Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics

In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.

Specific Protein Properties of Setcreasea pupurea Boom under Copper Stress

[Objective] This study was to investigate the expression of the specific protein in Setcreasea purpurea Boom under copper stress, with the aim to clarify the copper tolerance mechanism of S. purpurea. [Method] Methods of water culture, elec- trophoresis and chromatography were used to analyze the molecular weight of the specific protein in the copper hyperaccumulator S. purpurea, as well as its expression time and the minimum copper concentration for the expression. And the specific protein was isolated and purified. [Result] Under copper stress, the minimum concentra- tion of copper to induce the expression of the specific protein from S. purpurea was 50 umol/L, and the expression time of the protein was in the 4th week with the molecular weight of 89.4 kDa. [Conclusion] The results show that the copper tolerance of S. purpurea is closely related with the expression of the specific protein.

Expression and role of specificity protein 1 in the sclera remodeling of experimental myopia in guinea pigs

AIM：To study the expression of collagen I and transcription factor specificity protein 1（Sp1）,a transforming growth factor-β1（TGF-β1） downstream target,and reveal the impact of the TGF-β1-Sp1 signaling pathway on collagen remodeling in myopic sclera.METHODS：Seventy-five 1-week-old guinea pigs were randomly divided into normal control,form deprivation myopia（FDM）,and self-control groups.FDM was induced for different times using coverage with translucent latex balloons and FDM recovery was performed for 1wk after 4wk treatment;then,changes in refractive power and axial length were measured.Immunohistochemistry and reverse transcription-polymerase chain reaction were used to evaluate dynamic changes in collagen I and Sp1 expression in the sclera of guinea pigs with emmetropia and experimental myopia,and the relationship between collagen I and Sp1 levels was analyzed.RESULTS：In the FDM group,the refractive power was gradually changed（from 2.09±0.30 D at week 0 to-1.23±0.69 D,-4.17±0.59 D,-7.07±0.56 D,and-4.30±0.58 D at weeks 2,4,6,and 1wk after 4wk,respectively;P〈0.05）,indicating deepening of myopia.The axial length was increased（from 5.92±0.39 mm at week 0 to 6.62±0.36 mm,7.30±0.34 mm,7.99±0.32 mm,and 7.41±0.36 mm at weeks 2,4,6,and 1wk after 4wk;P〈0.05）.The m RNA and protein expression of Sp1 and collagen I in the sclera of the FDM group was lower than that of the control groups（P〈0.05）,and the reduction was eye-coverage time-dependent.Furthermore,correlation between Sp1 and collagen I down-regulation in the myopic sclera was observed.CONCLUSION：Our data indicate that transcription factor Sp1 may be involved in the regulation of type I collagensynthesis/degradation during myopic sclera remodeling,suggesting that TGF-β1 signaling plays a role in the development and progression of myopia.

AtKP1, a kinesin-like protein, mainly localizes to mitochondria in Arabidopsis thaliana

Kinesins and kinesin-like proteins (KLPs) constitute a large family of microtubule-based motors that play important roles in many fundamental cellular and developmental processes. To date, a number of kinesins or KLPs have been identified in plants including Arabidopsis thaliana. Here, a polyclonal antibody against AtKP1 (kinesin-like protein 1 in A.thaliana) was raised by injection the expressed AtKP1 specific C-terminal polypeptides in rabbits, and immunoblot analysis was conducted with the affinity-purified anti-AtKP1 antibody. The results indicated that this antibody recognized the AtKP1 fusion proteins expressed in E. coli and proteins of ～125 kDa in the soluble fractions of Arabidopsis extracts. The molecular weight was consistent with the calculated molecular weight based on deduced amino acids sequence of AtKP1. To acquire the subcellular localization of the protein, AtKP1 in Arabidopsis root cells was observed by indirect immunofluorescence microscopy. AtKP1 was localized to particle-like organelles in interphase or dividing cells, but not to mitotic microtubule arrays. Relatively more AtKP1 was found in isolated mitochondria fraction on immunoblot of the subcellular fractions. The AtKP1 protein could not be released following a 0.6 M KI washing,indicating that AtKP1 is tightly bind to mitochondria and might function associated with this kind of organelles.

Secondary Structure and Neurotrophic Effect of a 33.1 kDa Specific Protein (SSP-33.1) in Spinal Sensory Ganglia

Aim To analyze the secondary structure and neurotrophic effect of a specific protein in sensory neurons. Methods Comparison of the proteins expressed in the rat spinal sensory neurons and motor neurons was made by two dimensional electrophoresis. One specific protein in sensory neurons was isolated and purified by DEAE Sephacel ion exchange chromatography and high performance liquid chromatography. A primary analysis of its secondary structure by circular dichroism, and its neurotrophic effects were investigated using the model of dorsal root ganglia(DRG) cultured in vitro . Results The molecular weight and isoelectric point of the protein were 33 1 kDa and 5 52, respectively. Its circular dichroism showed that there were 20 8% α helix, 54 8% β sheet, 7 3% turn, and 17 1% random coil in its secondary structure. Biological experiments showed that the protein could promote the neurite outgrowth of DRG. Conclusion A specific protein in spinal sensory tissue with molecular weight of 33 1 kDa has been purified. There is mainly β sheet in the secondary structure of the protein. And the protein has neurotrophic effects in the model of DRG.

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

Neuron-specific Enclose and Myelin Basic Protein in Cerebrospinal Fluid of Patients with First Episode Schizophrenia

In order to study whether patients with schizophrenia have cerebral injury, neuron-specific enolase （NSE） and myelin basic protein （MBP）in cerebrospinal fluid （CSF） of 33 patients with first episode schizophrenia and 9 from the control group were determined by double antibody sandwich enzyme immunoassay method. The results showed that there was significant difference in the NSE contents between the experimental group and control group （P〈0.01）. The NSE contents in CSF in the experimental group were positively correlated with MBP in schizophrenia patients （P〈 0.05）. These findings suggested that patients with schizophrenia had cerebral injury.

Using position specific scoring matrix and auto covariance to predict protein subnuclear localization

The knowledge of subnuclear localization in eukaryotic cells is indispensable for under-standing the biological function of nucleus, genome regulation and drug discovery. In this study, a new feature representation was pro-posed by combining position specific scoring matrix (PSSM) and auto covariance (AC). The AC variables describe the neighboring effect between two amino acids, so that they incorpo-rate the sequence-order information;PSSM de-scribes the information of biological evolution of proteins. Based on this new descriptor, a support vector machine (SVM) classifier was built to predict subnuclear localization. To evaluate the power of our predictor, the benchmark dataset that contains 714 proteins localized in nine subnuclear compartments was utilized. The total jackknife cross validation ac-curacy of our method is 76.5%, that is higher than those of the Nuc-PLoc (67.4%), the OET- KNN (55.6%), AAC based SVM (48.9%) and ProtLoc (36.6%). The prediction software used in this article and the details of the SVM parameters are freely available at http://chemlab.scu.edu.cn/ predict_SubNL/index.htm and the dataset used in our study is from Shen and Chou’s work by downloading at http://chou.med.harvard.edu/ bioinf/Nuc-PLoc/Data.htm.

Protein phosphatases and chromatin modifying complexes in the inflammatory cascade in acute pancreatitis

Acute pancreatitis is an inflammation of the pancreas that may lead to systemic inflammatory response syndrome and death due to multiple organ failure. Acinar cells, together with leukocytes, trigger the inflammatory cascade in response to local damage of the pancreas. Amplification of the inflammatory cascade requires up-regulation of proinflammatory cytokines and this process is mediated not only by nuclear factor κB but also by chromatinmodifying complexes and chromatin remodeling. Among the different families of histone acetyltransferases, the p300/CBP family seems to be particularly associated with the inflammatory process. cAMP activates gene expression via the cAMP-responsive element (CRE) and the transcription factor CRE-binding protein (CREB). CREB can be phosphorylated and activated by different kinases, such as protein kinase A and MAPK, and then it recruits the histone acetyltransferase co-activator CREB-binding protein (CBP) and its homologue p300. The recruitment of CBP/p300 and changes in the level of histone acetylation are required for transcription activation. Transcriptional repression is also a dynamic and essential mechanism of down-regulation of genes for resolution of inflammation, which seems to be mediated mainly by protein phosphatases (PP1, PP2A and MKP1) and histone deacetylases(HDACs) .Class HDACs are key transcriptional regulators whose activities are controlled via phosphorylationdependent nucleo/cytoplasmic shuttling. PP2A is responsible for dephosphorylation of class HDACs, triggeringnuclear localization and repression of target genes, whereas phosphorylation triggers cytoplasmic localization leading to activation of target genes. The potential benefit from treatment with phosphodiesterase inhibitors and histone deacetylase inhibitors is discussed.

The expression and localization of a novel protein phosphatase inhibitor 2810408A11Rik in mouse testis and sperm

This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique. A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik. The results of the bioinformatic analysi and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis, and 2810408AllRik protein included a protein phosphatase inhibitor domain. Western blotting assays, immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis, especially in post-meiosis round and long spermatids, and that it is localized in the acrosome and the post-nucleus area of sperm. Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis, sperm capacitation and fertilization.

Expression and role of specificity protein 1 and collagenⅠin recurrent pterygial tissues

AIM:To investigate the expression profiles of the transcription factor specificity protein 1(Sp1)and collagenⅠin recurrent pterygial tissues.What is more,to compare the changes of Sp1 and collagen I among primary pterygial tissue,recurrent pterygial tissue and conjunctival tissue.METHODS:In the prospective study,we collected the pterygial tissues of 40 patients who underwent resection of primary pterygial tissue and recurrent pterygial tissue,and the conjunctival tissues of 10 patients with enucleation due to trauma.The relative expression levels of Sp1 and collagen I were analyzed by reverse transcription quantitative-polymerase chain reaction and Western blot.Paired t-test was performed to compare the Sp1 and collagen I of recurrent pterygial tissues,as well as the primary pterygial tissues and conjunctival tissues.In further,Pearson’s hypothesis testing of correlation coefficients was used to compare the correlations of Sp1 and Collagen I.RESULTS:The content of Sp1 and collagen I m RNA and protein was significantly greater in recurrent pterygial tissue than that was in primary and conjunctival tissue(P<0.05).There was a positive correlation between the m RNA and protein levels of Sp1 and collagen I in recurrent pterygial tissues(protein:r=0.913,P<0.05;m RNA:r=0.945,P<0.05).CONCLUSION:Sp1 and collagen I are expressed in normal conjunctival,primary,and recurrent pterygial tissues,but expression is significantly greater in the latter.Sp1 and collagen I may be involved in the regulation of the development of recurrent pterygium.

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer

Specificity protein(Sp)transcription factors(TFs)Sp1,Sp3 and Sp4,and the orphan nuclear receptor 4A1(NR4A1)are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival.Results of knockdown and overexpression of Sp1,Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members.NR4A1 is also a prooncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth,survival,migration and invasion.There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin,epidermal growth factor receptor,PAX3-FOXO1,α5-andα6-integrins,β1-,β3-andβ4-integrins;this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites.Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells,and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.

Modeling the Tensile Properties of Soybean Protein Yarns

The tensile properties of a series of soybean protein yarns are tested in USTER THINKPAID Ⅲ. A nonlinear viscoelastic model has been proposed to describe the tensile behavior of soybean protein yarns. The model is composed of a Maxwell element, a linear spring and a nonlinear spring. The tensile properties of soybean protein yam are analyzed. The stress-strain curves of the yams are fitted. The average breaking tenacity and specific work of rupture are calculated using the average breaking strain. Comparisons indicate that theoretical predictions conform the experimental results very well.

Establishing an ELISA,Based on the HA1 Protein,for Detecting Antibodies against H3N2 Subtype Swine Influenza Virus

The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.

Purification and Analysis of Abscisic Acid-Specifically-Inducible Proteins from Rice Callus

Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65-100% saturated （NH4）2SO4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-electric points （pl） of the proteins with the same molecular mass （24.5 kD） were 6.1 and 6.9, respectively. The Western blot analysis indicated that the proteins expressed in different tissues were obviously different. The A1 （pl 6.1） protein was only detected in calli treated with ABA and seed embryos （SE）. However, the A2 （pl 6.9） protein was found not only in the calli treated with ABA and SE, but also in the white dry callus. Thus it suggested that the two proteins might play some important roles in the processes of seed embryo （or somatic embryo） formation.

Specific Proteins in the Indirect Somatic Embryogenesis of Freesia Refracta

Using the young inflorescence segments of Freesia refracta as explants, indirect somatic embryogenesis of somatic cells was induced in a N6 medium supplemented with some exogenous hormones. SDS-polyacrylamide gel electrophoresis（SDS-PAGE） was used for the analysis of soluble proteins produced during the somatic embryogenesis of this plant. There are six polypeptides, which might play some roles in the process of somatic embryo development. Tltree polypeptides（45, 53 and 55 kD） were detected in the stages of embryogenic callus, globular embryoid, and embryoid with coleoptiles, except the embryoid with leaf. One polypeptide（ 83 kD） was specific for the stages of embryoid with eoleoptiles and embryoid with leaf. One polypeptide（37 kD） was detected in the first two stages, namely, embryogenic callus and globular embryoid. One polypeptide（35 kD） was regularly synthesized in each stage, from embryogenic callus to embryoid with leaf.

Preparation of Polyclonal Antibody against Human MxA Protein and Its Specificity to Diversified Myxovirus Resistant Protein A

Objective To study the human myxovirus resistant protein A （MxA）, a specifically induced peptide by interferon I, and to use its level as a diagnostic criterion for viral infections. Methods Anti-MxA antisera from immunized mice were prepared with the expressed MxA protein of pET32a-MxA in E. coli BL-21（DE3）. To confirm the antiserum activity and specificity, the expression product of BL21, wild type MxA pEGFP-CI-wMxA and site-directed mutant MxA pEGFP-Cl-mMxA（N589S） stably transfected 3T3 cells and induced A549 cells were detected by Western blot with the antisera using non-MxA transfected or non-IFN-[3 induced cells, intact A549, NIH 3T3 cells transfected with pEGFP-CI and pET32a （＋）-transformed BL-21 as controls. Results The antisera had specific positive immunoreactivity to the NIH3T3 cells transformed with pEGFP-CI-wMxA and pEGFP-CI-mMxA, INF-β induced A549 cells and BL21 proteins expressed with pET32a （＋）-MxA. The hybridization signals from IFN-β induced A549 cells depended on the IFN-β inducing concentrations. Meanwhile, immunohistochemical assay showed that NIH 3T3 cells with pEGFP-C 1-wMxA and pEGFP-C 1-mMxA had 〉 98% of positive cells at 1：50 dilution of the serum and A549 cells induced by 20 ng/mL IFN-[3 for 48 h showed 95% positive cells. pEGFP-Cl-transfected NIH 3T3 cells were all negative. Conclusion Anti-sera are highly specific to diversified MxAs. The antibody is detectable by Western blot, immunocytochemistry and immunofluorescence assay.

Detection of cardiac myosin binding protein-C (cMyBP-C) by a phospho-specific PKD antibody in contracting rat cardiomyocytes

Protein phosphorylation plays an important role in physiological processes, such as muscle contraction. Phospho-specific antibodies have become powerful tools to study these processes. Cardiac myosin binding protein-C (cMyBP-C) is one of the proteins that make up the contractile apparatus of cardiomyocytes. Phosphorylation of cMyBP-C is essential for normal cardiac function, since dephosphorylation of this protein leads to its degradation and has been associated with cardiomyopathy. One of the upstream kinases, which phosphorylate cMyBP-C, is protein kinase D (PKD). While studying the role of PKD in cMyBP-C phosphorylation, we tried to analyze phosphorylation of PKD with a phospho-specific PKD-Ser744/748 antibody. Contrary to the expected 115 kDa, a signal was found for a 150-kDa protein. By MALDI-TOF mass spectrometry, we identified this protein to be cMyBP-C. These data were confirmed by immunostaining using the p-PKD-Ser744/748 antibody, which displayed a striated pattern similar to the one observed for a regular cMyBP-C antibody. To our knowledge there are no antibodies commercially available for phosphorylated cMyBP-C. Thus, the p-PKD-Ser744/748 antibody can accelerate research into the role of cMyBP-C phosphorylation in cardiomyocytes.

Effects of Propofol combined with remifentanil on the levels of MBP,NSE and S100B protein,D-D and inflammatory factors in patients with acute craniocerebral trauma

Objective: To investigate the effects of Propofol combined with remifentanil on serum levels of MBP, NSE and S100B protein, D-D and inflammatory factors in patients with acute craniocerebral trauma. Methods: A total of 100 patients were selected with traumatic brain injury who underwent emergency surgery from August 2014 to May 2017 in our hospital, then randomly divided them into the control group and the experimental group, 50 cases each. The control group received isoflurane combined with remifentanil to maintain anesthesia, and the experimental group received propofol and remifentanil to maintain anesthesia. The inflammatory factors and the levels of MBP, NSE, S100B and D-D in the two groups before and after anesthesia (T0), 1H (T1) and postoperative 1H (T2) were detected and compared. Results: There was no significant difference between the two groups in the levels of TNF-α. The serum level of hs-CRP in two groups of T1, T2 increased significantly, the difference was statistically significant compared with T0, in the experimental group, serum level of hs-CRP at T1 and T2 was significantly higher than the control group, the difference was statistically significant. Conclusion: Propofol combined with remifentanil anesthesia for acute craniocerebral trauma can maintain the balance of inflammatory cytokine levels during the perioperative period, inhibit the elevation of serum MBP, NSE, S100B protein and D-D levels, reduce brain cell damage. It has a good protective effect on brain cells and is worthy of clinical application.

Preliminary Identification of Human Nonserum Oviduct Specific Proteins by Using Electrophoresis and Immunoblotting Analysis

The present paper reported the preliminary results of identification of humannonserum oviduct specific proteins. The 1D-SDS-polyacrylamide gel electrophoresis (PAGE) and 2D-SDS-PAFE in conjunction with the immunoblotting assay were used in the present study. The results showed that the nonserum oviduct specific proteins with MW130, 100 and 80 kD existed in human oviduct fluid or oviductal extract. In addition, the antibody against pig oviduct antigens could more strongly cross-react with human oviduct antigens, mainly recognizing 130,116 and 100 kD proteins from human oviduct. It is suggested that in human oviduct there are some specific antigens possessing some similar epitopes to those in pig oviducts. This result seems to be consistent with predominant cross reactivity existing in antigens of porcine and human zona pellucida.