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Immunogenicity regulatory guidance and industry recommendations have evolved over the last two decades since unexpected immune reactions were first reported with erythropoietin. Since then, the guidelines and practices for immunogenicity have stemmed from a reaction to a high-risk molecule causing significant clinical impact. Similar thinking is often applied to all biotherapeutic drugs, even when a well-defined risk assessment suggests otherwise. In recent years, the current testing paradigm for immunogenicity has been challenged with more informative approaches being proposed. In a Focus Workshop held by the European Bioanalysis Forum in September 2023, the current immunogenicity testing paradigm was challenged based on the experience and learning of 20+ years of immunogenicity strategies. The workshop recommendations proposed a new paradigm, challenging the value of multiple tiers depending on the immunogenicity risk assessment based on context of use and moving toward treating immunogenicity as a pharmacodynamic biomarker for the drug. Such rethinking ultimately results in the appropriate and efficient focusing of resources on immunogenicity testing strategies that benefit patients most, moving to a new paradigm where implementation of appropriate and truly informative immunogenicity testing strategies, depending on the context-of-use, become the norm .

The European Bioanalysis Forum, in collaboration with several key industry stakeholders, has recently led discussions that address international immunogenicity guidance documents, specifically the three tier approach for immunogenicity testing strategies, after more than 20 years of experience with biotherapeutics. As part of this, the strategy and methods used to assess drug tolerance across all immunogenicity assays are challenged, emphasizing that bioanalytical scientists need to consider the context-of-use of each assay. Here, recommendations for drug tolerance assessments, driven by strong scientific rationale and subject to reevaluation as needed, are provided. This includes carefully considering the drug and positive control concentrations considered to be appropriate and which tiers are most relevant for performing drug tolerance assessments.

A new mechanism is described for DNA amplification using nucleic acid sequence-based amplification (NASBA) including a restriction enzyme digestion and P1 primer binding directly upstream of the digestion. For hepatitis B virus (HBV), herpes simplex virus (HSV) and methicillin resistant Staphylococcus aureus (MRSA) DNA, which all show very poor amplification with normal NASBA, assay sensitivity was improved by a factor 100-1000 when restriction enzyme digestion was performed prior to amplification. For the quantitative HBV assay, in combination with the NucliSENS Extractor (bioMérieux, Boxtel, The Netherlands), a 95% target detection rate of 242 WHO IU/ml and 50% detection rate of 35 WHO IU/ml was achieved. The lowest detectable HBV concentration was 10 WHO IU/ml. HBV DNA could be quantified with an algorithm comparable to that used for RNA quantitation and by using a two step approach a dynamic range of 10(2)-10(9)WHO IU/ml (>6 log) was shown to be quantifiable. For the qualitative HSV assay, in combination with the NucliSENS miniMAG (bioMérieux, Boxtel, The Netherlands), the 95% detection rate was determined to be 84 and 138 copies/isolation for HSV 1 and HSV 2, respectively, which corresponds to approximately 10 copies per amplification for both targets. For MRSA, the limit of detection was <10 equivalent CFU per amplification.

A model of the alveolar air-fluid boundary was developed for a future study of the influence of several gases and gas mixtures on the surfactant film of the lung. Since the surface behaviour of the film material changed after denaturation, we had to use it in its native state. It was collected as foam during lung lavage and applied upon a hypophase of concentrated lavage fluid. This combination showed a constant physical behaviour during repeated compression and expansion of its surface. After the excess of applied material had left the surface there was hardly any change of gammamin and gammamax or of the shape of the area versus surface-tension curve (A-gamma curve) during several hours. Mean values of gammamin and gammamax were 5.5 +/- 2.0 and 55.0 +/- 2.0 dyne/cm respectively. The mean value of the hysteresis area was 847 +/- 82 dyne cm. Its shape was similar in all experiments. The stability of this model makes it useful for the intended investigations, but also seems to offer useful data for calculations in relation to the actual behaviour of lung alveolar surfactant in vivo.

A model of the gas-liquid barrier (surfactant film) of the lung was used to investigate the influence of temperature and of humidification of the gas upon the behaviour of films during rhythmic compression and expansion of their surface. Film behaviour was studied at room temperature or 37 degrees C, and at ambient humidity or high relative humidity. Changes of minimal surface tension (gamma min) and maximal surface tension (gamma max) were followed up to 1000 cycles. At 37 degrees C we measured higher values for gamma min and lower values for gamma max. At room temperature complete humidification does not influence surface tension, but at 37 degrees C gamma min increases. Thus both high temperature and high relative humidity result in sharp rise of gamma min, whereas high temperature alone is responsible for the temporary drop of gamma max. Considering these effects, investigations to study influences of foreign gases and of air pollution upon the surfactant film should be performed under conditions which approach those of the lung as close as possible. The possible influence of CO2 is discussed.

Surface tension (s.t.) of lung surfactant (l.s.) was measured at 37 degrees C in a closed box, filled with completely humidified gas (mixtures) such as room air, O2, N2, or alveolar gas. The film was compressed to 20% of the original area and expanded, with cycling times of 10 to 20 sec. Switching from one gas mixture to another nearly always caused a fall of s.t., but in general only temporarily. Only a gas phase containing CO2, like alveolar gas, caused a permanent fall of s.t. and a rise after its withdrawal. This was not due to a direct influence of CO2, but rather to the induced change of hypophase pH; the reaction of s.t. at end-expansion (gamma max) decreased and that at end-compression (gamma min) disappeared with a buffered hypophase. By using buffered or unbuffered hypophases, we were able to create pH values in a wide range and relate them to s.t. and CO2 concentration. With pH 5.5, the mean values of s.t. at end-compression and end-expansion were 20.2 and 51.2 mN/m, respectively. They reached the significantly higher values of 25.3 and 55.4 mN/m with pH = 8.5. With 5.6% CO2 in the atmosphere, which is comparable to alveolar gas, gamma min and gamma max had mean values of 22.9 and 52.8 mN/m, respectively. Such values correspond with a pH in the range of 6.7 to 7.1, which is supposed to be the range of pH of the pulmonary interstitium.