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Commercial specifications for a new biotherapeutic product are a critical component of the product's overall control strategy that ensures safety and efficacy. This paper describes strategies for setting commercial specifications as proposed by a consortium of industry development scientists. The specifications for some attributes are guided by compendia and regulatory guidance. For other product quality attributes (PQAs), product knowledge and the understanding of attribute criticality built throughout product development should drive specification setting. The foundation of PQA knowledge is an understanding of potential patient impact through an assessment of potency, PK, immunogenicity and safety. In addition to PQA knowledge, the ability of the manufacturing process to consistently meet specifications, typically assessed through statistical analyses, is an important consideration in the specification-setting process. Setting acceptance criteria that are unnecessarily narrow can impact the ability to supply product or prohibit consideration of future convenient dosage forms. Patient-centric specifications enable appropriate control over higher risk PQAs to ensure product quality for the patient, and flexibility for lower risk PQAs for a sustainable supply chain. This paper captures common strategic approaches for setting specifications for standard biotherapeutic products such as monoclonal antibodies and includes considerations for ensuring specifications are patient centric.

A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Early-phase specifications are established to ensure that materials used in clinical studies have appropriate product quality, reducing the risk of harm to patients. Currently, guidance is available for specification setting practices at commercial phase. With very limited data and manufacturing experience available, it is not possible to fully align to these expectations at the start of clinical trials. A survey was performed among 19 biopharmaceutical companies to gather information about the current practices for setting specifications in early-phase development. The results indicate that most companies develop platform approaches to support setting specifications at the first-in-human clinical trial stage of development. Based on shared learning across multiple companies, example specification approaches for monoclonal antibodies and antibody-drug conjugates are included. General principles of the example specifications can also be applied to other protein therapeutics and vaccines. Strategies for justification of acceptance criteria are described, along with discussion of considerations for some specific tests. Options for use of non-numerical acceptance criteria are also discussed. While specifications for each molecule must be set considering available molecule-specific information, the presented information leverages shared learning from multiple companies, to provide guidance for early phase specification setting strategies.

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.

Treatment of rats with phenobarbital has been shown to result in an enormous increase in CYP2B1/2 mRNA levels in the liver. This study was performed in order to relate this effect of phenobarbital treatment to changes in the chromatin structure of the CYP2B1/2 gene. Phenobarbital treatment was shown to be associated with a twofold increase in DNase I sensitivity of the CYP2B1/2 gene in the liver. In addition, induction of the CYP2B1/2 gene transcription by phenobarbital resulted in a marked enrichment in the liver of CYP2B1/2 sequences in the nuclear matrix as compared to the matrix-free fraction. Control studies with the CYP1A2 gene, of which the transcriptional activity is not influenced by phenobarbital, did not show any changes in both DNase I sensitivity and matrix association upon phenobarbital treatment. Furthermore, after phenobarbital treatment, no changes in DNasel sensitivity or nuclear matrix association of the CYP2B1/2 gene were observed in the kidney. These data indicate a direct, positive correlation between the structure of the rat CYP2B1/2 gene and its activity.

This paper presents kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamster ovary cells incubated with sugar nucleotides. Permeabilized cells released 30 times more oligosaccharide material than metabolically labelled cells, normalized to the amount of labelled glycoprotein acceptor, making this an amenable system for study. Fifteen to forty per cent of the oligosaccharide material released by permeabilized cells was oligosaccharide-phosphate, depending on the nature and amount of the oligosaccharide-lipids synthesized. The oligosaccharide-phosphates released were recovered in the cytosol, and were exclusively Man2Glc-NAc2P and Man5GlcNAc2P, released from oligosaccharide-lipids thought to be facing the cytosol. In contrast, the structures found as neutral oligosaccharide material were similar to those attached to newly synthesized glycoproteins, indicating that the oligosaccharides were subjected to the same processing enzymes whether or not they were protein bound. Importantly, the kinetics of the transfer to protein and the release of free neutral oligosaccharide were parallel, suggesting that the same enzyme was responsible for both processes. Structural analyses demonstrated that the same Man5GlcNAc2 structure was transferred to protein and released as free oligosaccharide. Neutral oligosaccharides were found in both the cytosol and the pellet; however, oligosaccharides with one GlcNAc residue at the reducing end (OS-Gn1) were found exclusively in the supernate. The major neutral oligosaccharide produced after 2 h of metabolic labelling was Man5GlcNAc and it was found in the cytosol.

A significant problem in structure determination of complex carbohydrates, especially for bacterial polysaccharides, is determination of the absolute configuration of the component monosaccharides. A number of analytical methods have been used for this purpose but, as a result of the wide variety of chemical properties of sugars found in complex polysaccharides, no single method is universally applicable. High-resolution gas chromatography of volatile derivatives with chiral reagents is the most widely used method. Optical activity, although direct and simple, lacks sensitivity generally requiring a large quantity of pure monosaccharide. We report a combination of high-performance anion-exchange chromatography (HPAEC) with combined electrochemical pulsed amperometric detection and in-line detection of optical rotation with an in-line laser polarimeter for analysis of a number of sugars found in complex polysaccharides. We show that application of the method for analysis of capsular polysaccharides of several gram-positive and gram-negative pathogenic bacteria provides useful information simultaneously on carbohydrate composition and the enantiomeric configuration of component sugars.

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.

The human epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein having 11 potential N-glycosylation sites in its extracellular domain. N-Glycosylation is needed for proper membrane insertion, EGF binding and receptor functioning. The human epidermoid carcinoma A431 cell line secretes a soluble 105 kDa glycoprotein (sEGFR) that represents the extracellular domain of the membrane-bound form, and its glycosylation pattern has been investigated. After liberation of the oligosaccharides from sEGFR with PNGase F, the glycans were fractionated along different routes, including Concanavalin A affinity chromatography, anion-exchange chromatography, HPLC and high-pH anion-exchange chromatography. The oligosaccharide fractions were characterized by 500- and 600-MHz 1H-NMR spectroscopy and mass spectrometry (FAB, ESI, and MALDI-TOF). The oligomannose-type glycans range from Man5GlcNAc2 to Man8GlcNAc2 and account for 17% of the total carbohydrate moiety. Furthermore, di-, tri'- and tetraantennary complex-type structures are present, both neutral and (alpha2-3)-sialylated (up to tetrasialo), comprising 24 and 59%, respectively, of the total carbohydrate moiety. In this study, 32 new complex-type glycans are characterized containing the Le(x), Le(Y), and sialyl-Le(x) determinants, the bloodgroup A and H antigens, as well as the ALe(Y) determinant. This first comprehensive glycosylation study on a human nonrecombinant receptor shows the immense heterogeneity of the glycosylation of sEGFR.

N-Glycans linked to the human secreted form of epidermal growth factor receptor were isolated from A431 cells after swainsonine treatment. Analysis of the oligosaccharides by (1)H NMR spectroscopy and mass spectrometry shows the presence of oligomannose- and (alpha2-3)-sialylated hybrid-type glycans. The major hybrid-type oligosaccharide chains are fucosylated at the Asn-bound GlcNAc residue. Smaller amounts of the hybrid-type structures are also fucosylated at peripheral GlcNAc residues, constituting the sialyl-Le(x) antigen. No complex-type glycans are found, suggesting the absence of alpha-mannosidase III. An assay for alpha-mannosidase III on the A431 cells in the absence and presence of 6 microM swainsonine shows that Man(5)GlcNAc(2) is not converted into Man(3)GlcNAc(2), thereby confirming that these cells do not contain alpha-mannosidase III activity.

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.