Subcutaneous Injection Site Pain of Formulation Matrices.

PURPOSE: The objective of this work was to systematically evaluate the effects of formulation composition on subcutaneous injection site pain (ISP) using matrices comprising of common pharmaceutical excipients. METHODS: Two randomized, blinded, crossover studies in healthy subjects were conducted at a single site, where subjects received 1 mL SC injections of the buffer matrices. ISP intensity was measured using a 100 mm visual analogue scale (VAS), which was then analyzed via heatmap, categorical grouping, subgroup analysis, and paired delta analysis. RESULTS: Buffer type, buffer concentration and tonicity agent showed a substantial impact on ISP. Citrate buffer demonstrated a higher ISP than acetate buffer or saline). The 20 mM citrate buffer was more painful than 10 or 5 mM citrate buffers. NaCl and propylene glycol were significantly more painful than sugar alcohols (mannitol, sucrose, trehalose or glycerol). Histidine buffers exhibited ISP in the descending order of 150 mM > 75 mM > 25 mM > 0 mM NaCl, while histidine buffers containing Arginine-HCl at 0, 50, or 150 mM all showed very low ISP. Histidine buffer at pH 6.5 showed a lower ISP than pH 5.7. CONCLUSIONS: This systematic study via orthogonal analyses demonstrated that subcutaneous ISP is significantly influenced by solution composition.

A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies.

Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection, and quantitation purposes is most commonly used to monitor HCP removal during process development, but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs in biopharmaceutical samples. We describe a simple and powerful methodology to characterize residual HCPs in (monoclonal) antibodies by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics approach. Differing from the traditional methodology, the sample preparation approach maintains nearly intact antibody while HCPs are digested. Thus, the dynamic range for HCP detection by MS is 1 to 2 orders of magnitude less than the traditional trypsin digestion sample preparation procedure. HCP spiking experiments demonstrated that our method could detect 0.5 ppm of HCP with molecular weight >60 kDa, such as rPLBL2. Application of our method to analyze a high-purity NIST monoclonal antibody standard RM 8670 derived from a murine cell line expression system resulted in detection of 60 mouse HCPs; twice as many as previously reported with 2D-UPLC/IM/MSE method. A control monoclonal antibody used for 70 analyses over 450 days demonstrated that our method is robust.

Endotoxin recovery using limulus amebocyte lysate (LAL) assay.

A phenomenon initially reported by Chen and Vinther in 2013 [1], and now commonly referred to as low endotoxin recovery (LER), has prompted the Food and Drug Administration (FDA) to request specific data demonstrating the capability of the LAL BET method (i.e., USP <85>) to recover endotoxin from spiked samples over time. The results of these spike/hold recovery studies are expected to be included in the Biologics License Applications (BLA) for review by the Center for Drug Evaluation and Research (CDER) Hughes (2014) and Hughes et al. (2015) [2,3]. Such studies involve spiking a known amount of a surrogate endotoxin, such as purified lipopolysaccharide (LPS), into undiluted biological products and then testing at different time points to determine the recovery over time. We report here the experience and learning gained from conducting spike/hold recovery studies for a monoclonal antibody (Mab) product. Results from initial hold studies spiked with purified LPS showed rapid loss of endotoxin activity in the drug substance (DS) and significant batch-to-batch variation in the drug product (DP). After careful review and examination of the experimental details, it was determined that the study design and execution differed from the routine batch release USP <85> BET method with regard to mixing time and sampling scheme. The hold study design was subsequently revised so that the mixing time and sampling were the same as the verified USP <85> BET method used for routine batch release testing. The spike/hold recovery studies were repeated and the results demonstrated that LPS could be consistently recovered over time. These findings highlight the importance of carefully controlling sample preparation procedures in a spike/hold recovery study in order to demonstrate the suitability of using the LAL BET method for endotoxin detection.

Method for characterization of PEGylated bioproducts in biological matrixes.

PEGylation of peptides and proteins has been widely used to enhance stability and reduce immunogenicity of biotherapeutics. Characterizing the degradation of these PEGylated products in biological fluids can yield essential information to support pharmacokinetic evaluations and provide clues about their in vivo properties useful for further molecular optimization. In this paper, we describe a novel and uncomplicated approach to characterize PEGylated peptides or proteins and their related degradation products in biological matrixes. The method involves direct liquid chromatography/mass spectrometry (LC/MS) analysis of animal sera containing low nanograms to low micrograms per milliliter of PEGylated product with or without an acetonitrile precipitation sample treatment. Applying the methodology to analyze the model PEGylated peptides, 20K PEGylated-Pancreatic Polypeptide analogue (PPA) and 20K PEGylated-glucagon, we elucidated the decomposition pathways occurring in animal sera. The data provided direct evidence of cleavages within the peptide backbone. The identified degradation products were unambiguously confirmed by tandem mass spectrometry with high-energy C-trap dissociation (HCD) analysis, followed with in-source fragmentation. Additional spiking studies demonstrated nearly full recovery of PEGylated products, linear detection when the spiked concentration of PEGylated product was ≤1000 ng/mL, and a low ng/mL limit of quantitation (LOQ).

Identification of racemization sites using deuterium labeling and tandem mass spectrometry.

Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples. Immunoglobulin G subclasses IgG1, IgG2, and IgG4 samples were first stressed in protonated or deuterated buffer (pH 8 or 9) at 40 or 50 degrees C storage for days or weeks. These forced degraded samples were reduced, S-carbamidomethylated, and digested with trypsin in protonated solution, and the tryptic digests were then analyzed via liquid chromatography/mass spectrometry (LC-MS) or sequenced via liquid chromatography/tandem mass spectrometry (LC-MS/MS) to detect racemized peptides and elucidate the location of racemized amino acid residues. The methodology successfully identified several racemized amino acid residues in the constant region of the heavy chains of the three IgG subclasses. Although the IgG subclasses have very similar primary protein sequences, our results interestingly indicated different racemization rates for specific amino acid residues.

Non-native intermediate conformational states of human growth hormone in the presence of organic solvents.

PURPOSE: Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions. METHODS: The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering. RESULTS: CD data suggested that the secondary structure of hGH remained unchanged up to 50\% (v/v) ethanol, but the tertiary structure was perturbed at a20% ethanol. Fluorescence anisotropy, however, showed that the mobility of the buried Trp residue was restricted even at 30% ethanol, suggesting a differently packed structural core in 30% ethanol relative to the native structure. Consistent with this result, thermal unfolding of hGH in 30% ethanol was more facile compared to that in 0% and 20% ethanol. At >40% ethanol, fluorescence data were consistent with increased solvent exposure of the tryptophan. CONCLUSIONS: The results point to progressive unfolding of hGH that increases solvent exposure of the hydrophobic core as a function of ethanol concentration and suggest that non-native intermediate states are populated in 30-60% ethanol.