Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.     

Low level sequence variant analysis of recombinant proteins: an optimized approach

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.     

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells: A novel therapeutic modality

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP/GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.Key words: prostate cancer, proteasome inhibitor, non-steroidal modulator, apoptosis, ER stress     

Trends in CD4 Count Testing,Retention in Pre-ART Care,and ART Initiation Rates over the First Decade of Expansion of HIV Services in Haiti

Background

High attrition during the period from HIV testing to antiretroviral therapy (ART) initiation is widely reported. Though treatment guidelines have changed to broaden ART eligibility and services have been widely expanded over the past decade, data on the temporal trends in pre-ART outcomes are limited; such data would be useful to guide future policy decisions.

Methods

We evaluated temporal trends and predictors of retention for each step from HIV testing to ART initiation over the past decade at the GHESKIO clinic in Port-au-Prince Haiti. The 24,925 patients >17 years of age who received a positive HIV test at GHESKIO from March 1, 2003 to February 28, 2013 were included. Patients were followed until they remained in pre-ART care for one year or initiated ART.

Results

24,925 patients (61% female, median age 35 years) were included, and 15,008 (60%) had blood drawn for CD4 count within 12 months of HIV testing; the trend increased over time from 36% in Year 1 to 78% in Year 10 (p<0.0001). Excluding transfers, the proportion of patients who were retained in pre-ART care or initiated ART within the first year after HIV testing was 84%, 82%, 64%, and 64%, for CD4 count strata ≤200, 201 to 350, 351 to 500, and >500 cells/mm³, respectively. The trend increased over time for each CD4 strata, and in Year 10, 94%, 95%, 79%, and 74% were retained in pre-ART care or initiated ART for each CD4 strata. Predictors of pre-ART attrition included male gender, low income, and low educational status. Older age and tuberculosis (TB) at HIV testing were associated with retention in care.

Conclusions

The proportion of patients completing assessments for ART eligibility, remaining in pre-ART care, and initiating ART have increased over the last decade across all CD4 count strata, particularly among patients with CD4 count ≤350 cells/mm³. However, additional retention efforts are needed for patients with higher CD4 counts.     

Establishment of a Canine Rabies Burden in Haiti through the Implementation of a Novel Surveillance Program

The Republic of Haiti is one of only several countries in the Western Hemisphere in which canine rabies is still endemic. Estimation methods have predicted that 130 human deaths occur per year, yet existing surveillance mechanisms have detected few of these rabies cases. Likewise, canine rabies surveillance capacity has had only limited capacity, detecting only two rabid dogs per year, on average. In 2013, Haiti initiated a community-based animal rabies surveillance program comprised of two components: active community bite investigation and passive animal rabies investigation. From January 2013 –December 2014, 778 rabies suspect animals were reported for investigation. Rabies was laboratory-confirmed in 70 animals (9%) and an additional 36 cases were identified based on clinical diagnosis (5%), representing an 18-fold increase in reporting of rabid animals compared to the three years before the program was implemented. Dogs were the most frequent rabid animal (90%). Testing and observation ruled out rabies in 61% of animals investigated. A total of 639 bite victims were reported to the program and an additional 364 bite victims who had not sought medical care were identified during the course of investigations. Only 31% of people with likely rabies exposures had initiated rabies post-exposure prophylaxis prior to the investigation. Rabies is a neglected disease in-part due to a lack of surveillance and understanding about the burden. The surveillance methods employed by this program established a much higher burden of canine rabies in Haiti than previously recognized. The active, community-based bite investigations identified numerous additional rabies exposures and bite victims were referred for appropriate medical care, averting potential human rabies deaths. The use of community-based rabies surveillance programs such as HARSP should be considered in canine rabies endemic countries.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.