests, the frequency sweep and creep-recovery tests were carried out, respectively. The suspensions containing vacuum freeze dried nanoparticles showed higher apparent viscosity within shear rate range (0.1-100s(-1)) and temperature range (25-90°C). The suspensions containing vacuum freeze dried nanoparticles were found to have more shear thinning and less thixotropic behavior compared to those containing spray dried nanoparticles. In addition, the suspensions containing vacuum freeze dried particles had stronger elastic structure. However, the suspensions containing spray dried nanoparticles had more stiffness and greater tendency to recover from the deformation.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

Application of Chitosan in Veterinary Vaccine Production

Probability-based surveys of the application of chitosan succinate as an adjuvant added to the vaccine against animal necrobacteriosis have been performed. The addition of 3.7% chitosan succinate (MM 330 kDa) to the standard-dose vaccine formulation could contribute to improvement of the vaccine immunogenicity, as measured by the hemagglutination test (HA): 1 : 512 as compared to 1 : 128 in the control range. The use of 0.5-% chitosan succinate solution as a protective medium for drying in the production of a dried inactivated vaccine against infectious bovine rhinotracheitis could ensure the preservation of the biopreparation’s high immunogenic activity. Chitosan succinate was shown to have quite good solubility in water, allowing the possible use of saline solution to reconstitute the vaccine into the suspension for injection and to abandon expensive solvents.     

EVALUATION OF PIGMENT EXTRACTION METHODS AND A RECOMMENDED PROTOCOL FOR PERIPHYTON CHLOROPHYLL a DETERMINATION AND CHEMOTAXONOMIC ASSESSMENT1

Many methods have been proposed to extract and quantify algal pigments. Comparative studies have found that pigment extraction efficiency varies among solvent and mechanical disruption protocols due to differential cellular resistance, thereby, leading to potential misinterpretation of pigment data. When the type or resistance of algae are unknown, a method is required that efficiently extract pigments from all taxonomic groups. The objective of this study was to develop a simple and efficient one stage periphyton pigment extraction protocol by comparing the extractability of four solvents (acetone, methanol, methanol/acetone, and methanol/acetone/N,N‐dimethylformamide), the effects of grinding, and the effects of freeze‐drying. The best overall extraction was obtained using freeze‐dried samples extracted with methanol/acetone/DMF/water (MAD). Eighty‐six percent more chlorophyll was extracted when the sample was freeze‐dried relative to fresh/frozen samples extracted with 90% acetone. Freeze‐drying greatly improved the extraction of both polar and non‐polar (lipophilic/hydrophobic) pigments while MAD increased the extractability of polar pigments and improved peak resolution of all pigments. Chemotaxonomic assessment differed between samples that were fresh/frozen or freeze‐dried before extraction. The relative abundance of cyanobacteria was greater for freeze‐dried material compared with fresh/frozen due to the improved extractability of cyanobacterial pigments. Based on the results of this study, the traditional approach of 90% acetone as a solvent is not recommended for periphyton samples containing cyanobacteria or when the composition of the mat is unknown. The combination of freeze‐drying and MAD was sufficient for the extraction of pigments from a periphyton mat containing filamentous cyanobacteria, green algae, and diatoms.     

Survival of Freeze Dried Bacterial Cultures

The survival of 100 strains of bacteria, representing 15 genera, on freeze drying and during storage in the freeze dried state for 10 years has been assessed. Gram-positive organisms tended to survive better than Gram-negative. Viable organisms were recovered from all but one of the cultures.